Partial purification of extracellular toxic material of fish Vibrio

Hemolytic activity of 3 pathogenic strains, of fish Vibrio commonly associated with vibriosis (V anguillarum NCMB6 and NCMB571 strains, and Vibrio sp N7802 strain) was examined, using chicken and mammalian erythrocytes. Vibrio strains NCMB6 and NCMB571 and their culture filtrates had hemolytic activity against 8 kinds of erythrocytes tested, whereas Vibrio strain N7802 produced only a little amount of hemolysin. Strain NCMB571 culture filtrate and its material partially purified by column chromatography were lethal in mice. From 2 peaks of protein, hemolysin was recovered from the 1st peak, which coincided with toxicity in mice. Heat-inactivation of culture filtrate indicated that hemolytic materials may be thermolabile proteins, but that toxic material may be comparatively thermostable.     

Characterization of Vibrio anguillarum Strains Isolated from Diseased Fish in Finland

Characterization of V anguillarum strains (n=109) isolated from diseased salmonids was performed. Eight O serovars were found among the strains. Serovar Ol was predominant (90 %), while serovars O2, O3, O5, O8, O9, and a new serovar Va NT2, were represented by 1 or 2 strains. Two strains remained non-typeable. One of these was cross-reactive with several antisera, but had a LPS profile identical to that of serovar O8. All serovars showed specific LPS profiles. All but 1 of the Ol strains had a plasmid comparable in size to the pJMl virulence plasmid, while plasmids of different sizes were found in O2, Va NT2 and the non-typeable strains. Apart from a single strain resistant to tetracycline, all the strains were sensitive to oxolinic acid, tetracycline, and trimethoprim-sulfonamides. By their biochemical and antigenic properties strains causing vibriosis among salmonids in Finland closely resemble Scandinavian strains. Predominance of the serovars Ol and O2 suggests that commercial vaccines containing these serovars should afford sufficient protection against vibriosis in Finland.     

Isolation and Characterization of Large Marine Bacteriophage (Myoviridae), VhKM4 Infecting Vibrio harveyi

Abstract

The causative agent responsible for vibriosis in tropical fish aquaculture, Vibrio harveyi, has become a major bacterial pathogen. Studies suggest that this bacterium has developed resistance to antibiotics commonly used in aquaculture. In view of this situation and the requirement for the proposed postantibiotic era, bacteriophage therapy seems to be a promising control strategy for fish vibriosis. In this study, a lytic Vibrio phage VhKM4 belonging to a member of large, marine Myoviridae was successfully isolated. It exhibited bacteriolysis to both V. harveyi VHJR7 and V. parahaemolyticus ATCC 17802. The latent period of the VhKM4 phage was recorded at 60 min. It also recorded average burst size of approximately 52 plaque-forming units per infected cell. A strong bacteriolytic activity at low multiplicity of infection of 0.01 indicates the effectiveness of this large marine myovirid against fish pathogenic strain of V. harveyi VHJR7.

Received June 16, 2016; accepted October 7, 2016 Published online February 6, 2017     

Virulence, cytotoxic and inflammatory activities of Vibrio anguillarum and Aeromonas salmonicida isolated from cultivated salmonid fish in Sweden

Extracellular products in culture filtrates of Aeromonas salmonicida subsp. achromogenes and Vibrio anguillarum isolated from infected fish have been shown to possess skin inflammatory factor. The extracellular products from Vibrio anguillarum were cytotoxic in HeLa and CHO cells. In addition to the skin lesions, the culture filtrates of V. anguillarum caused necrotic reaction on the rabbit skin. Five of 6 strains of V. anguillarum were lethal to mice after intraperitoneal administration of 3×10⁷ CFU. Only 1 strain of 5 A. salmonicida subsp. achromogenes produced extracellular products which elicited cytotoxic effects in the CHO cells. None of the A. salmonicida subsp. achromogenes strains were lethal to mice. The cytotoxins were inactivated when heated at 65°C for 30 min. The results indicate that the thermolabile exotoxins are non-enterotoxic since they failed to stimulate fluid accumulation in the rabbit ileal loop and did not cause elongation of the CHO cells. The rounding off of CHO cells, as well as of HeLa cells indicate that the exotoxins may play an important role in fish diseases.     

