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1.
柱状黄杆菌是柱形病的病原体,可感染全球的淡水鱼类。自然和养殖条件下的海水鱼类以及观赏鱼类也可感染发病。全世界每年由柱形病所造成的经济损失极其严重。文章回顾和总结了柱状黄杆菌的发现、病理变化、超微结构特点和血液学特征等方面的研究近况,对柱状黄杆菌的分离鉴定、柱形病的爆发条件及防治措施进行了概述,并提出了柱状黄杆菌研究的发展方向。  相似文献   

2.
Flavobacterium columnare is a fresh water bacterium that causes columnaris diseases in over 36 fish species. Intra-species typing of F. columnare can be performed by pulsed-field gel electrophoresis (PFGE). However, this method is hampered by the degradation of chromosomal DNA in about 10% of strains. In the current study, DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. The results substantiate that after problems due to DNases are overcome, PFGE analysis is a reproducible highly discriminating epidemiological method for studying F. columnare isolates regardless of fish host.  相似文献   

3.
The ability of a high virulence strain (AJS 1) and a low virulence strain (AJS 4) of Flavobacterium columnare (Flexibacter columnaris) to attach to the gills of black mollies (Poecilia sphenops) was investigated. For that purpose, two groups of 25 black mollies each were immersed in a bath containing 10(6) CFU/ml of F. columnare AJS 1 or AJS 4. At regular intervals from 1 to 12 h after the contact infection, fish were sacrificed and gills, skin, spleen and heart were sampled for bacteriology. Samples of the gills were taken for immunohistochemical and electron microscopic examination. Bacteriological examination proved that the number of gill-associated F. columnare was higher for AJS 1 than for AJS 4. Strain AJS 1 was isolated from the heart and spleen of 6 and 1 of the 16 examined animals, respectively. Strain AJS 4 was not isolated from the internal organs of any fish. When examined immunohistochemically, strain AJS 1 was found closely associated with gill epithelium whereas this was not the case for strain AJS 4. The adherence of bacteria to the gill tissue challenged with the virulent strain AJS 1 was also clearly demonstrated using scanning and transmission electron microscopy. These results indicate that adhesion of F. columnare to the gill tissue constitutes an important step in pathogenesis.  相似文献   

4.
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5.
Objective   To determine if juvenile pearl oysters ( Pinctada maxima ) infected with Haplosporidium hinei are also infected with another haplosporidian parasite, Minchinia occulta .
Design   Archived samples of pearl oysters infected with H. hinei were examined using polymerase chain reaction (PCR) assays and in situ hybridisation (ISH) to analyse and identify haplosporidians. A 144-bp and 220-bp region of Minchinia DNA were targeted by PCR and amplified DNA from formalin-fixed H. hinei -infected pearl oyster samples was sequenced. A 25-bp oligonucleotide probe targeting a variable section of the parasite's small subunit rRNA gene was used in ISH.
Results   The results of DNA-based diagnostic assays supported each other. The sequences obtained by PCR were found to be almost identical to M. occulta from rock oysters and the ISH assay demonstrated infection with M. occulta in affected pearl oysters. ISH indicated a prevalence of infection of 26.7% in one of the previous outbreaks.
Conclusion   Pearl oyster spat are susceptible to infection by a Minchinia parasite, most likely M. occulta , which was recently identified in rock oysters within the pearl-producing zones of Western Australia and is associated with mortalities of up to 80% in this species. The occurrence of haplosporidian co-infections in pearl oysters suggests the immunocompetence of juvenile oysters may be an important factor in preventing infection and therefore preventing mortalities such as those occurring in the recent outbreaks of pearl oyster oedema disease.  相似文献   

6.
Industrial fish production exposes fish to potentially stressful conditions, which in turn may induce infections by opportunistic pathogens. Probiotics appear to be a promising way to prevent opportunistic infections in aquaculture. In this study, we tested the inhibitory potential of endogenous bacterial communities found in the mucus of brook charr (Salvelinus fontinalis) against two major pathogens Flavobacterium columnare and Flavobacterium psychrophilum. Nine bacterial strains were isolated from brook charr skin mucus and tested for potential antagonistic activity. Results from both agar diffusion assays and broth co-culture assays showed the presence of antagonism. We identified seven bacterial strains, collected from unstressed fish, which exerted strong antagonism against F. psychrophilum and/or F. columnare. These strains were mixed and used to treat columnaris disease in an in vivo experiment in which four distinct fish families were tested. This treatment resulted in a decrease of mortality (54-86%) across fish families indicating that candidates from the host microbiota are potentially suitable for probiotic development. This would allow for the efficient (ability to adhere and colonize the host mucus) and durable management (antagonistic effect against pathogens which would be harmless for the host and safe for its environment) of opportunistic diseases in aquaculture.  相似文献   

