De-Hai Xu

Parasitism by protozoan Ichthyophthirius multifiliis enhanced invasion of Aeromonas hydrophila in tissues of channel catfish.

Protozoan Ichthyophthirius multifiliis Fouquet (Ich) and bacterium Aeromonas hydrophila are two common pathogens of cultured fish, which cause high fish mortality. Currently there is no information available for the effect of parasitism by Ich on survival of channel catfish and invasion of A. hydrophila in fish tissues following exposure to A. hydrophila. A trial was conducted in this study to: (1) determine whether A. hydrophila increased fish mortality in Ich-parasitized channel catfish; and (2) compare the bacterial quantity in different tissues between non-parasitized and Ich-parasitized catfish by real-time polymerase chain reaction (qPCR). The results demonstrated that the Ich-parasitized catfish showed significantly (P<0.05) higher mortality (80%) when exposed to A. hydrophila by immersion than non-parasitized fish (22%). Low mortality was observed in catfish exposed to Ich alone (35%) or A. hydrophila alone (22%). A. hydrophila in fish tissues were quantified by qPCR using a pair of gene-specific primers and reported as genome equivalents per mg of tissue (GEs/mg). Skin, gill, kidney, liver and spleen in Ich-parasitized fish showed significantly higher load of A. hydrophila (9400-188,300 GEs/mg) than non-parasitized fish (4700-42,100 GEs/mg) after exposure to A. hydrophila. This study provides evidence that parasite infections enhance bacterial invasion and cause high fish mortality.

Molecular responses of calreticulin genes to iron overload and bacterial challenge in channel catfish (Ictalurus punctatus)

Infection and inflammation are often accompanied by oxidative stress caused by the accumulation of reactive oxygen species which can be deleterious to the health of the host. Antioxidant defense mechanisms and components are crucial in limiting cellular and tissue-level damage and restoring homeostasis. In mammals, calreticulin is a 46-kDa multifunctional calcium binding protein of the endoplasmic reticulum that has many critical functions in the eukaryotic cell including regulation of intracellular calcium homoeostasis, lectin binding and chaperoning, and oxidative stress responses. In previous studies from our lab, the calreticulin gene was observed to be strongly upregulated in catfish during challenge with infectious Gram-negative bacteria. However, little is known about the function of this gene in teleost fish. The objective of this study, therefore, was to characterize the calreticulin gene from channel catfish, to determine its genomic organization, to profile its patterns of tissue expression, and to establish its potential for physiological antioxidant and immune responses in catfish after bacterial infection with Edwardsiella ictaluri and iron treatment. Our results indicate that there are at least three calreticulin related genes in the catfish genome. The three calreticulin genes are widely expressed in various tissues under homeostatic conditions and their expression showed significant upregulation following infection and/or iron level changes.

Enhanced susceptibility of channel catfish to the bacterium Edwardsiella ictaluri after parasitism by Ichthyophthirius multifiliis.

Bacterium Edwardsiella ictaluri and parasite Ichthyophthirius multifiliis (Ich) are two common pathogens of cultured fish. The objective of this study was to evaluate the susceptibility of channel catfish Ictalurus punctatus to E. ictaluri and determine bacterial loads in different fish organs after parasitism by Ich. Fish received the following treatments: (1) infected by I. multifiliis at 5000 theronts/fish and exposed to E. ictaluri; (2) infected by I. multifiliis alone; (3) exposed to E. ictaluri alone; and (4) non-infected control. E. ictaluri in fish organs were quantified by quantitative real-time polymerase chain reaction and reported as genome equivalents per mg of tissue (GEs/mg). The results demonstrated that the Ich-parasitized catfish showed significantly (P<0.05) higher mortality (91.7%) when exposed to E. ictaluri than non-parasitized fish (10%). The bacterial loads in fish infected by 5000 theronts/fish ranged from 6497 to 163,898 GEs/mg which was between 40 and 2000 fold higher than non-parasitized fish (49-141 GEs/mg). Ich infection enhanced the susceptibility of channel catfish to bacterial invasion and increased fish mortality.

The Early Development of Ichthyophthirius multifiliis in Channel Catfish in Vitro

Abstract This study established ex vivo conditions suitable for studying early trophont development in excised tissues of channel catfish Ictalurus punctatus within the first 48 h. Infective theronts of Ichthyophthirius multifiliis were added to freshly excised channel catfish fin, gill, and skin and established fin cells (BF-2) of bluegill Lepomis macrochirus. The early development of trophonts, including growth, rotation speed, attachment, and survival (%), was studied in different tissues and at different times. In all, 86–92% of the theronts attached to fish tissues in an enter, leave, and reenter pattern within the first 10 min. Theront attachment was more than 88% in fins, gills, and skin 1 h post exposure (PE). Attachment and shape transformation of theronts were not observed on BF-2 cells. Trophonts grew to a size of 30.0 ± 3.1 μm in diameter 4 h PE in the gills, an increase of 9.6%/h. The size increase of trophonts was 1.2%/h in the gill between 4 and 8 h. Trophonts grew slowly and increased in d...

