Genotypic and phenotypic analysis of Elizabethkingia meningoseptica in bullfrog Rana catesbeiana isolated in Taiwan.

Elizabethkingia meningoseptica is a hazardous bacterium for agriculture production and human health. The present study identified E. meningoseptica from the bullfrog, human and reference strain BCRC 10677 by API 20NE, 50S ribosome protein L27 sequencing and pulse field gel electrophoresis to differentiate isolates of E. meningoseptica from aquatic animals and humans. All isolates from bullfrogs and humans were identified as E. meningoseptica by DNA sequencing with 98.8%-100% sequence identity. E. meningoseptica displayed significant genetic diversity when analysed using pulsed-field gel electrophoresis (PFGE). There were six distinct pulsotypes, including one pulsotype found in bullfrog isolates and five pulsotypes found in human isolates. However, E. meningoseptica from bullfrog exhibited one genotype only by PFGE. Overall, molecular epidemiological analysis of PFGE results indicated that the frog E. meningoseptica outbreaks in Taiwan were produced by genetically identical clones. The bullfrog isolates were not genetically related to other E. meningoseptica from human and reference isolates. This research provided the first comparisons of biochemical characteristics and genetic differences of E. meningoseptica from human and bullfrog isolates.

Skin Cortisol and Acoustic Activity: Potential Tools to Evaluate Stress and Welfare in Captive Cetaceans.

As people's focus broadens from animals on farms to zoos and aquaria, the field of welfare science and the public's concern for animal welfare continue to grow. In captive animals, stress and its causes are topics of interest in welfare issues, and the identification of an objective method that can be used to assess animals' stress as a physiological state is essential. Both behavioral and physiological parameters can be used as indicators in order to assess animal stress quantitatively. To validate this approach, acoustic activity and the sloughed scrape skin cortisol concentration were used to evaluate the animal welfare of captive beluga whales (Delphinapterus leucas). The acoustic activity (5 min at 10:00 am) of three captive D. leucas was routinely recorded by a transducer and analyzed using audio editing software. The calls were separated into three main categories: whistles, pulses, and combo calls. The sloughed scrape skin samples were collected non-invasively once a week from all three animals' fluke and/or flipper. Cortisol was extracted using a modified skin steroid extraction technique, and detected via commercially available enzyme immunoassays. The results showed that the cortisol concentration increased by varying levels when the whales encountered the same event. In addition, the number and distribution of the calls changed along with the events. This indicated that the changes in the cortisol concentration and acoustic behavior may have reflected the fluctuations in the environment and body condition. Therefore, the scrape cortisol measurement and acoustic recordings could be used to monitor stress levels in captive beluga whales. We recommend that aquaria consider incorporating skin scrape cortisol and acoustic activity monitoring into their standards for animal welfare.

Preparation of recombinant glycoprotein B (gB) of Chelonid herpesvirus 5 (ChHV5) for antibody production and its application for infection detection in sea turtles.

The Chelonid herpesvirus 5 (ChHV5) infection possibly associated to the fibropapillomatosis (FP) disease in sea turtles worldwide remains largely unknown and limited studies have used serological approaches to detection of antibodies against ChHV5 in sea turtles with or without FP. We aimed to develop diagnostic platforms based on the viral glycoprotein B (gB) for ChHV5 infection. In this study, five recombinant sub-fragments of the gB protein were successfully expressed and subsequently served as antigens for both seroprevalence and antibody production. The results indicated that the five expressed proteins harbored antigenicity, shown by the results of using sera from sea turtles that were PCR-positive for ChHV5. Moreover, seropositive sea turtles were significantly associated with FP (p < 0.05). We further used the expressed protein to produce antibodies for immunohistochemical analysis, and found that the in-house-generated sera specifically stained FP lesions while normal epithelium tissues remained negative. Of major importance, the reactivity in the ballooning degeneration area was much stronger than that in other regions of the FP lesion/tumour, thus indicating ChHV5 viral activities. In summary, the developed serological test and specific anti-gB antibodies for IHC analysis could be applied for further understanding of epidemiological distributions of ChHV5 infection in sea turtles, and studies of ChHV5 pathogenesis.

