Study On Etiology, Pathology And Diagnostic Method Of The Streptococcus Iniae Infection In Channel Catfish Ictalurus Punctatus

Streptococcus iniae (S.iniae, Streptococcus shiloi), which belongs to Lactobacillales, Streptococcaceae, and Streptococcus, is one of the most important aquatic pathogens. Since first identified from a freshwater dolphin in 1976, the infected host range of S.iniae has constantly expanded in several decades, mainly including astilapia (Oreochromis spp.), rainbow trout (Oncorynchus mykiss), coho salmon (Oncorhynchus kisuth), and other finfish in temperate waters. Since human infection dolphins streptococcus clinical cases were successively reported by Canada, America, Hong Kong, Singapore etc., the public health implications of S.iniae also has been gradually recognized. Channel Catfish (Ictalurns Punctatus) is a global famous freshwater aquaculture species, and also an important export-oriented economic fishes, which is widely cultured in more than 30 provinces, such as Sichuan, Hunan, Anhui et al. province. Channel catfish was once recognized to be resistant to infection streptococcal. However, there emerged sporadic clinical reports on channel catfish streptococcal infection in recent years. It lacks systematic research about characteristic of pathogen, pathology, pathopoiesis mechanism and diagnostic methods. So in this study, we sampled spontaneous infection channel catfish with cage cultured in Guangxi Province, China. Through research of aetiology, pathology, tissue distribution and diagnostic methods, it aimed to elucidate the etiology of the channel catfish infection, the pathogenicity of this bacteria strain and pathology lesions, and to find out the injury mechanism of S.iniae infection of channel catfish, as well as to provide an effective diagnosis method. This is a basic research, which is the foundation of the pathogenic mechanism of virulence factors in channel catfish S.iniae infection.A pathogenic bacterium (DGX07) was isolated from the liver, spleen, kidney and brain of the spontaneous infection channel catfish. Artificial infection test proved that the bacterium was the pathogen of the disease. The isolated strain was a Gram-positive cocci, with (3-hemolytic activity, with capsule, catalase (-), PRYA (+) et al. A phylogenetic tree was constructed by comparing the 16S rDNA sequences of the isolated strian (FJ951434) with other relative bacteria Strepotococcus sp. in GenBank databases. The homologies between 16S rDNA Gene and S.iniae-ATCC29178 (AF335572) was 99.7%. According to the morphological, biochemical characteristics and phylogenetic analysis, the isolated strain (DGX07) was identified as S.iniae. We preceded antimicrobial susceptibility testing of the S.iniae-DGX07 strain with the rapid amplified polymorphic DNA (RAPD) technique, and the result showed that the strain S.iniae-DGX07 was sensitive to most drugs of chloromycetin,β-lactams, macrolides, tetracyclines and quinolones, while insensitive to many drugs of aminoglycosides, sulfonamides and other drugs. The results of etiology highlighted that the host range of S.iniae was greater than originally documented.The strain of S.iniae-DGX07 could survive in the condition of 10℃-45℃, NaCl 0%-4%and pH6.0-9.0, and grow on many culture medium, especially on Blood-TSA,THA and BHIa. The half lethal dose (LD50) in channel catfish and mice were 4.93×106CFU and 3.51×105CFU, separately. S.iniae-DGX07 has the virulence related gene, including pgmA (JF795256), cpsD (JF795257), cpsK (JF795255) and sagA, which sequence similarities with S.iniae were 99.9%,99.3%,100%and 100%respectively.The pathogenicity of channel catfish infected by S. iniae was evaluated through light microscope, election microscope and blood index examinations. The symptom and pathological changes of artifical infection were similar to spontaneous S.iniae infected fish. Gross changes examination showed marked petechial hemorrhages of skin and congestion of internal organs, particularly in the liver, spleen and kidneys. Other features included color fading in edge of fin ray along with enteritis and ascites. Histopathological examination showed oedema, degeneration and necrotic changes in many organs. Spleen, kidney and liver, as target organs of infection, appeared metamorphic necrosis. Further, hepatitis, splenitis, interstitial nephritis, meningitis, myocarditis and catarrhal enteritis with numerous monocytes and neutrocytes infiltrates were evident. Intact S.iniae cells were seen in macrophages. Cytopathological changes showed swelling and ultrastructure destory in visceral organs'cells. The mitochondria swelled with disintegration and dissolution of the cristaes. The chromatin showed condensation and margination. The blood chemical index showed that hemoglobin (Hb), red blood cell count (RBC), total protein (TP), albumin (ALB), cholesterol (CHO), creatinine (Cr), sodium (Na+), chlorine (Cl-), calcium (Ca2+) decreased, white blood cell count (WBC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (BUN), potassium (K+) increased. These results indicated that S.iniae infection caused septicemia in channel catfish, with anemia increasing the deaths of fish.Quantitative inoculation method and immunohistochemical method to detect bacteria in tissue distribution showed that the spleen, liver and kidney is the main distribution organs of bacteria. The strain S.iniae-DGX07 could proliferate in the spleen and kidney. Antigen in various organs within the positive signal mainly scattered in phagocytic cells, blood and interstitial cells. The results tissue distribution confirmed that S.iniae infection of channel catfish causes septicemia to build the foundation of pathogenic mechanism of single virulence factor.The polyclonal antiserum against S.iniae-DGX07 was obtained to establish the indirect ELISA assay to detect S.iniae. This ELISA method has high specificity, and has not cross reaction with the Stenotrophomnas maltophilia, Aeromonas hydrophila, Edwardsiella ictaluri, Aeromonas veronii, Yersinia Rucker, Aeromonas caviae and Streptococcus agalactiae. The coefficients of variation inner-plate or inter-plate were 4.54%and 5.62%severally. The lowest detectable limit of S.iniae was 1.5×105 CFU/mL, equally 1.5 x104 CFU. The critical OD450 values of liver, kidney, spleen and brain were 0.467,0.454,0.630 and 0.594 respectively. In those organs, the detection time were 12h, 24h,48h and 48h, and the detection rate were 75%,50%,100%and 40%separately.A double PCR method for diagnosing the disease of S.iniae with two pair of primers based on 16S rRNA gene and lactate oxidase (IctO) gene. Its optimum reaction conditions were as follows:dNTP (10mM) 1.0μL, MgC12(25mM) 1.5μL, 10pM 16S rRNA F/R:10pM lctO F/R (1:5) 2μL, Taq enzyme (5U/μL) 0.4μL,10×PCR Buffer 2.5μL, Templete DNA1μL, ddH2O to 25μL. The lowest detection limit of S.iniae was 650pg. The detection rate of artificial infection channel catfish after 24h was 100%in liver, spleen and kidney samples.