Studies on adhesion, haemagglutination and other biological properties of Vibrio cholerae O139

Abstract The adhesive capabilities of eight Vibrio cholerae O139 epidemic strains to isolated rabbit intestinal epithelial cells (RIEC) were observed to be high similar to those observed with a Vibrio cholerae O1 strain isolated from patients. Toxin production by the strains, measured by accumulation of fluid in rabbit ileal loop model, was high and the toxin was lethal as the animal expired within 6 h. Culture filtrates of the strains exhibited the presence of vascular permeability factor which produce induration and necrosis in the adult rabbit and guinea pig skin. All the strains showed high to moderate haemagglutinin titres against chicken erythrocytes and produced El Tor-like haemolysin. SDS-PAGE of the outer membrane preparation of the strains showed the presence of major protein component at 38 kDa region. The lethality of the toxin, high adhesive activity, shifting of the major outer membrane protein band and production of thermolabile haemolysin on Wagatsuma agar were the major variations of these epidemic strains from V. cholerae O1 and V. cholerae non-O1 strains isolated previously.     

Development of an improved synthetic medium for a better production of the new cholera toxin and its immunological relationship with the toxin produced by Vibrio cholerae O139 strains

Abstract An improved synthetic medium (M4) comprising syncase medium supplemented with sodium chloride (1%) and sucrose (0.5%) pH adjusted to 7.4 was developed for a better production of the new cholera toxin (NCT). The culture filtrates prepared in the M4 medium caused significantly ( P < 0.05) more fluid accumulation than that in syncase medium. Crude toxin, prepared in the M4 medium with V. cholerae O1 strains (X-392 and 2740-80) caused a reaction similar to that of the same amount of NCT (32 μg) prepared in the syncase medium. The neutralization of the optimal loop reacting dose of the NCT prepared in the M4 medium by anti-NCT raised against syncase prepared toxin indicates the release of the same kind of toxin in both media. These observations indicate that the modified M4 medium may be used for NCT preparation and further characterization. All the strains of Vibro cholerae O139 used in this study produced a toxin antigenically similar to NCT.     

Comparison of the host ranges and antigenicity of Cryptosporidium parvum and Cryptosporidium wrairi from guinea pigs.

Oocysts of a Cryptosporidium isolate from guinea pigs were not infectious for adult mice, but were infectious for two of three newborn calves and for suckling mice. However, oocysts isolated from calves or mice infected with guinea pig Cryptosporidium were not infectious for guinea pigs. Four isolates of C. parvum from calves were incapable of infecting weanling guinea pigs. Microscopic examination of tissue from the colon and cecum of suckling guinea pigs inoculated with C. parvum revealed sparse infection of some pups. These host range studies and previously described differences in 125I-labeled oocyst surface protein profiles between Cryptosporidium sp. from guinea pigs and C. parvum suggest they are distinct species. We propose the name Cryptosporidium wrairi be retained. Studies with monoclonal antibodies indicate that C. wrairi and C. parvum are antigenically related.     

Comparison of the Host Ranges and Antigenicity of Cryptosporidium parvum and Cryptosporidium wrairi from Guinea Pigs

ABSTRACT. Oocysts of a Cryptosporidium isolate from guinea pigs were not infectious for adult mice, but were infectious for two of three newborn calves and for suckling mice. However, oocysts isolated from calves or mice infected with guinea pig Cryptosporidium were not infectious for guinea pigs. Four isolates of C. parvum from calves were incapable of infecting weanling guinea pigs. Microscopic examination of tissue from the colon and cecum of suckling guinea pigs inoculated with C. parvum revealed sparse infection of some pups. These host range studies and previously described differences in ¹²⁵I-labeled oocyst surface protein profiles between Cryptosporidium sp. from guinea pigs and C. parvum suggest they are distinct species. We propose the name Cryptosporidium wrairi be retained. Studies with monoclonal antibodies indicate that C. wrairi and C. parvum are antigenically related.     

Heat-stable enterotoxin produced by Shigella flexneri.

Filtrates and ultrasonics extracts of Shigella flexneri showed rapid permeability factor (PF) test and proved positive in suckling mice and ligated rabbit loop tests within 4 hr. Delayed PF was not detected and the rabbit loop dilatation test read after 18 to 24 hr, the mouse pad oedema reaction, the test for elongation effect of CHO cells were also negative. In the delayed PF test a strong "blanching" effect was observed. A filtrate of an Ent-Escherichia coli strain was positive only in the rapid PF test, while filtrate and ultrasonic extract prepared from the Ent+ E. coli strain showed a positive reaction in all tests for enterotoxins (ST and LT) including the rapid PF test. Ultrasonic extracts of a S. flexneri and an Ent- E. coli strain concentrated by freeze-drying were fractionated on Sephadex G-100 column. S. flexneri fractions of 60--70ml were positive for rapid PF, dilation capacity in suckling mice, and the blanching effect in the delayed PF test. No positive reaction was found in the delayed PF test and in CHO cell culture. Similar fractions of Ent- E. Coli carried substances responsible for the rapid PF and the blanching effect, but without suckling mice positivity.     

