香鱼凝血因子X基因表达与鳗利斯顿氏菌感染的相关性

凝血途径的关键因子——凝血因子X(coagulation factor X,FX),是一种与免疫调控密切相关的维生素K依赖型丝氨酸蛋白酶.该研究克隆了香鱼(Plecoglossus altivelis) FX基因全长cDNA序列,它由1817个核苷酸组成,包含一个大的开放阅读框,编码一个由453个氨基酸组成的相对分子质量为5.07×104的蛋白.香鱼FX与已知的哺乳动物FX的结构相似,N端24个残基为信号肽序列.序列比较表明,香鱼FX与斑马鱼FX的氨基酸同一性最高,为53％.在健康香鱼中,FX基因mRNA主要在肝组织中表达,脑和鳃中也有少量表达.实时荧光定量PCR分析揭示,鳗利斯顿氏菌感染后香鱼肝组织中FX基因mRNA表达显著上调,16h时达到5.43倍.通过原核表达系统表达了香鱼FX丝氨酸蛋白酶结构域,并制备了抗血清.Western blotting分析显示,鳗利斯顿氏菌感染后香鱼血清中FX含量显著增加,并随着时间延长上调倍数不断增大,在36h时达到3.68倍.据此,FX基因mRNA及蛋白的表达与鳗利斯顿氏菌感染香鱼的过程紧密相关,揭示它可能在鱼类抗细菌感染的免疫反应中起重要作用.     

Isolation of bacteriophages specific to a fish pathogen, Pseudomonas plecoglossicida, as a candidate for disease control

Two types of bacteriophage specific to Pseudomonas plecoglossicida, the causative agent of bacterial hemorrhagic ascites disease in cultured ayu fish (Plecoglossus altivelis), were isolated from diseased ayu and the rearing pond water. One type of phage, which formed small plaques, was tentatively classified as a member of the family Myoviridae, and the other type, which formed large plaques, was classified as a member of the family Podoviridae. All 27 strains of P. plecoglossicida examined, which were isolated from diseased ayu from geographically different areas in 1991 to 1999, exhibited quite similar sensitivities to either type of phage. One strain of P. plecoglossicida was highly virulent for ayu, and the 50% lethal dose (LD(50)) when intramuscular injection was used was 10(1.2) CFU fish(-1); in contrast, phage-resistant variants of this organism were less virulent (LD(50), >10(4) CFU fish(-1)). Oral administration of phage-impregnated feed to ayu resulted in protection against experimental infection with P. plecoglossicida. After oral administration of P. plecoglossicida cells of this bacterium were always detected in the kidneys of control fish that did not receive the phage treatment, while the cells quickly disappeared from the phage-treated fish. Bacterial growth in freshwater was lower in the presence of phage, and the number of phage PFU increased rapidly. These results suggest that it may be possible to use phage to control the disease caused by P. plecoglossicida.     

Increased liver protein and mRNA expression of natural killer cell-enhancing factor B (NKEF-B) in ayu (Plecoglossus altivelis) after Aeromonas hydrophila infection

Natural killer cell-enhancing factor (NKEF) may mediate cellular responses to proinflammatory molecules. The liver proteins of Aeromonas hydrophila-infected ayu (Plecoglossus altivelis) and healthy control fish were analyzed by 2DE. A protein, which increased significantly in diseased fish, was identified as NKEF-B by MALDI-TOF-MS. A full-length cDNA clone of this protein was subsequently isolated. It contains 1092 bp with an open reading frame of 591 bp, coding for 197 amino acids with MW 21.9 kDa and pI 6.38, values similar to those determined by 2DE. Ayu NKEF-B had highest similarity (93.1% amino acid identity) to those of carp and zebrafish. Phylogenetic analysis showed that ayu NKEF-B falls into the fish NKEF-B cluster and is most closely related to that of carp and zebrafish. It was determined that ayu NKEF-B mRNA expression was significantly increased in many tissues at the early stage of bacterial infection. In conclusion, the increased NKEF-B mRNA and protein expression in ayu were closely associated with A. hydrophila infection.     

香鱼补体成分C9基因的克隆、序列分析及表达

补体成分C9是构成膜攻击复合体引起靶细胞溶解破坏的重要组成成分。该文测定了香鱼C9(aC9)基因的cDNA全序列,序列全长2125个核苷酸,编码一个由592个氨基酸组成、相对分子质量为6.56×104的前体蛋白,N端22个氨基酸为信号肽序列。序列分析表明,aC9与虹鳟C9的氨基酸同源性最高,达56.8%,与其它鱼类C9的同源性介于40.9%~53.8%之间。aC9在健康香鱼肝、脾、肠、鳃和肌肉有表达,其中在肝内的表达量最高。实时荧光定量PCR的结果显示,鳗利斯顿氏菌侵染4h后,肝中aC9mRNA表达量显著上调,并随着时间的推移在16h时达到峰值。Westernblotting分析的结果显示,鳗利斯顿氏菌侵染后香鱼血清中的aC9蛋白随着时间的推移呈显著上调。以上结果表明,香鱼肝组织C9基因表达变化与鳗利斯顿氏菌的侵染密切相关,揭示了C9在鱼类抗细菌免疫反应中具有重要的作用。     

