Identification of a secreted superoxide dismutase in Mycobacterium avium ssp. paratuberculosis.

Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis), the causative agent of Johne's disease, is an important animal pathogen that has also been implicated in human disease. The major proteins expressed by M. paratuberculosis were analyzed by two-dimensional gel electrophoresis, and a superoxide dismutase (Sod) was identified from this protein profile. The M. paratuberculosis Sod has a molecular mass of 23 kDa and an isoelectric point of 6.1. Sequence analysis of the corresponding sodA gene from M. paratuberculosis indicates that this protein is a manganese-dependent enzyme. We show that the M. paratuberculosis Sod is actively secreted, suggesting that it may elicit a protective cellular immune response in the host during infection.

Mycobacterium tuberculosis complex DNA from an extinct bison dated 17,000 years before the present.

In order to assess the presence of tuberculosis in Pleistocene bison and the origin of tuberculosis in North America, 2 separate DNA extractions were performed by 2 separate laboratories on samples from the metacarpal of an extinct long-horned bison that was radiocarbon dated at 17,870+/-230 years before present and that had pathological changes suggestive of tuberculosis. Polymerase chain reaction amplification isolated fragments of tuberculosis DNA, which were sequenced, and on which spoligotyping was also performed to help determine its relationship to the various members of the Mycobacterium tuberculosis complex. Extensive precautions against contamination with modern M. tuberculosis complex DNA were employed, including analysis of paleontologic and modern specimens in 2 geographically separate laboratories.

Mycobacterial infections in domestic and wild animals due to Mycobacterium marinum, M. fortuitum, M. chelonae, M. porcinum, M. farcinogenes, M. smegmatis, M. scrofulaceum, M. xenopi, M. kansasii, M. simiae and M. genavense.

The epidemiology and the natural distribution of Mycobacterium marinum, M. fortuitum, M. chelonae, M. porcinum, M. farcinogenes, M. smegmatis, M. scrofulaceum, M. xenopi, M. kansasii, M. simiae and M. genavense are described. In addition to the bacteriological, biochemical and genetic characteristics, the authors review the pathology of these species, including the natural and experimental diseases and the accompanying lesions, diagnosis, antibiotic sensitivities and treatment of animal infections caused by these mycobacteria.

Molecular epidemiology of Mycobacterium tuberculosis infection in Israel.

Restriction fragment length polymorphism analysis with IS6110 and DR-r probes was used to study 69 Mycobacterium tuberculosis isolates obtained from Israeli patients and new immigrants from the former Soviet Union and Ethiopia. DNA fingerprinting identified unique patterns for almost all isolates, indicating that most patients were infected with a unique strain imported from their country of origin and that their latent infection was reactivated in Israel.

Streptococcus agalactiae and Streptococcus difficile 16S-23S intergenic rDNA: genetic homogeneity and species-specific PCR.

Streptococcus difficile is a non-hemolytic Gram-positive bacterial coccus that causes septicemia and meningoencephalitis in farmed tilapia (Oreochromis sp.) and rainbow trout (Oncorhynchus mykiss). Recent studies have demonstrated S. difficile to be a group B, type Ib streptococcus with a whole cell protein electrophoretic profile indistinguishable from S. agalactiae and a biochemical profile similar to that observed for other group B, type Ib streptococci isolated from fish and frogs. The aim of this study was to expand on these findings by comparative nucleic acid sequence analysis of the 16S-23S ribosomal DNA (rDNA) intergenic spacers of S. difficile and S. agalactiae. The 97.7% sequence homology identified in these studies supports the taxonomic relationship of these two organisms. The sequence data generated were also used to construct a pair of species-specific PCR primers for use in molecular detection and identification schemes.

Acinetobacter baumannii at a tertiary-care teaching hospital in Jerusalem, Israel.

In a retrospective 10-year analysis of 3,536 patient-unique isolates, Acinetobacter baumannii imipenem susceptibility declined from 98.1 (1990) to 64.1% (2000), and ciprofloxacin susceptibility decreased from 50.5 to 13.1%. Imipenem median zone diameters decreased from 27. 7 (1997) to 18.8 mm (2000). No outbreaks were detected. Two clusters were identified for 41 strains genotyped by pulsed-field gel electrophoresis, but imipenem resistance was not clonal.