Immunogenicity,Protective Efficacy,and Non-Replicative Status of the HSV-2 Vaccine Candidate HSV529 in Mice and Guinea Pigs

HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine.     

Effect of aqueous leaf extract of Irvingia gabonensis on gastrointestinal tract in rodents

Effect of the aqueous leaf extract of I. gabonensis on the gastrointestinal tract was investigated on isolated rabbit jejunum, guinea pig ileum, gastrointestinal motility, castor oil-induced diarrhoea in mice and castor oil-induced fluid accumulation in rats. The results showed that the extract exhibited a concentration-dependent relaxation of spontaneous pendular movement of isolated rabbit jejunum and guinea pig ileum, and attenuated both acetylcholine-induced contraction of rabbit jejunum and histamine-induced contraction of guinea pig ileum. The extract (100, 200 and 400 mg/kg) also caused a significant dose-dependent decrease of gastrointestinal motility in mice (40.12, 39.45 and 37.45%), intestinal fluid accumulation in rats (71.43, 81.63 and 83.27%), and remarkably protected mice against castor oil-induced diarrhoea [58.33, 75 and 91.67% (Di Carlo score)] respectively. Preliminary phytochemical screening of the aqueous leaf extract of I. gabonensis revealed the presence of saponins, tannins, phenols and phlobatanins.     

Enterotoxicity ofVibrio furnissii isolated from eels

Fourteen strains ofVibrio furnissii, isolated from different ulcerated areas of eel, were tested to check their enterotoxicity in an animal model. Most strains caused fluid accumulation in ileal loop tests after serial passages and culture filtrates of most of the strains caused induration and increase in vascular permeability in rabbit skin. Production of extracellular haemolysin was also detected in all the culture filtrates. All of these observations clearly establish the enterotoxicity of these organisms.     

Virulence of Venezuelan Equine Encephalomyelitis Virus Subtypes for Various Laboratory Hosts

Mice, guinea pigs, and duck embryo cell cultures were inoculated with known subtypes of Venezuelan equine encephalomyelitis (VEE) virus and the attenuated (TC-83) strain of VEE. With the exception of TC-83, all strains were highly pathogenic for suckling mice by either intracranial or intraperitoneal routes of inoculation used. Virulence for older mice and guinea pigs provided a means to distinguish strains. In addition, virulence or lack of virulence for adult mice or guinea pigs provides a rapid method for separating epizootic subtype IB from TC-83 VEE virus isolates.     

Immunogenicity of B-antigen in experimental plague and pseudotuberculosis

The results of the evaluation of the immunogenic properties of B-antigen, earlier identified in the culture fluid of Yersinia pseudotuberculosis submerged culture, with respect to experimental plague and pseudotuberculosis are presented. B-antigen has been shown to produce protective effect in guinea pigs and, probably, hamadryas baboons, but not in white mice infected with the causative agent of plague. Immunizaton with B-antigen protects guinea pigs from primary pneumonic plague caused by both capsule-forming and noncapsular Y. pestis virulent strains. Passive immunization with antibodies to B-antigen induces limitedly pronounced protective effect in guinea pigs and is not effective for white mice with respect to experimental plague. No active or passive protection of white mice or guinea pigs, infected with Y. pseudotuberculosis cultures, has been achieved by the injection of B-antigen or antibodies to it.     

A comparative study on the biology of Cryptosporidium sp. from guinea pigs and Cryptosporidium parvum (Apicomplexa).

Cryptosporidium sp. from guinea pigs and C. parvum were compared morphologically, electrophoretically, and for the ability to infect suckling mice. Oocysts from guinea pigs measured 5.4 x 4.6 (4.8-5.6 x 4.0-5.0) microns and had a shape index (length/width) of 1.17 (1.04-1.33). Oocysts of C. parvum were similar and measured 5.2 x 4.6 (4.8-5.6 x 4.2-4.8) microns with a shape index of 1.16 (1.04-1.33). All suckling mice inoculated with oocysts of C. parvum became infected, whereas most, but not all, mice fed oocysts of the guinea pig isolate also became infected. However, mice inoculated with oocysts from guinea pigs produced on average 100-fold fewer oocysts by day 7 postinoculation than did mice infected with C. parvum, and the resulting infections were sparse and patchy along the ileum. Electrophoretic profiles were similar, but 125I surface labeling of outer oocyst wall proteins revealed striking differences between the two isolates. Cryptosporidium parvum had a wide molecular size range of 125I-labeled bands, whereas C. sp. from guinea pigs had a banding pattern clustered between 39 and 66 kDa, with a smaller number of bands greater than 100 kDa.     