Lymphocystis Disease Virus Infection in Clownfish Amphiprion ocellaris and Amphiprion clarkii in Taiwan.

Lymphocystic disease affects over 150 species of marine and freshwater fish worldwide. In this study, the lymphocystis pathogen was found in 2 (Amphiprion ocellaris and Amphiprion clarkii) of the 9 species of clownfish. Detection of lymphocystis disease virus (LCDV) was based on histopathological study, electron microscope observation of virus particles and gene sequence analysis from the MCP region. Infected A. ocellaris hosts showed sparse, multifocal, white, stiff, papilloma-like nodules on the body, skin, gills and fins; while, on A. clarkia, nodules were found on the operculum skin. Histopathologic study showed lymphocystic cells with an irregular nucleus, enlarged cytoplasm and intracytoplasmic inclusion bodies surrounded by the cell membrane. The viral particle presents virions 180-230 nm in diameter, hexagonal in shape with an inner dense nucleoid under transmission electron micrographs (TEM). From the ML polygenetic tree, the clownfish LCVD genotype was closely related to the LCDV strain from paradise fish, Macropodus opercularis (KJ408271) (pairwise distance: 92.5%) from China, then followed by the strain from Spain (GU320726 and GU320736) (pairwise distance: 90.8-90.5%), Korea (AB299163, AB212999, AB213004, and AB299164) (pairwise distance: 91.5-80.5%) and lastly Canada (GU939626) (pairwise distance: 83%). This is the first report of lymphocystis disease in A. clarkii in Taiwan.

Development and Validation of KASP Assays for the Genotyping of Racing Performance-Associated Single Nucleotide Polymorphisms in Pigeons.

Pigeon racing's recent upturn in popularity can be attributed in part to the huge prize money involved in these competitions. As such, methods to select pigeons with desirable genetic characteristics for racing or for selective breeding have also been gaining more interest. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for genotyping-specific genes is one of the most commonly used molecular techniques, which can be costly, laborious and time consuming. The present study reports the development of an alternative genotyping method that employs Kompetitive Allele Specific Polymerase Chain Reaction (KASP) technology with specifically designed primers to detect previously reported racing performance-associated polymorphisms within the LDHA, MTYCB, and DRD4 genes. To validate, KASP assays and PCR-RFLP assays results from 107 samples genotyped for each of the genes were compared and the results showed perfect (100%) agreement of both methods. The developed KASP assays present an alternative rapid, reliable, and cost-effective method to identify polymorphisms in pigeons.

Antimicrobial-resistance profiles of gram-negative bacteria isolated from green turtles (Chelonia mydas) in Taiwan.

The green turtle (Chelonia mydas) is listed as a globally endangered species and is vulnerable to anthropogenic threats, including environmental pollution. This study investigated the antimicrobial resistance of Gram-negative bacteria isolated from wild green turtles admitted to a sea turtle rehabilitation center in Taiwan. For this investigation, cloacal and nasal swab samples were collected from 28 green turtles between 2018 and 2020, from which a total of 47 Gram-negative bacterial isolates were identified. Among these, Vibrio spp. were the most dominant isolate (31.91%), and 89.36% of the 47 isolates showed resistance to at least one of 18 antimicrobial agents tested. Isolates resistant to one (6.38%), two (8.51%), and multiple (74.47%) antimicrobials were observed. The antimicrobial agents to which isolates showed the greatest resistance were penicillin (74.47%), followed by spiramycin, amoxicillin, and cephalexin. The antimicrobial-resistance profiles identified in this study provide useful information for the clinical treatment of sea turtles in rehabilitation facilities. The results of our study also imply that wild green turtles may be exposed to polluting effluents containing antimicrobials when the turtles traverse migratory corridors or forage in feeding habitats. To benefit sea turtle conservation, future research should focus on (1) how to prevent pollution from antimicrobials in major green turtle activity areas and (2) identifying sources of antimicrobial-resistant bacterial strains in coastal waters of Taiwan.

Establishment of loop-mediated isothermal amplification for rapid and convenient detection of Mycobacterium marinum complex.

