Serological Study of Bartonella henselae in Cat Scratch Disease in Japan

It has become clear that Bartonella henselae is a common cause of cat scratch disease (CSD). The indirect fluorescence antibody (IFA) test for detection of IgG and IgM antibodies to B. henselae concerning CSD showed that 5 (50%) of 10 patients with CSD had a serum IgG antibody titer of 1:128 or more and that 2 (20%) patients had a serum IgM antibody titer of 1:20 or more. One of 7 asymptomatic members of patients' families (14%) had IgG antibody to B. henselae at a titer of 1:256. IgM antibody to B. henselae was not detected in sera from the patients' families. Both IgG and IgM antibodies to B. henselae were not detected in sera from the healthy control group. These data suggest that B. henselae may be a cause of CSD in Japan.     

A factor in a wild isolated <Emphasis Type="BoldItalic">Neurospora crassa</Emphasis> strain enables a chromosome segment duplication to suppress repeat-induced point mutation

Repeat-induced point mutation (RIP) is a sexual stage-specific mutational process of Neurospora crassa and other fungi that alters duplicated DNA sequences. Previous studies from our laboratory showed that chromosome segment duplications (Dps) longer than ~300 kbp can dominantly suppress RIP, presumably by titration of the RIP machinery, and that although Dps <200 kbp did not individually suppress RIP, they could do so in homozygous and multiply heterozygous crosses, provided the sum of the duplicated DNA exceeds ~300 kbp. Here we demonstrate suppression of RIP in a subset of progeny carrying the normally sub-threshold 154 kbp Dp(R2394) from a cross of T(R2394) to the wild isolated Carrefour Mme. Gras strain (CMG). Thus, the CMG strain contains a factor that together with Dp(R2394) produces a synthetic RIP suppressor phenotype. It is possible that the factor is a cryptic Dp that together with Dp(R2394) can exceed the size threshold for titration of the RIP machinery and thereby causes RIP suppression.     

Does Coinfection of Bartonella henselae and FIV Induce Clinical Disorders in Cats?

It was found that Bartonella henselae (B. henselae) may induce clinical disorders in cats in natural conditions from a comparison of the serological status for B. henselae with the serostatus for feline immunodeficiency virus (FIV) and several clinical characteristics in 170 domestic cats. Seropositivity for B. henselae was not significantly different between FIV antibody-positive and -negative cats (18.4% vs 16.0%). The incidence of clinical characteristics were compared among four cat groups distinguished by the reactivity of sera against B. henselae and FIV. The incidence of lymph node swelling was lower in only FIV antibody-positive cats (3.0%), but higher in B. henselae antibody-positive cats (13.6%) and significantly higher in both B. henselae and FIV antibody-positive cats (42.9%) compared with the incidence of lymph node swelling in cats which were negative for both antibodies (5.5%). The same relation was also observed for the incidence of gingivitis among the 4 cat groups, suggesting that coinfection of B. henselae and FIV may be associated with gingivitis and lymphadenopathy in cats.     

Real‐time and multiplex real‐time polymerase chain reactions for the detection of Bartonella henselae within cat flea,Ctenocephalides felis,samples

Bartonella henselae (Rhizobiales: Bartonellacae), the agent of cat‐scratch disease, is an emerging bacterial pathogen which can be transmitted via infective faecal material of Ctenocephalides felis Bouché (Siphonaptera: Pulicidae). Worldwide, B. henselae has been identified in 1–53% of felines and 2.9–17.4% of fleas. Although culture is the routine method for detection, the procedure is time‐consuming and is rarely used for isolation directly from flea vectors. The current study reports the development of a quantitative real‐time polymerase chain reaction (qPCR) to detect and quantify B. henselae organisms from vector samples. The qPCR is specific and detects as few as 2.5 genome copies. To enable direct quantification of Bartonella organisms in different vector samples, we developed a qPCR to detect C. felis DNA that also acts as an extraction control. Combining both PCRs into a multiplex format validates B. henselae results when sampling flea populations, although there is a reduction in sensitivity. This reduction might be counteracted by a different combination of probe fluorophores.     