Characterization of an Enterotoxin Produced by Vibrio cholerae O139

A cholera-like enterotoxin was purified from Vibrio cholerae O139 strain AI-1841 isolated from a diarrheal patient in Bangladesh. Its characteristics were compared with that of cholera toxins (CTs) of classical strain 569B and El Tor strain KT25. Al-1841 produced as much toxin as O1 strains. The toxins were indistinguishable in terms of their migration profiles in conventional polyacrylamide gel disc electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectrofocusing as well as their affinity for hydroxyapatite. The skin permeability factor activity and the fluid accumulation induced in rabbit ileal loops of the toxin of AI-1841 were identical to those of the CTs. Three toxins equally reacted against anti-569B CT antiserum in Western blotting, and their B subunits formed a precipitin line against any anti-B subunit antiserum by double gel immunodiffusion. Anti-569B CTB antibody neutralized the three toxins in their PF activities and enterotoxicities. The amino acid sequence of 1841 toxin B subunit was identical with that of KT25 CTB, corresponding to the DNA sequence of ctxB from El Tor strains of the seventh pandemic. We concluded 1841 toxin was identical to CT of the seventh pandemic El Tor vibrios.     

On the toxinogenicity of Pseudomonas aeruginosa strains

Investigation of 367 P. aeruginosa strains primarily isolated from clinical and other biological material as well as from the environment yielded results suggesting a substantial toxinogenic potential. 92.6% of the assayed culture filtrates derived from the strains under investigation proved positive in the early skin tests on rabbits. 49.7% of the assayed material induced cytotoxic alterations on Vero cells, the rates for Y1 and CHO cells being 50.3% and 43.5% respectively. 54.3% culture filtrates caused haemolysis of rabbit RBC and 52.7% lysed horse RBC. Gelatinase activity was found in 96.3% of tested material, protease in 89.8%, lecithinase in 62.4% and elastase in 29.6%. 12.6% of tested material induced fluid accumulation in a ligated intestinal loop. None of the culture filtrates elicited a positive reaction in the suckling mice test suggesting the absence of the thermostable enterotoxin.     

Development and evaluation of a rapid, simple, sensitive, monoclonal antibody-based co-agglutination test for direct detection of Vibrio cholerae 01

Cholera epidemics caused by Vibrio cholerae 01 continue to represent a major public health concern in many developing countries. A rapid and simple test kit for the detection of V. cholerae 01 has been developed. The kit, CholeraScreen is a monoclonal antibody-based, co-agglutination test and is used directly with stool specimens. It does not include culturing the specimen and is performed without the need for sophisticated laboratory equipment. Specificity of the test was demonstrated, using 118 reference cultures, to which cross-reactions were not observed. Preliminary results of field trials carried out in Guatemala and Bangladesh demonstrated that the test is equally sensitive as conventional culture methods in detecting V. cholerae and, in many cases, more sensitive. The CholeraScreen test is simple, specific, and does not require culturing procedures, making it suitable for direct detection of cells of V. cholerae in clinical specimens, even in the field. Also, the test requires less than five minutes to complete.     

Vibrio mimicus are the reservoirs of the heat-stable enterotoxin gene (nag-st) among species of the genus Vibrio

Using a 0.27 kb DNA probe specific for the heat-stable enterotoxin gene (nag-st) of Vibrio cholerae non-O1, 1109 strains representing 17 species of the genus Vibrio, isolated from clinical and environmental sources were examined. The nag-st gene was preponderantly associated with strains classified as V. mimicus; 16.8% of these strains hybridized. It was more frequent in the clinical isolates (22.6%) than in the environmental isolates (13.7%). The incidence of nag-st gene-positive strains of V. mimicus isolated from different countries was uniformly high and ranged between 8.7% (Bangladesh) and 57.1% (environmental strains from USA). The incidence of the nag-st gene was much lower among strains of V. cholerae non-O1 (3.6%). Probe-positive and-negative strains of V. mimicus and V. cholerae non-O1 were used to evaluate the performance of the conventional suckling mouse assay for detection of the NAG-ST enterotoxin. Of the 31 probe-positive strains, only five (16.1%) yielded a positive fluid accumulation ratio (FA ratio) when neat heated culture supernatant was used to perform the suckling mouse assay. All the 31 probe-positive strains gave a positive FA ratio when 20-fold concentrated and heated culture supernatants of the strains were used to perform the suckling mouse assay. The need to concentrate (by at least 20-fold) the culture supernatant of strains of V. mimicus and V. Cholerae non-O1 was identified as an important step to obtain consistent results when using the suckling mouse assay for detection of NAG-ST.P. Yuan, A. Ogawa and T. Takeda are with the Department of Infectious Disease Research, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154, Japan; P. Yuan is also with the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. T. Ramamurthy and G.B. Nair are with the National Institute of Cholera and Enteric Diseases, Calcutta, India. T. Shimada is with the National Institute of Health, Tokyo 141, Japan. S. Shinoda is with the Faculty of Pharmaceutical Sciences, Okayama University, Japan.     

