The effect of mouse age on the determination of Rickettsia tsutsugamushi virulence

The effect of age on the susceptibility of ICR mice to lethal intraperitoneal (ip), Rickettsia tsutsugamushi infections was tested with five virulent strains--Karp, Kato, Gilliam, TA763, and TH1817--and three strains of reduced virulence--TA678, TA686, and TA716. Susceptibility differences were noted only in the ICR mice inoculated with two of the strains of reduced virulence, TA716 and TA678. With both strains, mice in the 12-weeks and younger age groups had lower death rates than did mice in the 21-weeks and older age groups. Also, CBA/CaJ mice of varying ages were inoculated intravenously with large doses of the Gilliam strain to determine the effect of age on susceptibility to acute death syndrome (ADS). A progressive increase in ADS resistance was seen in the 4-, 8-, 12-, and 16-week-old age groups. This study indicates that the age of mice used to test the virulence of R. tsutsugamushi strains may be an important consideration, especially when testing the ip lethality of strains of reduced virulence.     

Antigenic relationship among the eight prototype and new serotype strains of Orientia tsutsugamushi revealed by monoclonal antibodies.

Orientia tsutsugamushi, the etiologic agent of tsutsugamushi disease, exhibits great antigenic variation. Three classical strains (Karp, Gilliam, and Kato) and new antigenic types from Thailand (TA686, TA678, TA716, TA763, and TH1817) have been used as prototype strains of O. tsutsugamushi in many studies. In this study, monoclonal antibodies to the five Thailand strains were produced, and their reactivity against prototype strains and newly identified isolates from Korea and Japan was tested. With a panel of these monoclonal antibodies, we could analyze the antigenic relationship among various strains of O. tsutsugamushi from Thailand, Japan, and Korea. Twelve strains of the O. tsutsugamushi tested showed various reactivities to monoclonal antibodies, and no distinct pattern of reactivity was found according to their location of isolation. Although the Boryong and Kuroki strains were similar in reactivities to most monoclonal antibodies, several monoclonal antibodies could differentiate the two strains. These results indicate that the immunofluorescence antibody test using monoclonal antibodies used in this study is valuable for analyzing the antigenic relationship and classification of O. tsutsugamushi.     

Genetic typing, based on the 56-kilodalton type-specific antigen gene, of Orientia tsutsugamushi strains isolated from chiggers collected from wild-caught rodents in Taiwan

Orientia tsutsugamushi is the etiological agent of scrub typhus, a mite-borne, febrile illness that occurs in the Asia-Pacific region. We conducted strain characterization of O. tsutsugamushi isolates from chiggers obtained from rodents based the nucleotide sequence of the 56-kDa outer membrane protein gene. With the use of PCR, a total of 68 DNA sequences of 56-kDa antigen genes were amplified. Phylogenetic analysis revealed that there were at least six definable clusters among the 68 isolates: 37% Karp-related strains (25/68), 27% TA763 strains (18/68), 12% JG-related strains (8/68), 19% Kato-related strains (13/68), 4% divergent strains (3/68), and 1% representing a Gilliam prototype strain (1/68). Overall, the O. tsutsugamushi genotypes exhibited a high degree of diversity, similar to that seen in strains from the rest of the areas where scrub typhus is endemic. Moreover, the 56-kDa protein sequence similarity between O. tsutsugamushi isolates from mites and those from human patients (H. Y. Lu et al., Am. J. Trop. Med. Hyg. 83:658-663, 2010) were striking, thus highlighting potential risk factors for this emerging zoonotic disease.     

Prokaryotic expression and immunogenicity of 56-kDa protein of Orientia tsutsugamushi strain Karp

To express the 56-kDa protein of O. tsutsugamushi strain Karp, this protein gene was cloned into pET30a(+) before transforming into host bacteria, E. coli Rossetta. Specificity of the recombinant protein was assessed by ELISA using rabbit sera against common members of the order Rickettsiae and 10 other pathogenic bacteria. After IPTG induction, SDS-PAGE analysis of isolated protein demonstrated a band at approximately 46-kDa. Western blot and mass spectrometry analysis proved that the recombinant protein was expressed successfully. Specificity analysis demonstrated that all sera were negative, except sera against O. tsutsugamushi strains TA763, TH1817 and Kato, B. quintana, A. phagocytophilum, E. chaffeensis and B. bacilliformis. The purified protein was used to immunize BALB/c mice and polyclonal antisera were harvested. By examination of IFA and ELISA, the highest titer of the polyclonal antibodies reaches 1:1600. The recombinant 56-kDa protein in the study is valuable for developing a simple and rapid diagnostic test and vaccine for O. tsutsugamushi.     