An examination of Escherichia coli strains isolated from cases of bovine mastitis for possible virulence determinants

Ninety-five strains of Escherichia coli isolated from cases of bovine mastitis were examined for the possession of some of the possible virulence determinants. Ten strains which caused haemagglutination of bovine and ovine erythrocytes were considered to be fimbriated and an additional strain caused agglutination of chicken erythrocytes. Colicines were produced by fifteen strains and in three strains the colicine was identified as Col V. Forty-one of 71 strains that were examined serologically possessed capsular or envelope K antigens.     

Perceived vibriosis risk by Swedish rainbow trout net-pen farmers: Its effect on purchasing patterns and willingness-to-pay for vaccination

A mail questionnaire was submitted to all 38 brackish-water rainbow trout net-pen farmers on the south and east coasts of Sweden. One series of questions addressed farmers' willingness-to-pay pay (WTP) for vibriosis vaccination. The amount that experienced farmers are willing to pay for 100% vaccine efficacy (WTPF) was positively correlated with average annual vibrosis mortality rates. However, this correlation progressively decreased in magnitude as the annual posterior probability of vibriosis occurrence increased. Experienced farmers' WTP for partial vaccine efficacy (WTPP) was related to both average mortality rates and the range in the previous 2 years' annual vibriosis mortality rates. For farmers with low ranges, WTPP showed a strong negative correlation with mortality rates exceeding approximately 1–3%. For farmers with high ranges, the WTPP-mortality relationship was weakly negative. There was no significant linear relationship between WTP and the previous summer's vibriosis mortality rates for new farmers, but new farmers who had experience vibriosis were prepared to pay substantially more for vaccination than were vibriosis-free farmers.

Results of a second series of questions showed a majority of respondents indicating an initial preference for rearing older fish (yearlings or 2-year-olds) would instead purchase younger fish (fingerlings or yearlings, respectively), if the younger fish were guaranteed vibriosis-resistant. All such farmers were prepared to pay at least the full cost of vaccination for this protection. Most farmers indicated that they do not purchase surplus juvenile fish in anticipation of vibriosis losses.     

新城疫病毒F48E9株HN基因核苷酸序列

本研究采用 Ｓａｎｇｅｒ＇ｓ 双脱氧末端终止法测定了新城疫病毒国内标准强毒株 Ｆ４８ Ｅ９ 血凝素神经氨酸酶基因 ｃ Ｄ Ｎ Ａ 的核苷酸序列,推导出其氨基酸序列。 Ｆ４８ Ｅ９ Ｈ Ｎ 基因编码区全长为 １７１３ｂｐ,单一的开放阅读框编码５７１ 个氨基酸的糖蛋白。含有６ 个潜在的与天门冬酰胺（ Ｎ）连接的复合寡聚糖和高甘露糖寡聚糖结合位点,其中有 ５ 个簇集在蛋白分子的 Ｃ末端一半内,有１３ 个半胱氨酸残基。发现６ 个 Ｆ４８ Ｅ９ 特有的氨基酸残基,３７ 位 Ｆ（ Ｌ）、３８ 位 Ｓ（ Ａ）、６０ 位 Ｌ（ Ｐ）、１０５ 位 Ｓ（ Ｎ）、４４３ 位 Ａ（ Ｔ）、５７１ 位 Ｉ（ Ｖ）,这些位点除了 ６０ 位的 Ｐ有两株为 Ｓ以外,其它的在 １３ 株已知序列中均为高度保守序列。     

Active immunisation of black tiger prawn (Penaeus monodon) against vibriosis in Thailand.