7.
Degenerate primers were designed based on known sequence information for the circoviruses psittacine beak and feather disease virus and porcine circovirus and applied by polymerase chain reaction (PCR) to known virus-infected bursa of Fabricius (BF) from a pigeon. A 548-bp DNA fragment was amplified and shown to be specific to a novel circovirus, named pigeon circovirus (PiCV), and was used to produce sensitive and specific probes for detection of circovirus DNA by in situ hybridization (ISH). Using ISH on BF from 107 pigeons submitted for necropsy, infection was detected in 89%, compared with a histologic detection rate of 66%. Using the ISH technique, infected cells were also found in liver, kidney, trachea, lung, brain, crop, intestine, spleen, bone marrow, and heart of some birds. Large quantities of DNA were present in some of these tissues, and in the absence of BF, liver in particular is identified as a potentially useful organ to examine for presence of PiCV. This high prevalence of infection in diseased birds is noteworthy, emphasizing the need for studies to determine the precise role of this virus as a disease-producing agent.  相似文献   

8.
Taking advantage of the high sensitivity of polymerase chain reaction (PCR) and the cell-localizing ability of in situ hybridization (ISH), an indirect in situ PCR (ISPCR) method was developed for detecting the distribution of porcine circovirus type 2 (PCV2) in formalin-fixed and paraffin-embedded inguinal lymph nodes obtained from clinically healthy PCV2-carrier pigs and postweaning multisystemic wasting syndrome (PMWS)-affected pigs. Comparisons of the relative sensitivity of indirect ISPCR with other routinely used diagnostic methods for PCV2 indicated that nested PCR was the most sensitive method followed by indirect ISPCR, conventional PCR, ISH, and immunohistochemical (IHC) staining. Although indirect ISPCR, ISH, and IHC staining all revealed a similar signal distribution pattern of PCV2, using indirect ISPCR allowed specific amplification and detection of previously uneasily detected PCV2 signal than by routine ISH or IHC staining, particularly in those cells within the germinal center in clinically healthy PCV2-carrier pigs. Furthermore, six different PCV2 signal expression patterns in conjunction with the correlated lymphoid lesion stages were classified to describe the tissue morphological changes and viral infection. The result indicates that indirect ISPCR is a more effective, cell-based diagnostic tool with good specificity to detect limited PCV2 infection in formalin-fixed and paraffin-embedded tissue specimens and it would be a useful tool for further exploring the pathogenesis of PCV2 infection.  相似文献   

9.
DNA extraction and nested polymerase chain reaction (PCR) were developed for the detection of Haemophilus parasuis from formalin-fixed, paraffin-embedded tissues. The results for nested PCR were compared with those determined by in situ hybridization. The optimal results obtained show that use of xylene deparaffinization, digestion with proteinase K followed by nested PCR is a reliable detection method. A distinct positive signal was detected in 20 pigs naturally infected with H. parasuis by in situ hybridization. The rate of agreement between nested PCR and in situ hybridization for the detection of H. parasuis in formalin-fixed, paraffin-embedded tissues was 100%. The nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for the detection of H. parasuis with bacterial isolation.  相似文献   

10.
Porcine circovirus type 2 (PCV2), an economically important pathogen of swine, is the necessary cause of post weaning multisystemic wasting disease (PMWS); PCV2 infection is associated with porcine dermatitis and nephritis syndrome (PDNS). Current immunohistochemical (IHC) methodologies identify PCV2 antigens but are not capable of differentiating replicating virus from nonreplicating virion particles in tissue sections. In this paper, a combination of IHC using commercial monoclonal antibodies specific for single stranded (ss) and double stranded (ds) DNA and PCV2 specific in situ hybridization (ISH) was used to show the specificity of the former for PCV2 DNA in tissue sections from PCV2-infected gnotobiotic pigs. Cold-ethanol-fixed tissue sections were superior to formalin-fixed tissues for detection of PCV2 DNA, presumably due to the lack of protein cross-linking in the latter. These data demonstrate that conventional IHC detects PCV2 DNA forms in experimentally infected PCV2-positive gnotobiotic porcine tissue sections that are minimally compromised by either formalin fixation or the hybridization conditions needed for ISH.  相似文献   