A Herpesvirus Associated with Mass Mortality of Juvenile and Adult Koi, a Strain of Common Carp.

A herpesvirus was isolated from adult koi, a strain of common carp Cyprinus carpio, suffering mass mortality in two outbreaks-one in the mid-Atlantic region of the United States and the second in Israel. The principal external signs of dying fish were pale and irregularly colored gills. There were few consistent internal signs in either outbreak. The most prominent microscopic lesions were in the gills, where hyperplasia and necrosis of the epithelium were severe. Other lesions included interstitial nephritis, splenitis, and enteritis. Affected cells often contained nuclei with marginated chromatin and faint intranuclear inclusions. Typical herpesvirus particles were present in branchial epithelial cells, hepatocytes, and among circulating leukocytes. Inoculations of the koi fin (KF-1) cell line with tissue extracts from the gill and kidney-spleen resulted in cytopathic effects characterized by severe vacuolation first detected after 7 d incubation at 20°C. Exposures of adult koi to the herpesvirus as propagated in KF-1 cells by bath or intraperitoneal injections resulted in 80-100% mortality during a 26-d period, and the virus was reisolated from the gill, kidney, liver, spleen, intestine, and brain of dead fish. The viral agents from koi in Israel and the United States appear to be similar if not identical; both could be distinguished from Herpesvirus cyprini by indirect fluorescent antibody tests with rabbit anti-H. cyprini serum. Other factors should be examined but we strongly suspect that this newly recognized koi herpesvirus (KHV) has the potential to be a significant cause of mortality among koi and presumably common carp.

Red drum Sciaenops ocellatus mortalities associated with Streptococcus iniae infection.

We isolated for the first time Streptococcus iniae strains associated with diseased marine fish. Diseased red drum Sciaenops ocellatus were lethargic, and presented external signs (exophthalmia and loss of orientation) resembling those of freshwater fish infected by S. iniae. Skin lesions, extending to a necrotizing myositis, were typical of S. iniae infection of red drum. Histopathological findings indicate that S. iniae infection in red drum produces a chronic disease with systemic involvement characterized by multiple necrotic foci. Molecular epidemiology (RFLP [restriction fragment length polymorphism] ribotyping) revealed that 2 different ribotypes were involved in a single outbreak. The first is the EcoRI 'Israeli' trout and tilapine ribotype (Hind III type a strains), while the second is the EcoRI 'American' ribotype (Hind III type b strains), typical of tilapines farmed in Texas and Idaho.

Prevalence of anti-hepatitis A antibodies, hepatitis B viral markers, and anti-hepatitis C antibodies among immigrants from the former USSR who arrived in Israel during 1990-1991.

The goal of this study was to assess the susceptibility of the sub-population of over 500,000 immigrants from the former USSR who came to Israel during 1989-94 to HAV infection, and to provide military physicians with estimates of the prevalence of HBV and HCV carriage in this sub-population. 987 males aged 17-49 and 195 females aged 17-19, reporting to military recruitment offices between December 1991 and March 1992 were tested. Anti-HAV, anti-HBV antibodies and hepatitis B surface antigen (HBsAg) were detected by using standard enzyme immunoassay (EIA) tests, and anti-HCV antibodies by a second-generation EIA and confirmed by a third-generation INNO-LIA test. It was found that in the 17-19-year age-group the prevalence of anti-HAV antibodies was 37%, anti-HBV was 12.8%, HBsAg was 3.0% and anti-HCV 1.3%. All markers were higher among males. The prevalence of anti-HAV and anti-HBs antibodies increased with age among males. That of HBsAg and anti-HCV antibodies increased with age overall. In the multiple logistic regression analysis, HAV and HBV seropositivity were significantly associated with the mother's education and republic of origin. It was concluded that the prevalence of anti-HAV antibodies is similar to that among the local population, which should not be considered at a higher risk of infection during military service. On the other hand, the higher prevalence of HBsAg and anti-HCV antibodies in this sub-population should heighten the awareness of the possibility of chronic liver pathology.