Mechanisms of host defence in Crustaceae and vibriosis in shrimp and methods for its prevention are discussed as introduction. The own work deals with the development of a site-specific multivalent anti-vibrio vaccine and its application in Thailand. The vaccine was produced in a continuous bioreactor system from field strains of Vibrio alginolyticus, V. harveyi, V. parahaemolyticus and V. vulnificus, purified through ultrafiltration, and inactivated by formalin. It was applied to Penaeus monodon PL prior to release into the ponds via artemia larvae. As mean result from numerous trials carried out under commercial field conditions in Eastern Thailand it was shown that the PL tolerated the vaccine perfectly. Due to technical problems only few of the field trials could be monitored until harvest. The data available allow claiming an obvious protective effect of the vaccine.     

Antigenic variation among transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus strains detected with monoclonal antibodies to the S protein of TGEV.

Five nonneutralizing monoclonal antibodies (MAb) generated to the virulent Miller strain of transmissible gastroenteritis virus (TGEV) and specific for the S protein were characterized. Competition assays between purified and biotinylated MAb indicated that MAb 75B10 and 8G11 mapped near a new subsite, designated V and 2 MAb, 44C11 and 45A8, mapped to a previously designated subsite D. A fifth MAb mapped between subsites V and E. These MAb were tested with 3 previously characterized MAb to subsites A, E, and F in fixed-cell ELISA and cell culture immunofluorescent assays against 5 reference and 9 field strains of TGEV and 2 US strains (ISU-1 and ISU-3) of porcine respiratory coronavirus (PRCV). Subsites A, E, and F were conserved on all TGEV and PRCV strains examined. The 2 MAb to subsite V, 8G11 and 75B10, reacted only with the Miller TGEV strains (M5C, M6, and M60), except that 75B10 also recognized field strain U328. The MAb 11H8 did not react with 4 field strains or the Purdue strains of TGEV. The 2 MAb to subsite D reacted with all TGEV strains examined, but not with 2 US PRCV strains, 2 European PRCV strains, 1 feline infectious peritonitis virus strain, and 1 canine coronavirus strain. Because of this specificity for TGEV, but not PRCV, these latter 2 subsite D MAb may be useful for the development of competition ELISA to differentiate serologically between TGEV and PRCV infections in swine, similar to the currently used European subsite D MAb.     

IMMUNISATION OF CATTLE AGAINST VIBRIOSIS WITH VACCINES PREPARED FROM CAMPYLOBACTER FETUS SUBSP FETUS

Subcutaneous administration of vaccines prepared from cells of Campylobacter fetus subsp fetus strain A28 to heifers gave substantial protection against infertility due to C. fetus subsp venerealis strain B6. Strains A28 and B6 had different heat-stable antigens and conformed respectively to serotype B and serotype A of Berg et al (1971). The results suggested that the protective antigens were heat-labile antigens common to both strains. Although vaccines prepared from serotype B strains of C. fetus subsp fetus could be used to immunise cattle against vibriosis, the results did not suggest that their use in preference to those prepared from C. fetus subsp venerealis would offer any advantages.     

Purification and partial characterisation of a fish lethal extracellular protease from Vibrio pelagius

The purification, characterisation and lethal effect of an extracellular protease present in the extracellular products (ECPs) of a pathogenic Vibrio pelagius (7P) strain are described. The extracellular protease was purified by size-exclusion high-performance liquid chromatography and characterised by enzymatic assays. The lethal effect was evaluated by injection into fish. The native protease had a molecular mass of 39 kDa, was active on casein and L-leucyl-beta-naphthylamide (LNA) and its metalloprotease nature was shown by the LNA inhibition profile. Kinetic studies on the hydrolysis of casein and LNA confirmed a competitive inhibition of one substrate with respect to the other. The temperature assays showed that both aminopeptidolytic and caseinolytic activities were labile at 70 degrees C for 3 min. The N-terminal amino acid sequence of 7P protease revealed a high degree of homology with other metalloproteases of Vibrio species that are implicated in virulence. The purified 7P protease showed an LD(50) of 1.77 microgprotein/g fish for turbot. The quick lethal effect (<24h) and the macroscopic damage (external haemorrhagic areas, principally on fins and mouth, petechial haemorrhages in internal organs, but with no external or internal apparent necrotic areas) detected in the host were similar to those obtained by injection of total ECP and live cells of 7P strain.An extracellular protease with endopeptidolytic and exopeptidolytic activities, responsible for the lethal effect of ECP and clinical signs of vibriosis in turbot was purified from a pathogenic V. pelagius (7P) strain.     