11.
Ammonia concentrations in water can affect the severity of Flavobacterium columnare infections in fish. Two trials lasting 7 d each were conducted to determine the effect of a single immersion flush treatment of total ammonia nitrogen (TAN; 15 mg/L) on the survival of channel catfish Ictalurus punctatus infected with E columnare; the chemical was added while the water flowed continuously through the tanks. Both trials consisted of four treatments: (1) no ammonia exposure and no bacterial challenge (control), (2) ammonia exposure only, (3) bacterial challenge only, and (4) both ammonia exposure and bacterial challenge. Two hours after exposure to ammonia, the highest un-ionized ammonia level was 0.43 mg/L. The percent un-ionized ammonia is based on TAN, temperature, and pH. Caudal fins from three fish in each treatment were sampled at 24 h posttreatment to be analyzed by quantitative real-time polymerase chain reaction (qPCR). No significant difference in survival (mean +/- SE) was noted between the channel catfish in treatment 1 (95.2 +/- 1.2%) and those in treatment 2 (95.6 +/- 1.0%); however, survival in both treatments 1 and 2 differed significantly from that in treatments 3 (8.5 + 4.5%) and 4 (41.8 +/- 12.7%). Treatment 4 catfish had significantly higher survival than treatment 3 catfish. Quantitative PCR data showed that treatment 4 fish had significantly less F. columnare (7.6 x 10(5)) than did treatment 3 fish (1.2 x 10(7)), and treatment 2 fish (8.5 x 10(3)) had significantly less bacteria than did treatment 1 fish (6.9 x 10(4)), indicating that ammonia limited the F. columnare infection. The highest mean concentration of the bacteria (3.9 x 10(7)) was found on moribund fish. The ammonia concentrations tested did not negatively influence fish survival but interfered with the infection process. An in vitro assay was also conducted to evaluate the direct effects of ammonia on F columnare.  相似文献   

12.
A multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, and Yersinia ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The detection limits of the multiplex PCR was in the range of 2, 1, 1, 3, and 1CFU for A. hydrophila, A. salmonicida, F. columnare, R. salmoninarum, and Y. ruckeri, respectively. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria. The multiplex PCR assay was useful for the detection of the bacteria in naturally infected fish. This assay is a sensitive and specific and reproducible diagnostic tool for the simultaneous detection of five pathogenic bacteria that cause disease in fish. Therefore, it could be a useful alternative to the conventional culture based method.  相似文献   

13.
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14.
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15.
Flavobacterium columnare, causal agent of columnaris disease, is pathogenic to many species of freshwater fish throughout the world. The United States channel catfish (Ictalurus punctatus) aquaculture industry is severely impacted by columnaris disease. The majority of the F. columnare isolates recovered from diseased channel catfish belonged to either genomovars I or II. The objective of the present study was to determine if differences existed in the ability of these genomovars to induce mortality in channel catfish. Single strand conformation polymorphism analysis (SSCP) was used to ascribe the isolates used in this study to the appropriate genomovar. Immersion challenge experiments (15min immersion exposure to approximately 5x10(5) to 1x10(6) CFU/mL) were carried out to assess virulence of genomovar I and II isolates to channel catfish. The results demonstrated that genomovar II (n=4) isolates were significantly (P<0.05) more virulent to channel catfish fry (92-100% mortality) than genomovar I (n=3) isolates (0-46% mortality). In vivo adhesion of the genetically characterized F. columnare also correlated (r2=0.73) to increased mortality in the challenged fry. In fingerling channel catfish, significantly higher mortality (P<0.05) resulted with genomovar II isolates ALM-05-182 and ALG-00-530 as compared to all the genomovar I isolates (n=3). Mortality of genomovar II isolate BGFS-27 with similar to genomovar II isolate (ALG-00-530) and two genomovar I isolates (ALM-05-53 and 140). The results suggest that although both genomovars are present in the aquatic environment, genomovar II appears to be more pathogenic for channel catfish.  相似文献   

16.
Cyprinid herpesvirus-3 (CyHV-3) has spread worldwide and has had a major impact on koi and common carp production. Previous studies on the host range of the CyHV-3 found that fish species other than koi and common carp are fully resistant to natural virus exposure. Recently, CyHV-3 was detected in goldfish (Carassius auratus auratus) that were in contact with CyHV-3 infected koi. In the present study, a specific RT-PCR product was amplified from the viral thymidine kinase gene in gills, intestine and brain tissues of CyHV-3 infected goldfish. This implied that CyHV-3 replicated in these goldfish. Also, in the presence of a stress factor such as temperature fluctuation, the CyHV-3 infected goldfish transmitted the virus to cohabitated naïve SPF common carp. CyHV-3 DNA was detected in the cohabitated naïve carp tissues by PCR. The results of this study demonstrate that goldfish is a carrier for CyHV-3, permit virus propagation, and disseminate the virus to susceptible carp causing the disease.  相似文献   