Mycobacterium marinum is a zoonotic pathogen that can cause dermatological infection mainly from contaminated water or fish. Some well-known genetically similar species and subspecies are M. lifrandii and M. pseudoshottsii from amphibians and fish in aquaculture, and M. ulcerans, a causative agent of a neglected tropical disease (NTD), Buruli ulcer. They are believed to survive in water as their major niche, which might be related to their source of infection, but detailed ecological surveillance of the species complex remains to be done. Herein, we present a new detection system for M. marinum complex based on isothermal DNA amplification that can be conducted conveniently with high sensitivity and specificity. The target was a chromosomal gene, mrsA, including a restriction polymorphism between M. ulcerans (except for the most ancestral subspecies, M. ulcerans subsp. shinshuense) and the other species. The system was able to detect less than 500 fg (approximately 70 copies) of genomic DNA of M. marinum, within 60 min, and caused no amplification from mycobacterial species other than M. marinum complex species. It was also verified that restriction of the amplified DNA fragments was able to discriminate M. ulcerans as expected. This easy, quick, and convenient system is expected to facilitate detection of M. marinum complex from various resources.

A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas).

Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

A formalin-inactivated vaccine provides good protection against Vibrio harveyi infection in orange-spotted grouper (Epinephelus coioides).

Vibrio harveyi is one of the most common threats to farmed grouper, so considerable efforts are in practice to control the pathogen. This study presents a highly effective vaccine against V. harveyi in the orange-spotted grouper with the help of a single intraperitoneal immunization. The vaccine candidate was in form of whole, formalin-inactivated V. harveyi cells combined with a metabolizable ISA763 AVG adjuvant. Our results indicated that the vaccine triggered a remarkably higher expression level of interleukin (IL)-1ß, IL-6, IL-8, and IL-10 in the groupers' kidneys and spleens, as recorded after 1 and 3 days of immunization. Antibody titers were significantly elevated in the vaccinated fish. A pivotal observation was that the vaccine highly protected the grouper from a homologous V. harveyi strain challenge with relative percentage survival values of 100% and 91.7% at 6 and 12 weeks post-immunization, respectively. Vaccinated fish also demonstrated strong cross-protection against a heterologous bacterial isolate challenge. Therefore, the inactivated V. harveyi vaccine is a promising candidate that could stimulate good immune responses and confer remarkable protection in farmed groupers.

Transcriptome analysis of grey mullet (Mugil cephalus) after challenge with Lactococcus garvieae.

Grey mullet (Mugil cephalus) is an economically important fish species in Taiwan mariculture industry. Moreover, grey mullet are common hosts of a bacterial infection by Lactococcus garvieae. However, until now the information related to the immune system of grey mullet is unclear. Therefore, to understand the molecular basis underlying the host immune response to L. garvieae infection, Illumina HiSeq™ 2000 was used to analyse the head kidney and spleen transcriptome of infected grey mullet. De novo assembly of paired-end reads yielded 55,203 unigenes. Comparative analysis of the expression profiles between bacterial challenge fish and control fish identified a total of 7192 from head kidney and 7280 in spleen differentially expressed genes (P < 0.05), including 4211 upregulated genes and 2981 downregulated genes in head kidney, while in spleen 3598 genes were upregulated and 3682 downregulated. A significant enrichment analysis of these differentially expressed genes (DEG) in spleen and head kidney revealed major immune-related pathways, including complement and coagulation cascades, Toll-like receptor signalling, and antigen processing and presentation. Moreover, selected DEGs were validated using qPCR. Altogether, the results obtained on immune-related genes may allow for a better understanding of immunity in grey mullet to Lactococcus garvieae, carrying out detailed functional analysis of these genes and developing strategies for efficient immune protection against infections in grey mullet.

Identification, Molecular Cloning of IL-1ß and Its Expression Profile during Nocardia seriolae Infection in Largemouth Bass, Micropterus salmoides.