Mutational studies with diquat and paraquat in vitro.

Diquat and paraquat were assayed in the following tests. (1) Ames test in Salmonella typhimurium (strains TA1535, TA1537, TA1538, TA98 and TA100) with and without rat-liver microsomal fractions. (2) Resistance to 8-azaguanine in Salmonella typhimurium (strain hisG46, TA92 and TA1535. (3) Repair test in Salmonella typhimurium (strains TA1538 and TA1978). (4) Gene mutations in Aspergillus nidulans: 8-AG resistance and methionine suppression (meth A1 locus). (5) Lethal recessive damage in Aspergillus nidulans. (6) Unscheduled DNA synthesis (UDS) in human epithelial-like cells (EUE). Diquat and paraquat were positive in S. typhimurium (in the repair test and the 8-AG resistance system), in A. nidulans (for gene mutations and lethal recessive damage induction) and in EUE cells (UDS induction).     

Genotypic and phenotypic polymorphism of environmental strains of the moderately thermophilic bacterium Sulfobacillus sibiricus

Five cultures of moderately thermophilic spore-forming acidophilic chemolithotrophic bacteria were isolated from the zones of spontaneous heating of pyrrhotine-containing pyrite-arsenopyrite gold-arsenic sulfide ores in an operating open pit (strains B1, B2, B3, OFO, and SSO). Analysis of the chromosomal DNA structure revealed differences between these cultures at the strain level (apart from B3 and SSO, which had identical restriction profiles). All the strains had a similar G + C DNA molar content (47.4-48.3%). The level of DNA reassociation was 85 to 95%. The similarity between the DNA of the type strain Sulfobacillus sibiricus N1 isolated from arsenopyrite ore concentrate and that of these strains (83-93%) indicates that they belong to the same species. The strains had similar values of pH and temperature optimal for growth on ferrous iron (1.6-2.0 and 45-55 degrees C, respectively). They were mixotrophs; Fe(II), S0, and sulfide minerals along with organic compounds were used as energy sources and electron donors. However, the kinetic parameters of growth and substrate oxidation varied from strain to strain. Genetic variety of the strains from diverse ecosystems and environments is possibly the result of the different rates of microevolution processes.     

Virulence in Mice of Orientia tsutsugamushi Isolated from Patients in a New Endemic Area in Japan

Four strains of Orientia tsutsugamushi (KN-1, KN-2, KN-3 and GJ-1) isolated from patients in an area of Gifu Prefecture, Japan, in which tsutsugamushi disease is newly endemic, were examined for their virulence in mice. Among these, KN-1 (identified as Kawasaki type), GJ-1 (identified as Kuroki type) and KN-2 strains were found to be non-lethal for BALB/c mice as well as CH3/HeJ mice, even with high doses (10⁶ × being the 50% mouse infectious dose). On the other hand, the KN-3 strain was found to be sufficiently virulent to kill BALB/c mice. Among the prototype strains (Gilliam, Karp and Kato), the Karp and Kato strains exhibited high virulence to mice, while the Gilliam strain killed only a susceptible strain of mouse. BALB/c mice infected with KN-1 and KN-2 strains showed significant splenomegaly and moderate ascites accumulation in the first week of infection, while these symptoms became prominent during the second week of infection using KN-3, Karp and Kato strains. After infection with the GJ-1 strain, these symptoms were not observed. Antibody responses induced by infections with highly virulent strains were lower than that with low or intermediate virulent strains.     