Production of heat-stable enterotoxin II by chicken clinical isolates of Escherichia coli

Abstract Enterotoxigenic Escherichia coli isolated from diarrhea stools of chickens were examined for production of heat-stable enterotoxin II which is considered to be implicated only in diarrhea of pigs. Seven out of 38 strains examined were found to contain heat-stable enterotoxin II gene, determined by colony hybridization and the polymerase chain reaction. The culture supernatants of these strains caused fluid accumulation in the mouse intestinal loop test. This fluid accumulation activity was not lost by heating at 100°C and was neutralized by anti-heat-stable enterotoxin II antiserum. Purified heat-stable enterotoxin II caused fluid accumulation in the chicken intestinal loop assay. These results indicate that STII-producing E. coli is implicated in chicken diarrhea.     

Evaluation of enrichment media for improved PCR-based detection of V. cholerae and V. vulnificus from estuarine water and plankton

Aims: Pathogenic Vibrio spp., including V. cholerae and V. vulnificus, are commonly found along the estuaries of the south‐east United States; however, it is often difficult to recover these species directly from environmental samples. Pre‐enrichment assays are commonly used to improve the detection of pathogenic vibrios from environmental sources. Here, we evaluated a novel enrichment procedure using freshly collected and autoclaved natural estuarine water amended with 1% peptone (designated as estuarine peptone water, EPW) and compared it to traditional alkaline peptone water (APW) for detection by PCR of V. cholerae and V. vulnificus. Methods and Results: Of the 50 samples collected in total, V. cholerae DNA was detected in APW 10% of the time and in EPW 40% of the time. Likewise, the cholera toxin gene (ctxA) was detected in 4 vs 18% of the samples using APW and EPW, respectively. Conversely, APW showed improved recovery for V. vulnificus relative to EPW with respective detection frequencies of 46 and 20%. Results showed similar patterns across different sample types (water and plankton). Conclusions: While enrichment in traditional APW was adequate for the recovery of Vibrio vulnificius, use of sterile estuarine water amended with peptone significantly improved the detection of V. cholerae and the virulence gene ctxA from estuarine sources.     

口蹄疫病毒免疫活性串联片段FB的表达及免疫原性测定(英)

将口蹄疫病毒(FMDV)免疫串联片段FB克隆至原核表达载体pBAD/TOPO中,经鉴定后得到重组质粒pBAD-FB,将此重组质粒转化到受体菌TOP10中,用诱导剂阿拉伯醛糖分别以不同的浓度进行诱导,并在不同诱导时间进行采样,经处理后做SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹分析.结果发现以终浓度为0.002%的阿拉伯醛糖进行诱导,4 h后表达可达到高峰,其大小约为26 ku,软件扫描结果显示,FB融合蛋白的表达量占细菌总蛋白的28.9%,能与抗FMDV抗体发生特异性反应,融合蛋白以包涵体和可溶形式存在.将融合蛋白的可溶性组分用50% Ni-NTA树脂过柱纯化并抽提融合蛋白的包涵体,经过洗涤后分别制成油乳剂疫苗,经皮下注射免疫豚鼠,用乳鼠中和试验测定豚鼠血清中和指数,并用口蹄疫病毒对豚鼠进行攻毒.结果表明,用此融合蛋白的纯化产物和包涵体免疫豚鼠能诱导产生高滴度的中和抗体,对病毒的攻击提供100%的免疫保护.     

YNS益肤面霜对豚鼠皮肤变态反应及肠道菌群的影响

建立豚鼠皮肤变态反应模型,并使用YNS益肤面霜对皮肤变态反应模型进行干预,探讨这种面霜对改善及修复受损皮肤的作用及其对豚鼠肠道菌群的影响。

豚鼠24只,随机分成4组：对照组、阳性组、化妆品组和治疗组,每组6只。致敏接触后进行激发反应。提取4组豚鼠粪便标本DNA,使用16S rRNA高通量测序检测肠道菌群构成。

治疗组豚鼠皮肤致敏率为16.7%。益肤面霜治疗对肠道菌群alpha多样性影响不大,但有降低厚壁菌门（Firmicutes）与拟杆菌门（Bacteroidetes）比值（F/B）及粪球菌属（Coprococcus）相对丰度的趋势,有利于皮肤变态反应豚鼠肠道菌群组成的恢复。

YNS益肤面霜对皮肤具有改善及修复作用,并对豚鼠肠道菌群具有一定影响。