| Portunus trituberculatus, also known as swimming crab, gun crab, sea crab, belonging to Crustacea (Crustacea), Decapoda (decapoda), Portunidae (Portunidae).It is mainly distributed in East China Sea and Yellow Sea,which is a large marine economic crabs with rich nutritional and medicinal properties. However, in recent years, some epidemic diseases outkreaked and caused badly economic losses, which was attributed to many factors such as pollution of the marine environment, ecological environment deteriorated, particularly pathogenic microorganism. A preliminary study of the pathogens was taken to find the pathogen, pathogenesis, the effective prevention methods and means of detection.The typical symptoms of disease crabs were chosen as a material, through the isolation and identification of strains, back to a sense of artificial test, histopathologic observation and with the combination of morphological characteristics, physiological, and biochemical characteristics, and molecular biology and so on were investigated too. The PCR protocol amplifying cfa gene fragmengts of the Citrobacter freundii was established.With the purpose of the swimming crab early diagnosis and preventing of disease and providing scientific reference, the suitable drugs were selected to cure the bacterial disease.Between 2009 and 2010 the cultured Portunus trituberculatus suffered from serious disease in some farms of Qingkou Town,Ganyu County in Lianyungang City,Jiangsu Province. The main symptom displayed as drumble,no feeding, the tissue oedema of liver, gill and muscle; the liver and muscle were rotten of some crabs.Virtually pure dense cultures were obtained from liver, muscle, and lymph fluid of diseased Portunus trituberculatus, and strong pathogenicity of the isolate strain (JG091120-1) to the Portunus trituberculatus,Charybdis japonica, Penaeus chinensis, and non-pathogenic of the isolate strain (JG091120-2), which show that the bacteria JG091120-1 oriented crab death pathogen. The phenotypic characteristics of isolate strain was examined, including morphological characteristics, physiological, and biochemical characteristics, the 16S rRNA and gyrB genes were amplified and compared with those sequences deposited in databases. Phylogenetic analysis of 16S rRNA and gyrB genes indicated high homogeneity between the isolate strain and Citrobacter freundii from GenBank database. The 16S rRNA gene fragment length 1451bp(GeneBank Accession HQ170626) and gyrB gene fragment length 1186bp(GeneBank Accession HQ170627). The results showed that the identified isolate strain belonged to Citrobacter freundii. It is gram-negative, aerobic or facultative anaerobic, as the opportunistic pathogen, widely distributed in nature, human beings and animals (mammals, birds, reptiles and amphibians). It can lead crab, Channa argus, Cherax quadricarinatus, Nile tilapia and other aquatic animals to outbreaks of disease. In order to choose the appropriate antibiotic, the drug sensitive was tested, and testing results by paper extension methods for drug sensitive showed that among 48 antibiotics Ciprofloxacin, Fleroxacin, Ceftriaxone, Enoxacin, Enrofloxacin, Floxacin and norfloxacin Levofloxacin had the most inhibitive effects on the strain.The histopathology research conducted with tissue technology,hematoxylin and eosin staining.The organs samples such as gill, hepatopancreas, chelipeds muscle, intestine, heart and other organizations. At the same time carried out its preliminary analysis of pathogenesis. The results showed that the hepatopancreas were collected necrocyosis significantly serious,the nuclear were extence only in some areas,the muscle fibers were fractured or dying many cells karyolysis and nuclear disappeared and some cells to shrink or swelling, blood cell aggregation.The antimicrobial effects of 20 Chinese herbs including Galla chinensis, Rheum offcinale, Panax Notoginseng, Andrographis paniculata etc. to Citrobacter freundii were detected through disc diffusion method (punch method), at the same time the minimal inhibitory concentration and the minimum bactericidal concentration of some Chinese herbs which had significant inhibitory effects to Citrobacter freundii were detected by the tube dilution. The results showed that Caesalpinia sappan, Galla chinensis, Fructus Schisandrae Chinensis, Cortex Phellodendri Chinensis, Rhizoma Coptidis and Rheum offcinale had significant inhibitory and bactericidal effects, the minimal inhibitory concentrations of Caesalpinia sappan, Galla chinensis, Fructus Schisandrae Chinensis and Rheum offcinale bark to pathogenic Citrobacter freundii were among 0.780-3.125mg/mL, the minimal bactericidal concentrations were among 1.560-12.50mg/mL. Meanwhile,10 antibacterial fishery drugs including Enrofloxacin, Norfloxacin, Thiamphenicol etc. had been chosen to detect the antibacterial effect,the results showed that Cotrimoxazole, Enrofloxacin, Norfloxacin had significant inhibitory and bactericidal effects.Colonization factor antigens was a pathogenic factor of enterobacteriaceae which could product enterotoxin that was capable of causing death.In this paper, one pair of specific primers was designed that allowed amplification of 100bp gene fragments based on colonization factor antigen cfa gene sequence, then a novel simple polymerase chain reaction (PCR) that will detect Citrobacter freundii was established,and there was no cross reaction detected in other pathogenic species. The results of sensitivity test showed that the PCR primers could detect Citrobacter freundii at a level of as few as 6.47 X 103 CFU/mL, and detect purified chromosomal DNA at a level of as few as 0.1468ng/μL.Dominant Citrobacter freundii could be isolated from the positive sample of PCR through detecting aquatic products samples such as Penaeus chinensis, Tegillarca granosa, Placopecta magellanicus and tank water samples.The PCR protocol amplifying cfa gene fragments of the Citrobacter freundii was established and could be useful in the specific and rapid diagnose of the disease caused by Citrobacter freundii. |
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