Genotoxic evaluation of extracts from Aplysina fulva, a Brazilian marine sponge

A range of biologically active secondary metabolites with pharmacological application has been reported to occur in marine sponges. The present study was undertaken to provide a set of data on the safety of a hydro-alcoholic extract (ALE) and an aqueous fraction (AQE) from Aplysina fulva Pallas, 1766 (Aplysinidae, Verongida, Porifera). Salmonella typhimurium strains TA97, TA98, TA100 and TA102, Escherichia coli strains PQ65, OG40, OG100, PQ35 and PQ37 and Balb/c 3T3 mouse fibroblasts were used to detect induction of DNA lesions by ALE and AQE. Assays used for these analyses were a bacterial (reverse) mutation assay (Ames test), the SOS-chromotest and the comet assay. Both extracts presented identical infrared 2-oxazolidone spectra. ALE treatment induced a higher frequency of type-4 comets, indicative of increasing DNA migration, in the alkaline comet assay. ALE also induced a weak genotoxic effect, as expressed by the induction factor (IF) values in the test with E. coli strain PQ35 (IF=1.5) and by cytotoxic effects in strains PQ35, PQ65 and PQ37. Positive SOS induction (IF=1.7) was detected in strain PQ37 treated with diluted AQE. No genotoxic effects were observed in strains PQ35, PQ65, OG40 and OG 100 after treatment with AQE dilutions. Using the bacterial (reverse) mutation test and survival assays with or without S9 mix, after 60min of pre-incubation, we observed for strain TA97 treated with ALE a weak mutagenic response (MI=2.2), while cytotoxic effects were seen for strains TA98, TA100 and TA102. AQE did not show mutagenic activity in any of the strains tested, but a weak cytotoxic effect was noted in strain TA102. Our data suggest that both ALE and AQE from A. fulva induce DNA breaks leading to cytotoxicity and mutagenicity under the conditions used.     

Description of Oxalicibacterium horti sp. nov. and Oxalicibacterium faecigallinarum sp. nov., new aerobic, yellow-pigmented, oxalotrophic bacteria

Three strains of aerobic, Gram-negative, rod-shaped, non-spore-forming, yellow-pigmented bacteria (OD1^T, YOx^T and NS13), which were isolated in previous studies by enrichment in a mineral medium with potassium oxalate as the sole carbon source, were characterized. On the basis of 16S rRNA gene sequence similarity, strains OD1^T, YOx^T and NS13 belong to the Betaproteobacteria , most closely related to Oxalicibacterium flavum TA17^T (97.2–99.7% sequence similarity). The major whole-cell fatty acids were C_16:0, C_16:1ω7c and C_17:0 cyclo. The results of DNA–DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains OD1^T and YOx^T from O. flavum TA17^T and from each other. Therefore, it is concluded that the strains OD1^T and YOx^T represent novel species within the genus Oxalicibacterium , for which the names Oxalicibacterium horti sp. nov. (type strain OD1^T=DSM 21640^T=NBRC 13594^T) and Oxalicibacterium faecigallinarum sp. nov. (type strain YOx^T=DSM 21641^T=CCM 2767^T) are proposed.     

Three consistent patterns of response to substituted acridines in a variety of bacterial tester strains used for mutagenicity testing

91 substituted acridines were tested for mutagenicity in 1 strain of Escherichia coli (TA78) and 4 strains of Salmonella typhimurium (TA90, TA1537, TA98 and TA100). In general, compounds fall into 3 groups: (i) inactive in all strains, (ii) active in TA78, TA90 and TA1537, or (iii) active in TA98 and often one or more of the other frameshift strains. Compounds of class iii have previously been shown to differ from the others in causing excisible damage to DNA and in showing an enhanced mutagenic response when the plasmid pKM101 is present.     

Mutagenic and genotoxic effects of theophylline and theobromine in Salmonella assay and in vivo sister chromatid exchanges in bone marrow cells of mice.

The mutagenic and genotoxic effects of two methylxanthines, theophylline (TH) and theobromine (TB), were assessed in the Ames mutagenicity assay (in strains TA97a, TA100, TA102 and TA104) and in vivo sister chromatid exchanges (SCEs) in bone marrow cells of mice. These are the two most commonly used nervous system stimulators throughout the world. TH is used in the long-term treatment of asthma. Bacterial mutagenicity assay showed very weak mutagenic effects of both drugs in Salmonella strains TA102 and TA104 only in certain concentrations when S9 was added to it. No mutagenic effects were observed in any other strains used in this assay either with or without metabolic activation. But results of in vivo SCE assay indicate that these two drugs can induce significant SCE in bone marrow cells of mice.     