Molecular recognition of oligonucleotides and plasmid DNA by l‐methionine

The present study explores the effect of oligonucleotide composition on the mechanism of retention to l ‐methionine agarose support by chromatography and saturation transfer difference (STD)‐nuclear magnetic resonance (NMR) techniques. All chromatographic experiments were performed using 1.5 M (NH₄)₂SO₄. The binding profiles obtained by chromatography show that oligonucleotides with thymine had the highest retention time. In general, the larger homo‐oligonucleotides are more retained to the l ‐methionine agarose support. Moreover, the study with hetero‐oligonucleotides confirms that the presence of guanine reduces the retention on the l ‐methionine chromatographic support. These results are in accord with STD‐NMR experiments, which show that the strongest signals were observed for the methyl group of thymine, and no STD signals were observed for the guanosine protons. Finally, the retention behaviour of linear plasmid DNA (pDNA) with different sizes and base composition (2.7‐kbp pUC19, 6.05‐kbp pVAX1‐LacZ, 7.4‐kbp pVAX1‐LacZgag and 14‐kbp pcDNA‐based plasmid) was also evaluated by chromatography. The results indicate that the underlying mechanism of retention involves not only hydrophobic interactions but also other elementary interactions responsible for the biorecognition of pDNA molecules by l ‐methionine ligands. Copyright © 2014 John Wiley & Sons, Ltd.     

Molecular Cloning, Detection of Chromosomal DNA of the Mycoplasmalike Organism (MLO) Associated with Faba Bean (Vicia faba L.) Phyllody by Southern Blot Hybridization and the Polymerase Chain Reaction (PCR)

DNA from faba bean (Vicia faba L.) phyllody-diseased periwinle plants was separated from host plant DNA by bisbenzimid-CsCl buoyant-density gradient centrifugation. The mycoplasmalike organism (MLO) DNA was used for the construction of DNA probes. Two probes, 1.45 and 1.35 kbp, were selected and used for the detection of MLO DNA associated with faba pean (FBP) and for assessing the genetic relatedness of FBP-MLO with other mollicutes. The 1.45 kbp DNA probe hybridized with all MLO strains and, with Spiroplasma citri. The 1.35 kbp DNA probe specifically detected the MLO associated with FBP. Moreover, a specific primer pair (E1 and E2) selected from the partially sequenced 1.35 kbp probe allowed amplification of the 1.35 kbp fragment. DNA amplification was obtained also with Crotaltiana saltiana phyllody (Sudan), C. juncea, witches' broom (Thailand), and tomato big-bud (Australia), but no amplification was obtained in the cases of the healthy control, C. roseus phyllody (isolate n⁰) from Sudan, clover phyllody, Gladiolus aster yellow and yellow decline of lavender from France. The very strong signal observed in the case of FBP and C. saltiana phyllody agrees with previous results indicating that FBP and C. saltiana phyllody are caused by an identical MLO, and hence, C. saltiana acts as a reservoir of FBP-MLO in the Sudan. The weak signal obtained in the case of C. juncea witches' broom and tomáto big-bud indicates partial nucleotide homology. The major interest of this primer pair is the low quantity (as little as 100 pg) of the total DNA of diseased plant required for the detection of the FBP disease and the possibility of detecting genetic relatedness with other MLOs.     

Selective Excretion of Alkaline Xylanase by Escherichia coli Carrying pCX311

Plasmid pCX311, which we constructed, has two HindlU DNA fragments (2.6 kbp and 2.0 kbp) of alkalophilic Bacillus sp. strain C-125 in the HindlU site of pBR322.

These two fragments were essential not only for the xylanase production but also for the excretion of periplasmic proteins. The cloned 4.6 kbp fragment encodes some components that made the outer membrane of E. coli permeable. Some proteins such as xylanase and ²-lactamase were excreted, but alkaline phosphatase was not excreted into the culture broth.     