17.
H9N2亚型禽流感病毒原位杂交检测方法的建立   总被引：4，自引：1，他引：4  
根据已知禽流感病毒(Avian influenza virus，AIV)核蛋白(NP)基因序列，选取其中保守区域设计出1时引物。以RT-PR方法扩增出543bp的NP基因片段，采用地高辛标记制备探针。MDCK细胞人工感染H9N2亚型禽流感病毒后，不同时间取样制备细胞涂片，进行原位杂交，研究了禽流感病毒时MDCK细胞的感染。探讨了原位杂交的条件及其应用于检测AIV感染的可行性。结果显示，感染24h后，原位杂交可检测出细胞内的病毒RNA，并且杂交信号的强度与感染时间的长短有关。研究表明。原位杂交检测禽流感病毒，不但能很好地表现病毒和细胞的位置关系,而且具有较好的特异性和灵敏性，为禽流感病毒的组织原位杂交检测奠定了实验基础。  相似文献   

18.
Chelonid herpesvirus (ChHV) infection in tortoises associated with stomatitis-rhinitis complex is a severe, mostly epizootic disease characterized by proliferative and diphtheroid-necrotizing glossitis, pharyngitis, rhinitis, and tracheitis, often occurring with pneumonia and encephalitis. The UL5 gene from a German ChHV isolate was used to generate a digoxigenin-labeled 307-base-pair DNA probe by polymerase chain reaction (PCR). ChHV DNA was detected in paraffin-embedded tissues of five naturally infected tortoises (two Afghan tortoises [Testudo horsfieldii], USA; two Hermann's tortoises [Testudo hermanni], Switzerland; one T. hermanni, Germany) by means of in situ hybridization (ISH) and PCR. Distribution of ChHV DNA exhibits many characteristics of alphaherpesvirus but also some characteristics of betaherpesvirus infections. The amino acid sequence of a portion of the ChHV UL5 homolog exhibited more than 50% similarity to alphaherpesvirus UL5 proteins. Nuclear hybridization signals were detected in epithelial cells of the lingual mucosa and glands. Furthermore, ChHV DNA was observed in tracheal epithelium, pneumocytes, hepatocytes, the renal tubular epithelium, cerebral glia cells and neurons, and intramural intestinal ganglia. ChHV DNA in endothelial cells of many organs underlines the systemic character of the disease. Importantly, ChHV DNA was detected by ISH in multiple tissues of tortoises originating from different geographic provenances. This indicates a high degree of conservation of the UL5 gene fragment among viruses prevalent in tortoises on different continents. With the described ISH, a molecular biological tool is available for rapid and specific diagnosis of ChHV infections and, more importantly, comparative pathogenetic studies of ChHV isolates from geographically unrelated regions.  相似文献   

19.
Diagnosis of the cutaneous form of canine leishmaniosis is mostly performed by histological or immunohistological examination of skin biopsies. In modern histology, the polymerase chain reaction (PCR) has gained increasing importance as a complementary tool to directly demonstrate the presence of parasite DNA in the tissue sections. For the present study, a previously described Leishmania-PCR has been further developed and optimised in view of its practicability for routine histological application. Since formalin-fixation of histological specimens causes partial DNA-destruction, which may hamper diagnostic PCR analysis, primers specific for the highly conserved alpha-actin gene sequences were used to pre-diagnostically assess the isolated sample-DNA for its functionality in a PCR-reaction. This alpha-actin-specific PCR detects DNA from a large variety of mammalian species and thus exhibits relevance for both human and veterinary medical application. A recombinant internal positive control was introduced to monitor possible sample-related inhibitory effects during the amplification reaction. We performed a retrospective evaluative study with 18 formalin-fixed samples from dogs with suspected or proven leishmaniosis. Six samples were PCR-incompatible. In turn, 9 of the other 12 samples were PCR-positive, and immunohistochemical results matched these findings. Based on these technical achievements, the Leishmania-PCR proved to be a valuable tool to complement conventional histological and immunohistological methods for diagnosis of cutaneous leishmaniosis in formalin-fixed, paraffin-embedded skin biopsies.  相似文献   

20.
Flavobacterium columnare (F. columnare) is the causative agent of columnaris disease. This bacterium affects both cultured and wild freshwater fish including many susceptible commercially important fish species. F. columnare infections may result in skin lesions, fin erosion and gill necrosis, with a high degree of mortality, leading to severe economic losses. Especially in the last decade, various research groups have performed studies aimed at elucidating the pathogenesis of columnaris disease, leading to significant progress in defining the complex interactions between the organism and its host. Despite these efforts, the pathogenesis of columnaris disease hitherto largely remains unclear, compromising the further development of efficient curative and preventive measures to combat this disease. Besides elaborating on the agent and the disease it causes, this review aims to summarize these pathogenesis data emphasizing the areas meriting further investigation.  相似文献   

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