In the present study, IL-1ß cDNA was identified and analyzed from largemouth bass (Micropterus salmoides). Full length IL-1ß mRNA was obtained using Rapid Amplification of cDNA Ends (RACE), which contains 78 bp 3'-UTR, a 455 bp 5'-UTR, and an open reading frame (ORF) of 702 bp coding for 233 amino acid residues. The molecular weight and theoretical isoelectric point of largemouth bass IL-1ß protein was predicted to be 26.7 kDa and 6.08 respectively. A largemouth bass IL-1ß phylogenetic analysis showed a close relation to the IL-1ßs of striped trumpeter (Latris lineata), Chinese perch (Siniperca chuatsi), and Japanese sea bass (Lateolabrax japonicus). Peptidoglycan upregulated IL-1ß in the spleen and head kidney, while lipopolysaccharide upregulated detectable levels of IL-1ß in the spleen only. Largemouth bass, challenged with Nocardia seriolae (1.0 × 106 cfu/mL), showed a significant increase in IL-1ß at 3 and 5 days post infection (dpi) in the spleen, while in the head kidney significant expression was found at 2 and 3 dpi, peaking at 3 dpi. Furthermore, tumor necrosis factor α (TNF-α) showed significantly higher expression in the spleen at 3 and 5 dpi, and in the head kidney at 1 and 3 dpi, with expression decreasing at 5 dpi in both tissues.

De Novo Transcriptome Analysis of Differential Functional Gene Expression in Largemouth Bass (Micropterus salmoides) after Challenge with Nocardia seriolae.

Largemouth bass (Micropterus salmoides) are common hosts of an epizootic bacterial infection by Nocardia seriolae. We conducted transcriptome profiling of M. salmoides to understand the host immune response to N. seriolae infection, using the Illumina sequencing platform. De novo assembly of paired-end reads yielded 47,881 unigenes, the total length, average length, N50, and GC content of which were 49,734,288, 1038, 1983 bp, and 45.94%, respectively. Annotation was performed by comparison against non-redundant protein sequence (NR), non-redundant nucleotide (NT), Swiss-Prot, Clusters of Orthologous Groups (COG), Kyoto Encyclopaedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Interpro databases, yielding 28,964 (NR: 60.49%), 36,686 (NT: 76.62%), 24,830 (Swissprot: 51.86%), 8913 (COG: 18.61%), 20,329 (KEGG: 42.46%), 835 (GO: 1.74%), and 22,194 (Interpro: 46.35%) unigenes. Additionally, 8913 unigenes were classified into 25 Clusters of Orthologous Groups (KOGs) categories, and 20,329 unigenes were assigned to 244 specific signalling pathways. RNA-Seq by Expectation Maximization (RSEM) and PossionDis were used to determine significantly differentially expressed genes (False Discovery Rate (FDR) < 0.05) and we found that 1384 were upregulated genes and 1542 were downregulated genes, and further confirmed their regulations using reverse transcription quantitative PCR (RT-qPCR). Altogether, these results provide information on immune mechanisms induced during bacterial infection in largemouth bass, which may facilitate the prevention of nocardiosis.

Phosphoglycerate kinase enhanced immunity of the whole cell of Streptococcus agalactiae in tilapia, Oreochromis niloticus.

Streptococcus agalactiae is a Gram-positive bacterium and a severe aquaculture pathogen that can infect a wide range of warmwater fish species. The outer-surface proteins in bacterial pathogens play an important role in pathogenesis. We evaluated the immunogenicity of two of the identified surface proteins namely phosphoglycerate kinase (PGK) and ornithine carbamoyl-transferase (OCT). PGK and OCT were over-expressed and purified from Escherichia coli and used as the subunit vaccines in tilapia. Tilapia immunized with the S. agalactiae modified bacteria vaccine (whole cell preparations with recombinant PGK and OCT proteins) individually were tested for the efficacy. OCT and PGK combined with WC had a higher survival rate. A high-level protection and significant specific antibody responses against S. agalactiae challenge was observed upon the vaccinated tilapia with the purified PGK protein and S. agalactiae whole cells. The specific antibody titer against S. agalactiae antigen suggested that increased antibody titers were correlated with post-challenge survival rate. Il-1ß expression profile was higher in PGK + WC-treated group. Tnf-α expression in the PGK + WC group was significantly increased. Taken together, our results suggested the combinations of recombinant protein and whole cell may elicit immune responses that reach greater protection than that of individual S. agalactiae components.

Efficacy of a formalin-inactivated vaccine against Streptococcus iniae infection in the farmed grouper Epinephelus coioides by intraperitoneal immunization.