Biodiversity of Lactococcus garvieae strains isolated from fish in Europe, Asia, and Australia.

Lactococcus garvieae (junior synonym, Enterococcus seriolicida) is a major pathogen of fish, producing fatal septicemia among fish species living in very diverse environments. The phenotypic traits of L. garvieae strains collected from three different continents (Asia, Europe, and Australia) indicated phenotypic heterogeneity. On the basis of the acidification of D-tagatose and sucrose, three biotypes were defined. DNA relatedness values and a specific PCR assay showed that all the biotypes belonged to the same genospecies, L. garvieae. All of the L. garvieae strains were serotyped as Lancefield group N. Ribotyping proved that one clone was found both in Japan, where it probably originated, and in Italy, where it was probably imported. PCR of environmental samples did not reveal the source of the contamination of the fish in Italy. Specific clones (ribotypes) were found in outbreaks in Spain and in Italy. The L. garvieae reference strain, isolated in the United Kingdom from a cow, belonged to a unique ribotype. L. garvieae is a rising zoonotic agent. The biotyping scheme, the ribotyping analysis, and the PCR assay described in this work allowed the proper identification of L. garvieae and the description of the origin and of the source of contamination of strains involved in outbreaks or in sporadic cases.

Development of specific nested oligonucleotide PCR primers for the Streptococcus iniae 16S-23S ribosomal DNA intergenic spacer.

Streptococcus iniae is a cause of septicemia, meningoencephalitis, and death in farmed fish and of cellulitis in human beings. A set of nested oligonucleotide PCR primers that specifically amplified a 373-bp subunit from a variety of clinical isolates from farmed fish and human patients were constructed from a 524-bp consensus sequence of the S. iniae 16S-23S ribosomal DNA intergenic spacer.

Identification of Lactococcus garvieae by PCR.

Lactococcus garvieae junior synonym Enterococcus seriolicida) is an emerging zoonotic agent isolated from economically important fish (rainbow trout and yellowtail), from cattle, and from humans. Clindamycin susceptibility is the only phenotypic test which can differentiate L. garvieae from Lactococcus lactis, another emerging agent in humans. A PCR assay for the identification of L. garvieae was developed and resulted in an amplified fragment of 1,100 bp in size. The PCR assay was shown to be specific to L. garvieae. The PCR assay was positive for all the L. garvieae strains tested, which originated from three different continents (Asia, Australia, and Europe). The PCR assay was negative for the phenotypically similar L. lactis and for all the other fish pathogens tested, including Streptococcus iniae and Aeromonas salmonicida. The PCR assay was applied to plasma obtained from diseased animals and was found sensitive enough to detect bacteria from 1 microl of plasma. The PCR assay that was developed is the only practical test besides the clindamycin test which can specifically identify the zoonotic agent L. garvieae and which can differentiate it from L. lactis.

Immune status against diphtheria among immigrants from the former USSR who arrived in Israel during 1990-1991.

Large outbreaks of diphtheria occurred recently in the former USSR. Between 1989 and 1994, a total of about 600,000 Soviet immigrants arrived in Israel. The immune status against diphtheria in a sample of 992 men aged 17-49 and 195 women aged 17-19, who arrived in Israel during 1990-91, was studied in order to evaluate the need for vaccination. Participants completed a self-administered questionnaire and diphtheria antitoxin antibody levels were measured by means of ELISA. At age 17-19, the prevalence of antitoxin antibody levels below the protective level of 0.01 IU/ml was 4.8% in the men and 2.1% in the women. Among the men, the percentage lacking protection declined from 4.8% at age 17-19 years to 1.6% at age 20-24, and increased to 18.2% at age 35-49. In the oldest group, the prevalence of those lacking protection was considerably higher than for the general Israeli population. In the multivariate analysis, age, mother's education and republic of origin were significantly associated with the absence of protection. Immigrants from the former USSR appear to be more susceptible to diphtheria, thus increasing the possibility of clinical disease, and it is recommended that they receive booster doses of diphtheria toxoid.