Isolation of a monoclonal antibody with specificity for commonly employed vaccine strains of Newcastle disease virus

A monoclonal antibody, AVS-I, was produced from a hybridization of murine myeloma cells and splenocytes from mice immunized with the La Sota strain of Newcastle disease virus (NDV). The hybridoma producing AVS-I, selected from 184 NDV-positive supernatants, is one of two supernatants that reacted exclusively with lentogenic strains in an indirect enzyme-linked immunosorbent assay. AVS-I can also be assayed by hemagglutination-inhibition (HI), which was used to test selected reference avian paramyxovirus (PMV) strains of types 1 to 3. NDV vaccines La Sota and B1 and field isolates from chickens, turkeys, pigeons, and cockatoos were also used as antigens. AVS-I had a high binding affinity for all La Sota and B1 strains, including vaccines. The antibody bound with a lower titer to the Australian Queensland V4 and Ulster strains, but it did not bind to the F strain, a lentogenic strain from England. AVS-I was HI-negative against the other PMV reference strains. AVS-I may be valuable for identifying field isolates antigenically similar to La Sota and B1 and rapidly differentiate those vaccine strains from more virulent viruses.     

Macrolides and lincomycin susceptibility of Mycoplasma hyorhinis and variable mutation of domain II and V in 23S ribosomal RNA

A total of 151 strains of Mycoplasma hyorhinis isolated from porcine lung lesions (weaned pigs, n=71, and finishers, n=80) were investigated for their in vitro susceptibility to 10 antimicrobial agents. Thirty-one strains (28 from weaned pigs and 3 from finishers) showed resistance to 16-membered macrolide antibiotics and lincomycin. The prevalence of the 16-membered macrolide-resistant M. hyorhinis strain in weaned pigs from Japanese herds has approximately quadrupled in the past 10 years. Several of the 31 strains were examined for mutations in the 23S ribosomal RNA (rRNA). All field strains tested showed a transition of A to G at position 2059 of 23S rRNA-rendered Escherichia coli. On the other hand, individual tylosin- and lincomycin-resistant mutants of M. hyorhinis were selected in vitro from the susceptible type strain BTS7 by 3 to 9 serial passages in subinhibitory concentrations of each antibiotic. The 23S rRNA sequences of both tylosin and lincomycin-resistant mutants were compared with that of the radical BTS7 strain. The BTS7 mutant strain selected by tylosin showed the same transition as the field-isolated strains of A2059G. However, the transition selected in lincomycin showed mutations in domains II and V of 23S rRNA, G2597U, C2611U in domain V, and the addition of an adenine at the pentameric adenine loop in domain II. The strain selected by lincomycin showed an additional point mutation of A2062G selected by tylosin.     

Enterotoxin production at 4, 22, and 37 degrees C by Yersinia enterocolitica and Yersinia enterocolitica-like bacteria isolated from porcine tonsils and pork products

Altogether, 71 strains of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from porcine tonsils and pork products were examined for their ability to produce enterotoxin using the infant mouse assay. Of these, 37 strains (52.1 %) produced enterotoxin at 22 °C, 3 were positive at 4 and 22 °C, and 1 was enterotoxigenic at 22 and 37°C. No strain was positive at all 3 temperatures. The highest prevalence of enterotoxin production at 22°C was detected in serotype 0:11 (80.0%), followed by 0:3/ biotype 4 (74.2 %), and 0:12 (66.7 %). Enterotoxin production at 4°C was recorded in 2 (15.4 %) of the Yersinia kristensenii strains (0:11, 0:12) and 1 of the Yersinia enterocolitica strains, (0:3) examined. One Yersinia kristensenii strain (0:11) was enterotoxigenic at 37 °C. The results indicate that enterotoxin production is a common feature of yersiniae isolated from porcine tonsils and pork products in Norway and may represent a possible source of food borne intoxication.     