Vaccination is the most effective means of preventing infectious diseases; however, few vaccines are effective against Streptococcus iniae (S. iniae) in grouper. This work presents an efficacious and safe vaccine against S. iniae infections in the grouper Epinephelus coioides. The vaccine candidate was the S. iniae GSI-310 strain. The vaccination was administered by intraperitoneal injection, and consisted of formalin-inactivated antigens combined with an AS-F or ISA763A adjuvant. Peripheral blood samples were collected for RT-qPCR and phagocytosis and agglutination assays. Our results indicated that immunoglobulin M (igm) was maximally expressed in the two vaccinated groups at 3 months post-secondary vaccination (PSV). A significant upregulation of mRNA expression for interleukin-1ß (il-1ß) and tumor necrosis factor-α (tnf-α) was also observed in fish treated with antigens combined with ISA763A, which peaked at 3 months PSV. In fish treated with antigens combined with AS-F, il-1ß and tnf-α expression peaked at 14 days post-primary vaccination (PPV). Phagocytic activity and index increased significantly in the two vaccinated groups. Furthermore, fish in the two vaccinated groups exhibited significantly elevated agglutination titers compared to fish in the control group, in which almost no agglutination reaction was detected. In the efficacy test, the vaccinated and control groupers were treated with S. iniae at 1, 3, and 6 months PSV. The relative percentage survival (RPS) values of antigens with AS-F and antigens with ISA763A were both 100% at 1 and 3 months PSV; at 6 months PSV, the RPS values for these groups were 100% and 97.7%, respectively. Furthermore, the level of protection observed in the field trial closely resembled that achieved on a laboratory scale. Therefore, the proposed vaccine mixed with AS-F or ISA763A improved immune responses and provided safe and long-lasting protection in farmed groupers.

Vaccine efficacy of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Edwardsiella ictaluri against E. tarda in tilapia.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), derived from the outer-membrane protein (OMP) fraction, has been used as a potential candidate for vaccine development. The gene-encoding 37 kDa GAPDH outer membrane protein (OMP) from Edwardsiella ictaluri was amplified using polymerase chain reaction (PCR) and was cloned and expressed in Escherichia coli BL21 (DE3). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and nucleotide and amino acid sequencing were used to analyze the expressed antigenic protein and gene encoding this protein. Comparative DNA and protein sequence analysis of GAPDH from E. ictaluri GAPDHs from several Gram-negative bacterial species within the Enterobacteriaceae family revealed that the GAPDHs within this group are highly conserved and share a sequence similarity of 75-100% with E. ictaluri GDPDH. Rabbit antiserum raised against the E. ictaluri recombinant GAPDH (rGAPDH) protein recognized purified GADPH, indicating that it has a strong immunogenicity. Tilapia fish were intraperitoneally immunized with formalin-killed E. ictaluri whole cells, and rGAPDH (30 µg fish(-1)) from E. ictaluri, both of which were emulsified in ISA 763A adjuvant. At 3 months after immunization, fish were challenged with the E. tarda strain to assess vaccine efficacy; the relative percent survival (RPS) values were found to exceed 71.4%. The specific mean antibody titer log2 level of groups vaccinated with rGAPDH at 3 months was significantly higher than that of non-vaccinated fish (control group). Therefore, this recombinant protein can be considered a multi-purpose candidate vaccine against several pathogenic bacteria.

Immunoprotection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Lactococcus garvieae against Lactococcosis in tilapia.

In this study, the gene encoding 40 kDa GAPDH of L. garvieae was determined and overexpressed by using the Escherichia coli expression system. Analysis results indicated that the sequences of GAPDH of L. garvieae nucleotide and its amino acid are highly homologous (80.4-100%) to several products of GAPDH from L. garvieae and other Streptococcus-related bacteria. According to Western blotting results, rabbit antiserum and tilapia infection serum reacted strongly to the recombinant GAPDH protein. In another experiment, tilapia were immunized intraperitoneally with formalin-killed L. garvieae whole cells, recombinant GAPDH (50 µg fish(-1)) from L. garvieae or both. ISA 763A was used as an adjuvant for vaccine and saline was used as a negative control. The fish challenged at 4 weeks after immunization with GAPDH+WC+ISA had the highest survival rate at 100%, followed by fish immunized with WC+ISA or GAPDH+ISA, which had RPS values of 87.5% and 50%, respectively. Additionally, specific antibody responses against L. garvieae whole cells and GAPDH were based on enzyme-linked immunosorbent assay. Following 4 weeks of immunization, the specific antibody level of all vaccine groups significantly increased, except for antibody responses against L. garvieae GAPDH of those immunized with formalin-killed L. garvieae whole cells. Our results further demonstrated that GAPDH from L. garvieae protected tilapia from experimental L. garvieae infection, implying the potential use of L. garvieae GAPDH as a vaccine against L. garvieae.