Biological characteristics and pathogenicity of a highly pathogenic Shewanella marisflavi infecting sea cucumber, Apostichopus japonicus.

Shewanella marisflavi isolate AP629 is described as a novel pathogen of sea cucumber. The LD(50) values (14 days) in sea cucumber, mice and swordtail fish were 3.89 × 10(6) , 6.80 × 10(4) and 4.85 × 10(4) CFU g(-1) body weight, respectively. Studies on S. marisflavi were conducted, including morphology, physiological and biochemical characteristics, haemolysis, whole-cell protein and 16S rDNA gene sequence. Colonies of S. marisflavi appeared faint red on marine agar and green on thiosulphate-citrate-bile salt-sucrose media. Shewanella marisflavi had polar flagella. The cells were Gram-negative, oxidase- and catalase-positive and not sensitive to O/129. The bacterium exhibited β-haemolysis on sheep blood agar and produced H(2) S. Shewanella marisflavi survived and grew at 4-35°C, pH 6.0-9.2 and in the presence of 0-8% NaCl. The whole-cell proteins included 13 discrete bands, and proteins of molecular weight 87, 44 and 39 kDa were found in all five strains of Shewanella spp. The difference in 16S rDNA gene sequences in S. marisflavi was at the 446 bp site: S. marisflavi (KCCM 41822) - G, isolate AP629 - A. This is the first report that Shewanella is pathogenic to sea cucumber.

Parasiticidal effects of Morus alba root bark extracts against Ichthyophthirius multifiliis infecting grass carp

Ichthyophthirius multifiliis (Ich), an important fish parasite, can cause significant losses in aquaculture. To find efficacious drugs to control Ich, the root bark of white mulberry Morus alba was evaluated for its antiprotozoal activity. Bark was powdered and extracted with 1 of 5 organic solvents: petroleum ether, chloroform, ethyl acetate, acetone, or methanol. The extracts were concentrated, dissolved in 0.1% (v/v) DMSO, and used for anti-Ich trials. Acetone and ethyl acetate extracts significantly reduced the survival of Ich tomonts and theronts. In vitro, acetone extract at 25 mg l-1 killed all non-encysted tomonts, at 50 mg l-1 eradicated all encysted tomonts, and at 8 mg l-1 caused mortality of all theronts. Ethyl acetate extract at 50 mg l-1 eliminated all non-encysted tomonts, at 100 mg l-1 killed all encysted tomonts and terminated tomont reproduction, and at 8 mg l-1 killed all theronts. Low concentrations (2 and 4 mg l-1) of acetone and ethyl acetate extracts could not kill all theronts after 4 h exposure, but a significant decrease in theront infectivity was observed following 30 min of pretreatment with the extracts. The 96 h LC(50) values of acetone and ethyl acetate extracts to grass carp were 79.46 and 361.05 mg l-1, i.e. much higher than effective doses for killing Ich theronts (8 mg l-1 for both extracts) and non-encysted tomonts (12.5 and 25 mg l-1, respectively). Thus M. alba extract may be a potential new, safe, and efficacious drug to control Ich.

Phenotypic and genetic characterization of bacteria isolated from diseased cultured sea cucumber Apostichopus japonicus in northeastern China

During the winter-spring from 2004 to 2006 in northeastern China cultured Japanese sea cucumber Apostichopus japonicus suffered from a serious disease. Clinical signs included swollen mouth, skin ulceration and massive mortality. Clinical samples taken during this period were studied. Thirty-one bacterial samples were isolated from diseased sea cucumbers and identified through biochemical tests, 16S rRNA gene sequence analysis and PCR amplification, followed by pathogenicity determination. The results showed that the 31 isolates belonged to the genera Vibrio (64.5%), Shewanella (12.9%), Serratia (12.9%), Pseudoalteromonas (6.4%) and Flavobacterium (3.2 %). The 3 prominent strains were Vibrio splendidus (41.9%), Shewanella (12.9%) and Serratia odorifera biogroup I (12.9%). Pathogenicity tests demonstrated that 13 out of 31 isolates were pathogenic, including 8 strains of V splendidus, 3 strains of Shewanella sp. and 2 strains of Pseudoalteromonas tetraodonis. The pathogenic V splendidus showed the highest frequency of appearance. Median lethal dose (LD50) values (14 d) of V splendidus, Shewanella sp. and P. tetraodonis were 1.74 x 10(7), 7.76 x 10(6), 7.24 x 10(7) CFU g(-1) body weight of sea cucumber, respectively. The virulences differed by species: Shewanella sp. > V splendidus> P. tetraodonis. This is the first report of Shewanella sp. virulence in sea cucumber.