Bacteriophage control of Pseudomonas plecoglossicida infection in ayu Plecoglossus altivelis

Two previously isolated phages were used to examine the therapeutic effects against Pseudomonas plecoglossicida infection in ayu Plecoglossus altivelis. Phage PPp-W4 (Podoviridae) inhibited the in vitro growth of P. plecoglossicida more effectively than Phage PPpW-3 (Myoviridae), and a mixture (PPpW-3/W-4) of the 2 phages exhibited the highest inhibitory activity. In phage therapy experiments, ayu were fed P. plecoglossicida-impregnated feed (10(7) CFU fish(-1)) and then fed phage-impregnated feed (10(7) PFU fish(-1)). Mortalities of fish receiving PPpW-3, PPpW-4, PPpW-3/W-4, and a control fish receiving no phages were 53.3, 40.0, 20.0 and 93.3%, respectively. Phage (PPpW-3/W-4)-receiving fish also showed high protection against water-borne infection with P. plecoglossicida. In a field trial, when phage (PPpW-3/W-4)-impregnated feed was administered to ayu in a pond where the disease occurred naturally, daily mortality of fish decreased at a constant level (5% d(-1)) to one-third after a 2 wk period. The causal relationship of phages in this phenomenon was verified by the long-lasting appearance of administered phages in the kidneys of the fish, and a disappearance of P. plecoglossicida from apparently healthy fish. Neither phage-resistant organisms nor phage-neutralizing antibodies were detected in diseased fish or apparently healthy fish, respectively. These results indicate the potential for phage control of the disease.     

Pathogenesis of experimentally induced bacterial cold water disease in ayu Plecoglossus altivelis

Ayu Plecoglossus altivelis were experimentally infected with Flavobacterium psychrophilum, which is the causative agent of bacterial cold water disease (CWD). The fish infected by immersion usually died within an hour after they became moribund. The blood volume and haematocrit values of moribund fish were low, while those values of many infected fish that were not moribund were in the range of controls. Most of the affected fish in the immersion-infected groups had ulcerative lesions on their lower jaw. No histological evidence of haemolysis was observed. These results suggest that rapid bleeding occurred through ulcerative lesions, probably causing hypoxia which killed the fish. Ulcerative lesions developed on the dorsal skin when this area had been slightly abraded artificially prior to immersion challenge. Histologically, F. psychrophilum was initially found on the skin that had microscopic injuries, but not on normal skin. The bacterium then entered the dermis and migrated through connective tissues. The lesions subsequently expanded into the underlying musculature through the myosepta, developed necrotic myositis and formed externally open ulcers. Only in later stages of infection did mild lesions develop in the internal organs and the gill, probably caused by the bacterium migrating through blood vessels. This suggests that infection with CWD through the gill or digestive tract is unlikely. Virtually no open lesions were found in ayu challenged by intramuscular injections except at the injection sites. The results suggest that skin injuries are major portals of entry for F. psychrophilum in ayu, and the bacterium has affinity for collagenous connective tissues.     

Vibrio cholerae non-O1 isolated from ayu fish (Plecoglossus altivelis) in Japan.

A fish pathogen, Vibrio cholerae non-O1, was isolated from diseased ayu fish (Plecoglossus altivelis) collected from rivers in eight prefectural districts of Japan. This organism was found to have biochemical characteristics similar to those of V. cholerae non-O1, except that our isolates were negative for ornithine decarboxylase. Antiserum against an ayu isolate did not agglutinate with the majority of environmental V. cholerae non-O1 isolates, but a major O antigen was common among the ayu isolates. All strains were hemolytic to sheep erythrocytes, and oral administration of culture supernatants induced fluid accumulation in suckling mice. However, the crude toxin was not lethal to adult mice, and no cholera toxin-like enterotoxins were detected.     

Vibrio cholerae non-O1 isolated from ayu fish (Plecoglossus altivelis) in Japan.

A fish pathogen, Vibrio cholerae non-O1, was isolated from diseased ayu fish (Plecoglossus altivelis) collected from rivers in eight prefectural districts of Japan. This organism was found to have biochemical characteristics similar to those of V. cholerae non-O1, except that our isolates were negative for ornithine decarboxylase. Antiserum against an ayu isolate did not agglutinate with the majority of environmental V. cholerae non-O1 isolates, but a major O antigen was common among the ayu isolates. All strains were hemolytic to sheep erythrocytes, and oral administration of culture supernatants induced fluid accumulation in suckling mice. However, the crude toxin was not lethal to adult mice, and no cholera toxin-like enterotoxins were detected.