Comparison of genetic characteristics and pathogenicity of Lactococcus garvieae isolated from aquatic animals in Taiwan.

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 ApaI and SmaI 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.

Epidemiology and phylogenetic analysis of Taura syndrome virus in cultured Pacific white shrimp Litopenaeus vannamei B. in Taiwan.

Taura syndrome virus (TSV) has spread worldwide, causing significant economic losses since Taura syndrome was first described in Ecuador in 1992. To determine the prevalence and impact of TSV infection on the shrimp farming industry in Taiwan, Pacific white shrimp Litopenaeus vannamei B. were collected from 220 farms between 2004 and 2006 for viral detection by reverse transcription polymerase chain reaction. Data showed that the overall TSV prevalence rate was 20% (43/220 farms). Comparing shrimp growth stages, TSV prevalence rates were 4% for postlarvae, 24% for juveniles, 24% for subadults, 32% for adults, and 5% for brooders. Among TSV-positive farms, average infection incidence was 35% in postlarvae farms, 55% in juvenile farms, 39% in subadult farms, 31% in adult farms, and 20% in brooder farms. Notably, TSV was also detected in Exopalaemon orientis H. from 1 of 10 farms. Tail fans and appendages had red pigmentation, which is characteristic of TSV infection. Of shrimp with pathological lesions, 100% had lesions on tail fans, 88% on appendages, and 80% in gills. Sequence comparison using the TSV VP1 (structural protein) gene showed that 9 isolates from the farms had 92.3 to 99.5% nucleotide sequence identity with strains in the GenBank database from Taiwan (AF406789 and AY355310) and Venezuela (DQ212790). This is the first broad epidemiological study of TSV infection in L. vannamei in Taiwan.

Upregulation of actin-like gene expression in giant freshwater prawns Macrobrachium rosenbergii infected with Metschnikowia bicuspidata.

The giant freshwater prawn Macrobrachium rosenbergii is commercially cultured throughout the world including Taiwan. From 1992 to 1995, Taiwanese production decreased by approximately 50% due to disease. The yeast Metschnikowia bicuspidata is considered to be one of the major causes of white muscle disease, but the molecular mechanism of its pathogenesis is not known. Using RNA differential display (DD) with muscle and hepatopancreatic tissue, we identified a 324 nucleotide (nt) message specifically expressed by M. rosenbergii infected with M. bicuspidata but not in the controls. A ribonuclease protection assay (RPA) confirmed expression in both tissues. RPA data also revealed an additional 230 bp mRNA message that was not identified by DD. Using RNA ligase-mediated rapid amplification of 5' cDNA ends (5'-RACE), we successfully isolated a 1357 bp full-length gene (c57) that showed 92 and 87% sequence identity to the actin gene of the Kuruma shrimp Marsupenaeus japonicus (also called Penaeus japonicus) (GenBank accession number AB055975) and the beta-actin gene of the white shrimp Litopenaeus vannamei (also called Penaeus vannamei) (GenBank accession number AF300705), respectively. The deduced amino acid sequence of c57 showed 83 % sequence similarity to M. japonicus and L. vannamei actin proteins. Based on this high homology, we suggest that upregulation of actin expression in the muscle and hepatopancreas is part of the shrimp response to M. bicuspidata infection. Increased expression may be related to repair of tissues damaged by yeast infection.

Systemic infection of Kudoa lutjanus n. sp. (Myxozoa: Myxosporea) in red snapper Lutjanus erythropterus from Taiwan.

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 microm in width and 7.3 +/- 0.53 microm 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 microm in length and 2.2 +/- 0.49 microm 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.