Characterization of a virulent Lactobacillus sake phage PWH2

A virulent bacteriophage PWH2 was isolated from fermented sausage. Out of 14 strains of Lactobacillus sake and 5 strains of L. curvatus tested as potential hosts, only L. sake Ls2 was sensitive. The plaques had a diameter of 0.5-1 mm. One-step growth kinetics of bacteriophage PWH2 revealed the following characteristics: a latent period of 1.5 h, a rise period of 1 h and a burst size of 110 (⁺ _– 10) phages per cell. From electron micrographs it was deduced that bacteriophage PWH2 has an icosahedral head, a long non-contractile tail and a complex base plate. The genome is linear and 35 kbp in length. The structural proteins consist of three major and two minor proteins. After an infection of L. sake Ls2, isolates were obtained that were resistant to PWH2. By treatment with mitomycin C, isolate R4a was found to be lysogenic. DNA/DNA hybridisation proved homology of phage PWH2 with the chromosomal DNA of strain R4a. Correspondence to: W. P. Hammes     

Coexistence of Bartonella henselae and B. clarridgeiae in populations of cats and their fleas in Guatemala

Cats and their fleas collected in Guatemala were investigated for the presence of Bartonella infections. Bartonella bacteria were cultured from 8.2% (13/159) of cats, and all cultures were identified as B. henselae. Molecular analysis allowed detection of Bartonella DNA in 33.8% (48/142) of cats and in 22.4% (34/152) of cat fleas using gltA, nuoG, and 16S–23S internal transcribed spacer targets. Two Bartonella species, B. henselae and B. clarridgeiae, were identified in cats and cat fleas by molecular analysis, with B. henselae being more common than B. clarridgeiae in the cats (68.1%; 32/47 vs 31.9%; 15/47). The nuoG was found to be less sensitive for detecting B. clarridgeiae compared with other molecular targets and could detect only two of the 15 B. clarridgeiae‐infected cats. No significant differences were observed for prevalence between male and female cats and between different age groups. No evident association was observed between the presence of Bartonella species in cats and in their fleas.     

Prevalence of Bartonella henselae and Borrelia burgdorferi Sensu Lato DNA in Ixodes ricinus Ticks in Europe

Bartonella spp. can cause persistent bloodstream infections in humans and animals. To determine whether Bartonella henselae is present in questing Ixodes ricinus ticks, we analyzed the prevalence of B. henselae DNA among tick stages compared to the prevalence of DNA from Borrelia burgdorferi sensu lato, the pathogen most frequently transmitted by ticks. B. henselae DNA was present with a prevalence of up to ∼40% in tick populations sampled in four European sites (Eberdingen, Germany; Klasdorf, Germany; Lembach, France; and Madeira, Portugal). The odds of detecting B. henselae DNA in nymphal ticks was ∼14-fold higher than in adult ticks. No tick was found to be coinfected with B. henselae and B. burgdorferi sensu lato. Taken together, our data indicate that ticks might serve as a vector for the transmission of B. henselae to humans.In immunocompetent patients, Bartonella henselae infections often result in cat scratch disease (CSD), a self-limiting but often prolonged lymphadenitis; immunocompromised patients (e.g., AIDS patients) can suffer from vasculoproliferative disorders (bacillary angiomatosis, peliosis hepatis [1]). Cats are a confirmed reservoir host of B. henselae transmitting the pathogen by cat scratches or bites.Several Bartonella species (e.g., B. henselae, B. quintana, and B. vinsonii) cause a persistent intraerythrocytic bacteremia in their respective mammalian reservoir hosts (7). B. henselae was detected in the peripheral blood of a wide range of mammals including domestic (e.g., cats, dogs, and horses) and wild animals (e.g., porpoise, lions, cheetahs, and wild felids). Obviously, such an asymptomatic, persistent bacteremia with B. henselae represents an important factor for the spread of the pathogens via blood-sucking arthropods. Mechanistic details determining the intraerythrocytic presence of Bartonella spp. have been investigated in detail in a B. tribocorum rat infection model mimicking Trench fever (a human disease caused by B. quintana); here, the pathogen persists several weeks in the circulating blood in an immunoprivileged intraerythrocytic niche (28).Cat fleas are well established vectors for B. henselae (1). However, transmission by other arthropods, in particular ticks, has been suggested: B. henselae DNA was detected in questing Ixodes pacificus and I. persulcatus ticks in North America, Eastern Europe, and Russia, respectively (4, 13, 14, 22, 25) and in I. ricinus ticks feeding on people or domestic animals in Central Europe (24, 26). DNA of various Bartonella spp. has also been detected in keds, biting flies, and mites (reviewed in reference 2). Recently, ticks (I. ricinus) were experimentally infected with B. henselae. Inoculation of cats with salivary glands of infected ticks resulted in a B. henselae bacteremia (5). Nevertheless, controversial data about the prevalence of Bartonella spp. in ticks and their role as vectors for B. henselae exist (29).Here, we present data on the prevalence of B. henselae and Lyme disease spirochetes in 654 questing ticks (I. ricinus) collected at four locations in Europe, suggesting that ticks might serve as potential vectors for the transmission of B. henselae to humans.     