Pathogenicity of Escherichia coli O115:K"V165" strains isolated from pigs with diarrhea

Eighteen strains of Escherichia coli serogroup O115:K"V165" isolated from 1- to 8-week-old pigs with diarrhea were tested for toxigenicity, pathogenicity in pigs and mice, serum resistance, mannose-resistant hemagglutination (MRHA), F165 and other surface antigens, colicin V (Col V), aerobactin, and biotype. Twelve strains were positive for heat-stable enterotoxin (STb), MRHA-negative, and F165-negative; 5 strains were enterotoxin-negative, MRHA-positive, and F165-positive; and 1 strain was MRHA-positive, but F165- and enterotoxin-negative. Six of the 12 STb-positive strains moderately colonized the ileum of newborn colostrum-deprived pigs within 24 hours after inoculation. Two of the colonizing strains were able to induce watery diarrhea. All 12 STb-positive strains were nonpathogenic for adult mice and were serum-sensitive; 11 of 12 were Col V-negative, 9 of 12 did not produce aerobactin, and 10 of 12 belonged to biotypes other than 1 or 2. All 6 enterotoxin-negative strains colonized the small and large intestines, associated with peritoneal serosal surfaces, and induced septicemia and polyserositis in newborn colostrum-deprived pigs 1 to 2 days after inoculation. In contrast, 3 STb-positive strains poorly colonized the intestines and did not induce septicemia in pigs at 3 days after inoculation. All 6 enterotoxin-negative strains were Col V-positive, produced aerobactin, and belonged to biotype 1 or 2. Of the 5 enterotoxin-negative, F165-positive strains, only 4 were pathogenic for intraperitoneally inoculated adult mice and were serum-resistant. The enterotoxin-negative, F165-negative strain was neither serum-resistant nor mouse-pathogenic.(ABSTRACT TRUNCATED AT 250 WORDS)     

仔猪产气荚膜梭菌肠毒血症病原诊断及疫苗研究Ⅲ.7株A型产气荚膜梭菌菌株的产毒素试验

本试验对7株A型产气荚膜梭菌菌株在不同培养基、不同培养温度和不同培养时间的产毒力进行了试验。结果表明，培养基的种类和培养时间对菌株的产毒素能力影响较大，而培养温度则影响较小。所试菌株中C57-1菌株的产毒素能力最强，是制备疫苗的最佳菌株。     

Three statistical approaches for genetic analysis of disease resistance to vibriosis in Atlantic cod (Gadus morhua L.)

The objective of this study was to estimate and compare variance components and sire breeding values for disease resistance to vibriosis in Atlantic cod (Gadus morhua L.) using 3 statistical approaches. A total of 3,576 individually tagged juvenile cod from 50 full-sib families were infected with Vibrio anguillarum, which causes vibriosis, a frequently reported disease in cod aquaculture. The experimental fish were progeny of captured wild cod from populations of southern coastal cod (POP1), and northern coastal cod and northeast Arctic cod (combined as POP2 in the genetic analyses). Fish were randomly assigned to 1 of 3 test tanks, and daily mortality was recorded until the termination of the experiment at d 31 postinfection. Variance components were estimated separately for the 2 populations using a Cox regression model, univariate linear model, and a linear model that accounted for censoring. With all approaches, the additive genetic sire variance estimated from POP1 was greater than for POP2. Heritability estimates across populations varied from 0.08 to 0.17 depending on the method used. The Cox regression model and univariate linear model resulted in greater heritability estimates for POP1 (0.10 and 0.16) than for POP2 (0.08 and 0.13), whereas the contrary was true with a linear model that accounted for censoring (0.17 vs. 0.14). The predicted breeding values for the sires from the 3 approaches were highly correlated (0.97 to 0.99). This is likely due to the fact that censoring only occurred at the end of the test; i.e., observations of the most resistant fish were censored. The considerable genetic variation found in this study suggests that vibriosis resistance may be improved through selective breeding. The univariate linear model, even without censoring of the data, was robust for the estimation of breeding values using the present data. Therefore, inclusion of vibriosis resistance in the multivariate linear estimation of breeding values for the traits of economic importance in Atlantic cod seems appropriate.