Pentoxifylline and thalidomide fail to reduce hepatic steatosis during total parenteral nutrition and bowel rest in the rat.

BACKGROUND: We suggested that the continuous translocation of endotoxin from Gram-negative bacterial overgrowth during bowel rest and total parenteral nutrition (TPN) causes the release of tumor necrosis factor (TNF), resulting in liver damage and hepatic dysfunction. Because TPN-induced hepatic steatosis was significantly reduced by the monoclonal antibodies against TNF, we attempted a more clinically applicable approach using pentoxifylline and thalidomide. METHODS: A control group (group I) fed rat chow and four groups of rats receiving TPN were studied. Group II received TPN only; group III, TPN and 100 mg/kg/d pentoxifylline; group IV, TPN and 200 mg/kg/d pentoxifylline; and group V, TPN and 5 mg/kg/d thalidomide. On day 7, total liver fat was determined. RESULTS: Bowel rest and TPN resulted in a significant (p < .0005) increase in liver fat content that was unaltered by either pentoxifylline or thalidomide. CONCLUSIONS: Our results show no role for pentoxifylline or thalidomide in reducing TPN-associated hepatic steatosis.

Restriction fragment length polymorphisms of 16S rDNA and of whole rRNA genes (ribotyping) of Streptococcus iniae strains from the United States and Israel.

Streptococcus iniae (junior synonym S. shiloi) isolated from tilapia and trout in Israel and in the United States were subtyped by restriction length polymorphism (RFLP) based on PCR amplified 16S rDNA and by ribotyping. 16S rDNA RFLP discriminated between S. iniae and other fish pathogens but not between S. iniae strains. HindIII and EcoRI ribotypes of S. iniae discriminated American from Israeli strains rejecting the possibility of an epidemiological link between S. iniae infections in the two countries. Israeli strains isolated from tilapia and trout could not be completely differentiated. The S. iniae ATCC 29178T (T = Type strain) strain, isolated from a freshwater dolphin belonged to a ribotype different from those of all the fish isolates.

Development and efficacy of a vaccine against Streptococcus iniae infection in farmed rainbow trout.

Formalin killed bacteria were used as a vaccine against Streptococcus iniae infections in farmed rainbow trout. A single intraperitoneal injection of this vaccine in trout resulted in specific antibody production detectable for 6 months. Trout vaccinated at 50 g were protected under laboratory (experimental disease) and field conditions (natural disease) for at least 4 months against S. iniae infection. Passive transfer of S. iniae specific antibodies conferred protection. Under field conditions, mortality of non vaccinated trout exceeded 50%, whereas mortality of vaccinated trout did not reach 5%. In addition, vaccinated trout under field conditions gained 20% weight when compared with non vaccinated fish.

Delayed-type hypersensitivity activity of the Brucella L7/L12 ribosomal protein depends on posttranslational modification.

The ribosomal protein L7/L12 isolated from Brucella melitensis induces a delayed-type hypersensitivity (DTH) reaction in brucella-sensitized guinea pigs. Surprisingly, the recombinant brucella L7/L12 protein expressed in Escherichia coli as a fusion protein with a six-histidine tag cannot elicit such a reaction. The six histidines tagged to the recombinant L7/L12 protein were removed enzymatically, but the resulting protein did not induce a DTH reaction in sensitized animals. Incubation of the recombinant L7/L12 fusion protein in a B. melitensis lysate endowed the recombinant protein with a DTH activity, suggesting that the recombinant protein was modified by this treatment. Glycosylation or phosphorylation of the recombinant L7/L12 protein could not be detected. On the other hand, radiolabeled palmitic acid was found to be incorporated to the recombinant protein during its incubation in the brucella lysate. This incorporation was specific for the brucella L7/L12 protein and was inhibited when the brucella lysate was frozen and thawed before the incubation. The data reported here indicate that posttranslational modification of L7/L12 protein comprising at least an acylation step is required for the brucella L7/L12 DTH activity.