Genetic diversity of endophytic diazotrophs of the wild rice, Oryza alta and identification of the new diazotroph, Acinetobacter oryzae sp. nov

Thirty-three endophytic diazotrophs were isolated from surface-sterilized leaves, stem, and roots of wild rice Oryza alta. The SDS-PAGE profile of total protein and insertion sequence-based polymerase chain reaction (IS-PCR) fingerprinting grouped the isolates into four clusters (I-IV). The 16S rRNA gene sequence homology of the representative strains B21, B31, B1, and B23 of clusters I, II, III, and IV were assigned to Pseudomonas oleovorans (99.2% similarity), Burkholderia fungorum (99.4% similarity), Enterobacter cloacae (98.9% similarity), and Acinetobacter johnsonii (98.4% similarity), respectively. The results showed wide genetic diversity of the putative diazotrophic strains of the wild rice, O. alta, and the strains of cluster IV are the first report of nitrogen-fixing Acinetobacter species. The cell size, phenotypic characters, total protein profile, genomic DNA fingerprinting, DNA-DNA hybridization, and antibiotic resistance differentiated strain B23(T) from its closest relatives A. johnsonii LMG999(T) and Acinetobacter haemolyticus LMG996(T). The DNA-DNA hybridization also distinguished the strain B23(T) from the closely related Acinetobacter species. Based on these data, a novel species, Acinetobacter oryzae sp. nov., and strain B23(T) (=LMG25575(T)?=?CGMCC1.10689(T)) as the type strain were proposed.     

Genetic typing of the 56-kDa type-specific antigen gene of contemporary Orientia tsutsugamushi isolates causing human scrub typhus at two sites in north-eastern and western Thailand

Orientia tsutsugamushi is the causative agent of scrub typhus, a major cause of febrile illness in the rural areas of Southeast Asia. Twenty-three strains of O. tsutsugamushi were isolated from patients with scrub typhus in north-east (Udorn Thani province) and western Thailand (Tak province) between 2003 and 2005. The isolates were characterized by sequencing the entire ORF of the 56-kDa-type-specific antigen gene, followed by phylogenetic analysis. The majority (15/23) of isolates clustered with the Karp-type strain, six with a Gilliam-type strain and one each with the TA716- and TA763-type strains. Overall, there was considerable diversity in sequence, comparable to that seen in strains from across the rest of the scrub typhus-endemic world. There was no significant difference in the distributions of strains between the two provinces (P=0.08, Fisher's exact) nor a temporal change in distribution with year of isolation (P=0.80, Fisher's exact). Within this diversity there were also examples of isolates with identical 56-kDa genotypes that were cultured from patients from the same geographical areas.     

Genotoxic evaluation of extracts from Aplysina fulva, a Brazilian marine sponge

A range of biologically active secondary metabolites with pharmacological application has been reported to occur in marine sponges. The present study was undertaken to provide a set of data on the safety of a hydro-alcoholic extract (ALE) and an aqueous fraction (AQE) from Aplysina fulva Pallas, 1766 (Aplysinidae, Verongida, Porifera). Salmonella typhimurium strains TA97, TA98, TA100 and TA102, Escherichia coli strains PQ65, OG40, OG100, PQ35 and PQ37 and Balb/c 3T3 mouse fibroblasts were used to detect induction of DNA lesions by ALE and AQE. Assays used for these analyses were a bacterial (reverse) mutation assay (Ames test), the SOS-chromotest and the comet assay. Both extracts presented identical infrared 2-oxazolidone spectra. ALE treatment induced a higher frequency of type-4 comets, indicative of increasing DNA migration, in the alkaline comet assay. ALE also induced a weak genotoxic effect, as expressed by the induction factor (IF) values in the test with E. coli strain PQ35 (IF = 1.5) and by cytotoxic effects in strains PQ35, PQ65 and PQ37. Positive SOS induction (IF = 1.7) was detected in strain PQ37 treated with diluted AQE. No genotoxic effects were observed in strains PQ35, PQ65, OG40 and OG 100 after treatment with AQE dilutions. Using the bacterial (reverse) mutation test and survival assays with or without S9 mix, after 60 min of pre-incubation, we observed for strain TA97 treated with ALE a weak mutagenic response (MI = 2.2), while cytotoxic effects were seen for strains TA98, TA100 and TA102. AQE did not show mutagenic activity in any of the strains tested, but a weak cytotoxic effect was noted in strain TA102. Our data suggest that both ALE and AQE from A. fulva induce DNA breaks leading to cytotoxicity and mutagenicity under the conditions used.     