Molecular responses of ceruloplasmin to Edwardsiella ictaluri infection and iron overload in channel catfish (Ictalurus punctatus).

Ceruloplasmin is a serum ferroxidase that carries more than 90% of the copper in plasma and has documented roles in iron homeostasis as well as antioxidative functions. In our previous studies, it has been shown that the ceruloplasmin gene is strongly up-regulated in catfish during challenge with Edwardsiella ictaluri. However, little is known about the function of this gene in teleost fish. The objective of this study, therefore, was to characterize the ceruloplasmin gene from channel catfish, determine its genomic organization, profile its patterns of tissue expression, and establish its potential for physiological antioxidant responses in catfish after bacterial infection with E. ictaluri and iron treatment. The genomic organization suggested that the catfish ceruloplasmin gene had 20 exons and 19 introns, encoding 1074 amino acids. Exon sizes of the catfish ceruloplasmin gene were close to or identical with mammalian and zebrafish homologs. Further phylogenetic analyses suggested that the gene was highly conserved through evolution. The catfish ceruloplasmin gene was mapped to both the catfish physical map and linkage map. The catfish ceruloplasmin gene was mainly expressed in liver with limited expression in other tissues, and it was significantly up-regulated in the liver after bacterial infection alone or after co-injection with bacteria and iron-dextran, while expression was not significantly induced with iron-dextran treatment alone.

Classification of a Hypervirulent Aeromonas hydrophila Pathotype Responsible for Epidemic Outbreaks in Warm-Water Fishes

Lineages of hypervirulent Aeromonas hydrophila (vAh) are the cause of persistent outbreaks of motile Aeromonas septicemia in warm-water fishes worldwide. Over the last decade, this virulent lineage of A. hydrophila has resulted in annual losses of millions of tons of farmed carp and catfish in the Peoples Republic of China and the United States (US). Multiple lines of evidence indicate US catfish and Asian carp isolates of A. hydrophila affiliated with sequence type 251 (ST251) share a recent common ancestor. To address the genomic context for the putative intercontinental transfer and subsequent geographic spread of this pathogen, we conducted a core genome phylogenetic analysis on 61 Aeromonas spp. genomes, of which 40 were affiliated with A. hydrophila, with 26 identified as epidemic strains. Phylogenetic analyses indicate all ST251 strains form a coherent lineage affiliated with A. hydrophila. Within this lineage, conserved genetic loci unique to A. hydrophila were identified, with some genes present in consistently higher copy numbers than in non-epidemic A. hydrophila isolates. In addition, results from analyses of representative ST251 isolates support the conclusion that multiple lineages are present within US vAh isolated from Mississippi, whereas vAh isolated from Alabama appear clonal. This is the first report of genomic heterogeneity within US vAh isolates, with some Mississippi isolates showing closer affiliation with the Asian grass carp isolate ZC1 than other vAh isolated in the US. To evaluate the biological significance of the identified heterogeneity, comparative disease challenges were conducted with representatives of different vAh genotypes. These studies revealed that isolate ZC1 yielded significantly lower mortality in channel catfish, relative to Alabama and Mississippi vAh isolates. Like other Asian vAh isolates, the ZC1 lineage contains all core genes for a complete type VI secretion system (T6SS). In contrast, more virulent US isolates retain only remnants of the T6SS (clpB, hcp, vgrG, and vasH) which may have functional implications. Collectively, these results characterize a hypervirulent A. hydrophila pathotype that affects farmed fish on multiple continents.

Enhanced mortality in Nile tilapia Oreochromis niloticus following coinfections with ichthyophthiriasis and streptococcosis.

Ichthyophthirius multifiliis Fouquet (Ich) and Streptococcus iniae are 2 major pathogens of cultured Nile tilapia Oreochromis niloticus (L). Currently there is no information available for the effect of coinfection by Ich and S. iniae on fish. The objective of this study was to determine the effects of parasite load and Ich development size on fish mortality following S. iniae infection. Low mortality (< or =20%) was observed in tilapia exposed to Ich or S. iniae alone. Mortalities increased from 38% in tilapia exposed to Ich at 10,000 theronts fish(-1) to 88% in fish at 20,000 theronts fish(-1) following S. iniae exposure. The median days to death were significantly fewer (7 d) in fish exposed to Ich at 20,000 theronts fish(-1) than fish exposed to 10,000 theronts fish(-1) (10 d). A positive correlation (correlation coefficient = 0.83) was noted between tilapia mortality and size of Ich trophonts at the time of S. iniae challenge. Fish parasitized with well-developed trophonts (Day 4, 2 x 10(7) microm3 in volume) suffered higher mortality (47.5%) than fish (10.0%) infested by young trophonts (Hour 4, 1.3 x 10(4) microm3 in volume) after S. iniae challenge. The results of this study demonstrated that both parasite load and trophont size increased susceptibility and mortality of tilapia to S. iniae infection.