Bartonella henselae in eastern Poland: the relationship between tick infection rates and the serological response of individuals occupationally exposed to tick bites

To explore the potential role of Ixodes ricinus as the presumed vector of Bartonella henselae in eastern Poland, ticks collected in various geographic locations were examined for the presence of B. henselae, and the results were matched against the prevalence of anti‐B. henselae antibodies in individuals occupationally exposed to tick bites. The presence of Bartonella DNA was investigated by PCR in a total of 1,603 unfed Ixodes ricinus ticks. The presence of IgG antibodies against B. henselae was investigated in serum samples from 332 people occupationally exposed to tick bites (94 farmers and 238 forestry workers). The total prevalence of B. henselae in ticks was 1.7%; the infection rates in males (3.1%) and females (2.7%) were nearly ten times greater than in nymphs (0.3%). The prevalence of seropositive results in the risk group (30.4%), farmers (27.7%) and forestry workers (31.5%), was significantly greater compared to the control group (8.9%). The results showed a weak positive correlation between the degree of infection of ticks and humans living in the same geographic region. The lack of a direct relationship indicates that exposure to tick bites is only one of the factors contributing to the significant preponderance of a seropositive response to B. henselae in the forestry workers and farmers over the control group. Other factors must be considered, such as contact with cats, which are popular domestic animals in Polish villages, and exposure to cat fleas.     

Prevalence of Rickettsia and Bartonella species in Spanish cats and their fleas

The aim of this study was to determine the prevalence of Bartonella henselae, Rickettsia felis, and Rickettsia typhi in fleas and companion cats (serum and claws) and to assess their presence as a function of host, host habitat, and level of parasitism. Eighty‐nine serum and claw samples and 90 flea pools were collected. Cat sera were assayed by IFA for Bartonella henselae and Rickettssia species IgG antibodies. Conventional PCRs were performed on DNA extracted from nails and fleas collected from cats. A large portion (55.8%) of the feline population sampled was exposed to at least one of the three tested vector‐borne pathogens. Seroreactivity to B. henselae was found in 50% of the feline studied population, and to R. felis in 16.3%. R. typhi antibodies were not found in any cat. No Bartonella sp. DNA was amplified from the claws. Flea samples from 41 cats (46%) showed molecular evidence for at least one pathogen; our study demonstrated a prevalence rate of 43.3 % of Rickettsia sp and 4.4% of Bartonella sp. in the studied flea population. None of the risk factors studied (cat's features, host habitat, and level of parasitation) was associated with either the serology or the PCR results for Bartonella sp. and Rickettsia sp.. Flea‐associated infectious agents are common in cats and fleas and support the recommendation that stringent flea control should be maintained on cats.     

Transfer RNA gene mapping studies on cyanelle DNA from Cyanophora paradoxa

Summary The 4S RNA of cyanelles from Cyanophora paradoxa strain LB 555 UTEX was fractionated by two-dimensional gel electrophoresis. Individual tRNA species were identified by aminoacylation, labeled in vitro and hybridized to restriction endonuclease fragments of cyanelle DNA. Hybridization experiments, using individual tRNA species, have revealed the location of two tRNA genes, coding for tRNA^Ala and tRNA^Ile, in each of the two spacer segments separating the 16S and 23S rRNA genes on the two inverted repeats (10 kbp each) and three tRNA genes in the small single-copy region (17 kbp) separating the two inverted repeats. A minimum of 14 tRNA genes in the large single-copy region (88.5 kbp) has also been found.Heterologous hybridization studies, using cyanelle tRNAs and chloroplast DNA from spinach, broad bean, or maize, indicate a high degree of homology between some tRNAs from cyanelles and chloroplasts.Although cyanelles are often condisered as having evolved from endosymbiotic cyanobacteria, the organization of tRNA genes on cyanelle DNA and the results of heterologous hybridization studies show that cyanelles are related to higher plant chloroplasts.     