Biogenic amine-forming microbial communities in cheese

The aim of this study was to screen two cheese starter cultures and cheese-borne microbial communities with the potential to produce biogenic amines in cheese during ripening. Bacteria of the genera Enterococcus and Lactobacillus and coliform bacteria were isolated from Dutch-type semi-hard cheese at the beginning of the ripening period. Statistically significant counts of bacterial isolates were screened for the presence of specific DNA sequences coding for tyrosine decarboxylase (tyrDC) and histidine decarboxylase (hDC) enzymes. The PCR analysis of DNA from 14 Enterococcus and 3 Lactobacillus isolates confirmed the presence of the targetted DNA sequences. Simultaneously, 13 tyrDC- and 3 hDC-positive isolates were grown in decarboxylase screening medium and this was followed by HPLC analysis of the produced tyramine and histamine. Conventional and molecular taxonomic analyses of the above-mentioned isolates identified the following species: Enterococcus durans (7 strains), Enterococcus faecalis (3 strains), Enterococcus faecium (1 strain), Enterococcus casseliflavus (3 strains), Lactobacillus curvatus (1 strain), Lactobacillus lactis (1 strain) and Lactobacillus helveticus (1 strain). All of the above Enterococcus and two of the Lactobacillus strains originated from contaminating microbial communities. The L. helveticus strain, which was tyrosine decarboxylase-positive and exhibited tyramine production, originated from starter culture 1 used for cheese production. Comparison of partial tyrDC sequences of positive Enterococcus isolates revealed 89% sequence similarity, and that of hDC-positive Lactobacillus isolates revealed 99% sequence similarity.     

Antigenic analysis by direct immunofluorescence of 114 isolates of Rickettsia tsutsugamushi recovered from febrile patients in rural Malaysia.

One hundred and fourteen Rickettsia tsutsugamushi isolates, recovered from febrile patients in central Peninsular Malaysia, were antigenically analyzed by direct immunofluorescence using eight prototype strains. Twenty-nine antigenic types were detected. The TA763, TA716, Karp and TA686 strains were the most common and occurred singly or in combination with each other or other strains in 86% of the isolates.     

应用RAPD方法对近交系小鼠进行遗传检测的研究

目的　为实验室日常检测近交系小鼠的遗传背景提供一种分子生物学方法。方法　用 6条随机引物对 6个品系近交系小鼠基因组DNA进行PCR扩增。结果　 6条随机引物中p2、p3、p5和p6四引物扩增的条带差异较为明显。结论　RAPD方法是一种有效的近交系小鼠遗传检测手段     

Genotypic and phenotypic polymorphism of environmental strains of the moderately thermophilic bacterium <Emphasis Type="Italic">Sulfobacillus sibiricus</Emphasis>

Five cultures of moderately thermophilic spore-forming acidophilic chemolithotrophic bacteria were isolated from the zones of spontaneous heating of pyrrhotite-containing pyrite-arsenopyrite gold-arsenic sulfide ores in an operating open pit (strains B1, B2, B3, OFO, and SSO). Analysis of the chromosomal DNA structure revealed the differences between these cultures at the strain level (apart from B3 and SSO, which had identical restriction profiles). All the strains had a similar G+C DNA molar content (47.4–48.3%). The level of DNA reassociation was 85 to 95%. The similarity between the DNA of the type strain Sulfobacillus sibiricus N1 isolated from arsenopyrite ore concentrate and that of these strains (83–93%) indicates that they belong to the same species. The strains had similar values of pH and temperature optimal for growth on ferrous iron (1.6–2.0 and 45–55°C, respectively). They were mixotrophs; Fe(II), S^o, and sulfide minerals along with organic compounds were used as energy sources and electron donors. However, the kinetic parameters of growth and substrate oxidation varied from strain to strain. Genetic variety of the strains from diverse ecosystems and environments is possibly the result of the different rates of microevolution processes.     