Immunization with bacterial antigens: infections with streptococci and related organisms.

Streptococcal infections of fish have been reported from various parts of the world, including the Far East, the United States, South Africa, Australia, Israel and Europe. Classification of Gram-positive cocci (DNA-DNA hybridization studies coupled with 165 sequencing) has shown that at least five different defined species are pathogenic to fish, e.g. Streptococcus iniae (syn. S. shilot), Streptococcus difficile, Lactococcus garvieae (syn. Enterococcus seriolicida), Lactococcus piscium and Vagococcus salmoninarum. "Streptococcosis" of fish should therefore be regarded as a complex of similar diseases caused by different genera and species of Gram-positive cocci, each capable of inducing CNS damage, as well as various degrees of multisystem organ involvement. Panophthalmitis ("pop-eye") and meningitis/meningoencephalitis are the sole findings in trout infected by S. iniae and in tilapines infected by S. difficile. In contrast, L. garvieae-infected trout bear a systemic hyperacute infection with diffuse haemorrhages. Therapeutic measures are generally ineffective. Development of vaccines is therefore essential to control these diseases. In our studies, trout were vaccinated intraperitoneally with whole-cell formalin-inactivated S. iniae and L. garvieae and tilapines with whole-cell formalin-inactivated and acellular S. difficile extract. Under laboratory conditions, S. difficile-vaccinated tilapines were protected against a challenge of 100 LD50s. Protection was correlated with the development of specific agglutinins. Western blot analysis supported the hypothesis that only a few proteins act as protective antigens. S. iniae autovaccines were effective in preventing the disease in rainbow trout in Israel. Under field conditions, fish vaccinated at 50 g were protected for over four months. The qualitative analysis of the humoral response indicated that specific antibodies are directed against a few protein moieties. The fact that passive transfer of antibodies protected fish from experimental infection suggests that the basic mechanism of protection is antibody mediated. L. garvieae autovaccines developed for Italian trout farming were found to elicit a response similar to that of S. iniae. Despite the high virulence of L. garvieae (LD50 of 6 x 10(1) CFU/fish, compared with 3 x 10(4) CFU/fish of S. iniae), the protection against the experimentally induced disease lasted for five months under laboratory conditions, with survival rates of 80-90%. A single injection of the vaccine (0.1 ml/fish) resulted in specific antibody production detectable for six months. In the field, protection rates of 70-80% were obtained for a period of three months, in fish of 200-300 g reared at water temperatures of 18-21 degrees C.

Enterococcus seriolicida is a junior synonym of Lactococcus garvieae, a causative agent of septicemia and meningoencephalitis in fish.

The reference strains of Enterococcus seriolicida (ATCC 49156T) (T = type strain) and of Lactococcus garvieae (ATCC 43921T) and 30 field strains of Gram-positive cocci isolated from diseased rainbow trout in Italy were found to be phenotypically (API 20 STREPT and API 50 CH) and genetically (DNA-DNA hybridization) similar. The high DNA-DNA homologies (70-100%) and the low delta Tme (less than 1.1 degrees C) among these strains showed that Enterococcus seriolicida and Lactococcus garvieae are synonyms, describing a single bacterial species. E. seriolicida strains should be classified as L. garvieae, which must be considered as a major pathogen of freshwater and salt water fish with a world-wide distribution.

Vaccination with whole-cell vaccine and bacterial protein extract protects tilapia against Streptococcus difficile meningoencephalitis.

Formalin-killed Streptococcus difficile strains used as vaccines delivered intraperitoneally were able to protect tilapia against a challenge of 100 LD50. The protection obtained was not strain specific. A vaccine based on an S. difficile extract containing 50% protein conjugated to alum also protected tilapia challenged with a virulent S. difficile strain. Protection in tilapia was correlated with the development of specific agglutinins. Western blot analysis supported the hypothesis that only a few proteins act as protective antigens in both the whole-cell vaccine and the streptococcal extract. The high efficacy of these vaccines make them good candidates for the control of streptococcal fish meningoencephalitis.