Seroepidemiological Survey of Bartonella (Rochalimaea) henselae in Domestic Cats in Japan

A total of 199 domestic cat serum samples from 3 geographical areas (northeastern, central and southwestern) of Japan collected between 1992 to 1994 were examined for serum antibody against Bartonella henselae using an immunofluorescent assay. The antibody prevalence was 15.1% (30/199). A significant difference in the prevalence of B. henselae antibody was observed between the northeastern area (6.3%:3/48) and the central area (22.0%: 13/59) in Japan. There was no significant difference between the average age of seropositive cats (4.39 ±3.26 years) and that of seronegative cats (4.03 ±3.84 years), and also between the frequency of seropositive male cats (16.5%: 15/91) and that of seropositive female cats (11.8%:9/76). This is the first report of B. henselae antibodies in cats in Japan.     

Chloroplast genome organization of bromegrass,Bromus inermis Leyss

Summary A physical map of the Bromus inermis chloroplast genome was constructed using heterologous probes of barley and wheat chloroplast DNA (cpDNA) to locate restriction sites. The map was aligned from data obtained from filter hybridization experiments on single and double enzyme digests. Cleavage sites for the enzymes PstI, SalI, KpnI, XhoI and PvuII were mapped. The chloroplast genome of B. inermis is similar in physical organization to that of other grasses. The circular cpDNA molecule of B. inermis has the typical small (12.8 kbp) and large (81.3 kbp) single-copy regions separated by a pair of inverted repeat (21 kbp) regions. The cpDNA molecule of B. inermis is collinear in sequence to that of wheat, rye, barley and oats. No structural rearrangements or major deletions were observed, indicating that the cpDNA of Bromus is a useful tool in phylogenetic studies.     

Restriction site map of the Chlorella virus PBCV-1 genome

The virus PBCV-1, which replicates in a Chlorella-like green alga, has a dsDNA genome. The DNA was mapped for BamHI, HindIII, and PstI restriction sites. The resulting map has a size of 333 kbp and is circular—indicating either covalently closed circular DNA or circularly permuted linear DNA. Several regions of repetitive DNA were also identified and located on the restriction map.     

Cryptogenic hepatitis patients have a higher Bartonella sp.-DNA detection in blood and skin samples than patients with non-viral hepatitis of known cause

BackgroundThis study aimed to assess the prevalence of Bartonella sp.-DNA detection in blood and skin samples from patients with non-viral end-stage liver disease awaiting liver transplantation.Methodology/Principal findingsBlood samples and healthy skin fragments from 50 patients were tested using microbiological and molecular methods. Fifteen patients had cryptogenic hepatitis (CH) and 35 had alcoholic, drug-induced or autoimmune liver disease. DNA was extracted from whole blood and liquid culture samples, isolates, and skin fragments. Thirteen of the 50 patients (26%) had Bartonella henselae DNA detection in their blood (9/50) and/or skin (5/50) samples. Colonies were isolated in 3/50 (6%) and infection was detected in 7/50 (14%) of the 50 patients. B. henselae-DNA detection was more prevalent in patients with CH than in other patients (p = 0.040). Of 39 patients followed-up for at least two years, a higher mortality rate was observed among patients with CH infected with B. henselae (p = 0.039).Conclusions/SignificanceFurther studies assessing the role of B. henselae infection in the pathogenesis of hepatitis patients must be urgently conducted.     