Evaluation of the mutagenic and cytostatic potential of aristolochic acid (3,4-methylenedioxy-8-methoxy-10-nitrophenanthrene-1-carboxylic acid) and several of its derivatives

Aristolochic acid (1), a constituent of Aristolochia species, has been used for medicinal purposes since the Graeco-Roman period. Following the observation that the compound was mutagenic and carcinogenic, it was removed from pharmaceutical products. Consistent with previous reports, we have found that 1 serves as a direct-acting mutagen in Salmonella typhimurium strains TA100, TA102, TA1537 and TM677, but was not active in the nitroreductase-deficient strains TA98NR and TA100NR. However, aristolic acid (2), a compound that differs in structure only by the absence of the nitro group, was also found to be a direct-acting mutagen in Salmonella strains TA98, TA100, TA102, TA1537, and TM677, as well as strains TA98NR and TA100NR. Both compounds (1 and 2) were active mutagens when evaluated with cultured Chinese hamster ovary cells. Thus, in contrast to previous suggestions, the nitro group at position 10 is not required to induce a mutagenic response. Also, a series of structural relatives (the methyl esters of 1 and 2 (3 and 4, respectively), aristolochic acid-D (5), aristolactam (6), aristolactam A-II (7), and aristolactam-N-beta-D-glucoside (8)) were evaluated for mutagenic potential with Salmonella typhimurium strain TM677 and found to be inactive. Since compounds 3 and 4 were found to be active mutagens with Salmonella typhimurium strains TA98, TA100, TA102 and TA1537 (sufficient quantities of compounds 5-8 were not available for testing), differential sensitivity of the tester strains unrelated to mutagenic potential is suggested. Further, compounds 1, 2, and 6-8 were evaluated for potential to inhibit growth with cultured KB or P388 cells. P388 cells were substantially more sensitive, and compound 1 was the most active of the materials tested (ED5 = 0.58 microM). Compound 6 also demonstrated appreciable activity (ED50 = 4.2 microM), as did compound 8 (ED50 = 6.0 microM). It therefore appears that phenanthrene-ring substituents, in addition to the nitro group at position 10, serve important roles for biological potential. In considering the carcinogenic event induced by aristolochic acid, these functionalities should also be taken into account.     

Optimum associations of tester strains for maximum detection of mutagenic compounds in the Ames test

The Ames test is now widely used as a short-term test for the detection of mutagens. Different strains are available with various genetic characteristics, and in the past decade various authors have recommended different associations of strains to give maximum detection potential. However, few studies have been done to compare the sensitivity of individual strains towards a wide range of compounds in a single study. In order to define the best association of strains for screening or regulatory purpose, we have tested 103 direct mutagens (reference genotoxins or in-house compounds) on 7 strains of Salmonella typhimurium: TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102. 126 different associations of strains have been studied in terms of sensitivity and percentage overlap. Optimum associations of 2, 3, 4 or 5 strains included strains both with and without plasmid pKM101. However, the specificity of detection is greatly diminished by the presence of plasmid pKM101 in the strain, as shown by the high degree of overlap in associations constituted entirely of strains containing the plasmid. The association of strains TA1538 and TA100 detected 86% of the chemicals tested and is therefore recommended for large-scale screening. A rate of detection of 100% was obtained when 6 strains were used. The best associations of 4 and 5 strains, which detected 97 and 99% chemicals respectively, all contained strains TA1537, TA1538 and TA102. Finally, the associations of 4 strains (TA1537, TA1538, TA100, TA102) or 5 strains (TA1535, TA1537, TA1538, TA97, TA102) seemed well adapted to the optimum detection of mutagenic compounds.