A Translocation Motif in Relaxase TrwC Specifically Affects Recruitment by Its Conjugative Type IV Secretion System

Type IV secretion system (T4SS) substrates are recruited through a translocation signal that is poorly defined for conjugative relaxases. The relaxase TrwC of plasmid R388 is translocated by its cognate conjugative T4SS, and it can also be translocated by the VirB/D4 T4SS of Bartonella henselae, causing DNA transfer to human cells. In this work, we constructed a series of TrwC variants and assayed them for DNA transfer to bacteria and human cells to compare recruitment requirements by both T4SSs. Comparison with other reported relaxase translocation signals allowed us to determine two putative translocation sequence (TS) motifs, TS1 and TS2. Mutations affecting TS1 drastically affected conjugation frequencies, while mutations affecting either motif had only a mild effect on DNA transfer rates through the VirB/D4 T4SS of B. henselae. These results indicate that a single substrate can be recruited by two different T4SSs through different signals. The C terminus affected DNA transfer rates through both T4SSs tested, but no specific sequence requirement was detected. The addition of a Bartonella intracellular delivery (BID) domain, the translocation signal for the Bartonella VirB/D4 T4SS, increased DNA transfer up to 4% of infected human cells, providing an excellent tool for DNA delivery to specific cell types. We show that the R388 coupling protein TrwB is also required for this high-efficiency TrwC-BID translocation. Other elements apart from the coupling protein may also be involved in substrate recognition by T4SSs.     

Comparison between the organization of nuclear ribosomal DNA unit of Euglena gracilis Z and var. Bacillaris

Summary We have characterized the nuclear rDNA unit of Euglena gracilis var. bacillaris and compared it to that of the Z strain. We have localized restriction sites for Eco R1, Sal 1, Sma 1, Hind III, Bam H1 and Bgl II on this unit as well as the coding region for 20 S and 25 S rRNAs. For both strains, results suggest an homogeneity of the 11.6 kbp rDNA units. Comparison between strains shows differences characterized by two additional Sal 1 sites in bacillaris and the likely methylation of one Sma 1 site in Z. Both differences are localized in a non-coding region of the rDNA unit. Analyses of 18 Euglena strains from various origins confirm these differences and allow easy recognition of bacillaris and Z type strains.Abbreviations kb kilo base - kpb kilo base pair - plasmids pRH 59 and pRH 57 contain a Hind III-HInd III nuclear DNA fragment from W₃BUL of 5.9 and 5.7 kbp respectively, pRB 48 and pRB 35 contain a Bam H1-Bam H1 nuclear DNA fragment from wild-type Z of 4.8 and 3.5 kbp respectively - SDS sodium dodecyl sulfate - UV ultra-violet     

Identification of the Mitochondrial Genome in the Chrysophyte Alga Ochromonas danica

ABSTRACT. Analysis of total DNA isolated from the Chrysophyte alga Ochromonas danica revealed, in addition to nuclear DNA, two genomes present as numerous copies per cell. The larger genome (?120 kilobase pairs or kbp) is the plastid DNA, which is identified by its hybridization to plasmids containing sequences for the photosynthesis genes rbcL, psbA, and psbC. The smaller genome (40 kbp) is the mitochondrial genome as identified by its hybridization with plasmids containing gene sequences of plant cytochrome oxidase subunits I and II. Both the 120- and 40-kbp genomes contain genes for the small and large subunits of rDNA. The mitochondrial genome is linear with terminal inverted repeats of about 1.6 kbp. Two other morphologically similar species were examined, Ochromonas minuta and Poteriochromonas malhamensis. All three species have linear mitochondrial DNA of 40 kbp. Comparisons of endonuclease restriction-fragment patterns of the mitochondrial and chloroplast DNAs as well as those of their nuclear rDNA repeats failed to reveal any fragment shared by any two of the species. Likewise, no common fragment size was detected by hybridization with plasmids containing heterologous DNA or with total mitochondrial DNA of O. danica; these observations support the taxonomic assignment of these three organisms to different species. The Ochromonas mitochondrial genomes are the first identified in the chlorophyll a/c group of algae. Combining these results with electron microscopic observations of putative mitochondrial genomes reported for other chromophytes and published molecular studies of other algal groups suggests that all classes of eukaryote algae may have mitochondrial genomes < 100 kbp in size, more like other protistans than land plants.