Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis, we detected 2 strains of T. vaginalis; the virus-infected ($V^+$) and uninfected ($V^-$) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in $V^+$ compared with $V^-$ isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in $V^+$ isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in $V^+$ and $V^-$ isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.
Klebsiella pneumoniae is the leading cause of nasocomial infection and the most commonly isolated from clinical specimens. $Extended-spectrum-{\beta}-lactamase$ (ESBL) producing K. pneumoniae infection was associated with a significantly longer duration of hospital stay and greater hospital charges. The purpose of this study is to investigate the antibiotic resistant patterns and the DNA fingerprint types of extended-spectrum ${\beta}-lactamase$ (ESBL) producing K. pneumoniae. 223K. pneumoniae strains were collected from three general hospitals with more than 500 beds in Busan, Korea from September 2004 to October 2005. The minimum inhibitory concentration (MIC) of antibiotics was measured using the Gram negative susceptibility (GNS) cards of VITEK (Vitek system, Hazelwood Inc., MO). Random amplified polymorphic DNA method was used to detect DNA fingerprint of the organisms. Of the 226 K. pneumoniae isolates 65 ESBL-producing K. pneumoniae strains were detected by the Vitek system and confirmed by the double-disk synergy test. All the 65K. pneumoniae strains were resistant cefazolin, cefepime, ceftriaxone and aztreonam, and 83.0% of the organisms were resistant to ampicillin/sulbactam, 66.1% to tobramycin, 67.6% to piperacillin/tazobactam, 61.5% to ciprofloxacin, and 47.6% to trimethoprim/sulfamethoxazole and 43.0% to gentamicin. The RAPD patterns were distincted as 10 types by three random 10-mer primers (208, 272, 277). Among ten type patterns, three types (Ic, IIb, IIIe) were remarkably represented at patient of internal department, nerve surgery department, general surgery department, and neonatal room. These results indicate that RAPD can be useful for DNA of strains typing in the epidemiological investigations. Therefore more investigation are needed in order to prevent the ESBL type-producing K. pneumoniae from spreading resistance to oxyimino cephalosphorin antibiotics.
The endemic status of clonorchiasis and metagonimiasis along the Geum gang (River) in Okcheon-gun (County) in Korea was examined. From February to December 7000, stools of total 1,081 inhabitants living in 5 villages were examined. Each stool specimen was examined by both the cellophane thick smear method and the formalin-ether sedimentation technique. Egg-positive cases were further analyzed by Stoll's egg-counting technique. and praziquantel was administered to positive cases. The egg-positive rates for Clonorchis sinensis and Metafonimus species were 9.3% and 5.5%, respectively, and the double infection rate was 3.5%. The numbers of eggs per gram (EPG) of feces of C. sinensis and Metafoninus sp. were $918{\pm}1,463$ and $711{\pm}947$, respectively. The egg-positive rates for C. sinensis and Metagoninus sp. in the riverside area were 14.2% and 8.4%, respectively, which were significantly higher than those of the inland area (3.2% and 1.7%. respectively). The egg-positive rates of C. sinensis and Metagoninus sp. in males (16.7% and 10.0%) were significantly higher than those of females (3.5% and 1.8%) However, there were no significant differences of EPG values between localities and sexes. The prevalence of clonorchiasis and metagonimiasis in this survey was significantly lower than that in the previous reports. However. there is still a high prevalence of infection with C. sinensis and Metagoninus sp. in this region. especially in the riverside area.
Infection with high-risk human papillomavirus (HR-HPV) is an essential cause of cervical cancer. Because of substantial geographical variation in the HPV genotype distribution, data regarding HPV type-specific prevalence for a particular country are mandatory for providing baseline information to estimate effectiveness of currently implemented HPV-based cervical cancer prevention. Accordingly, this review was conducted to evaluate the HR-HPV genotype distribution among Thai women with precancerous cervical lesions i.e. cervical intraepithelial neoplasia grade 2-3 (CIN 2-3), adenocarcinoma in situ (AIS), and invasive cervical cancer by reviewing the available literature. The prevalence of HR-HPV infection among Thai women with CIN 2-3 ranged from 64.8% to 90.1% and the three most common genotypes were HPV 16 (38.5%), HPV 58 (20.0%), and HPV 18 (5.5%). There were high squamous cell carcinoma/CIN 2-3 prevalence ratios in women with CIN 2-3 infected with HPV 33 and HPV 58 (1.40 and 1.38, respectively), emphasizing the importance of these subtypes in the risk of progression to invasive cancer among Thai women. Data regarding the prevalence and genotype distribution of HR-HPV in Thai women with AIS remain unavailable. Interesting findings about the distribution of HPV genotype in cervical cancer among Thai women include: (1) a relatively high prevalence of HPV 52 and HPV 58 in invasive squamous cell carcinoma; (2) the prevalence of HPV 18-related adenocarcinoma is almost double thepreviously reported prevalence, and (3) 75% of neuroendocrine carcinomas are HPV18-positive when taking into account both single and multiple infections.
Many antibiotic monographs cite the induction of vaginal infections as a possible side effect. Invariably, this is believed to be due to Candide albicans, and empirical therapy is given. However, recent studies raise the question of the extent to which yeast do infect the host after antibiotic use. A double-blind, randomized, placebo-controlled study was undertaken on female patients to determine how many yeast infections occurred following 10 days antibiotic use. In addition, the study was designed to examine whether oval use of probiotic lactobacilli can reduce the risk of vaginal infection. Twenty four patients diagnosed with respiratory, oval or throat infections received one of several types of antibiotic for 10 days, and two capsules containing 10$^{9}$ dried Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 from the day of commencement of antibiotic therapy for 21 days. The most commonly prescribed antibiotic was biaxin (clarithromycin). All but one patient had lactobacilli in the vagina upon entry to the study, and none developed yeast vaginitis or diarrhea during treatment or 20 days after completion of antibiotics. The mean Nugent score was higher in the placebo than the lactobacilli group (4.1 versus 2.4), and three cases of bacterial vaginosis arose (25 % incidence compared to 0% in the lactobacilli group) in the placebo group (2 receiving cefuroxime, 1 on biaxin). The study suggested that current antibiotic use is not necessarily associated with either diarrhea or yeast infection, as is often surmised. Nevertheless, daily use of probiotics was safe and could potentially reduce the risk of patients developing bacterial vaginosis after antibiotic use.
From the view point of phytopathological anatomy, the author has tried to study the effect of the shoot cluster disease virus on the internal structure of vascular tissues of chinese date tree (Ziziphus jujuba var. inermis Rehd.) comparing healthy checks and diseased plants. The materials were collected at the several sites, Kumgock-Ri, Masuc-Ri, Kyungi-Do, and near the campus of Korea University and around the area of Chongam-Dong, Seoul City, from August 15th to September 5th 1959. The leaf materials of healthy and diseased plants are fixed and aspirated in two kinds of killing solutions, formalin-acetic acid alcohol solution and Craf III solution. Sections were cut at 5-10$\mu$ thickness and stained with the double staining reagents of safranin and fast green. In this experiment the author has observed that there are marked structural changes in the infected plants in contrast of healthy checks. As figures 3-7 show that the following characteric changes have taken place on infected plants: 1) the arrangement of irregularly developed sieve elements in phloem, 2) the degeneration of phloem elements, 3) the irregular arrangement of epidermis in mid-vein, 4) more necrosis is observed among the parenchymatous cells, 5) abundant accumulatin of starch grains in parenchymatous cells, . In contrast to the above irregularities caused by the virus disease, the healthy checks appear normal structures as shown in figures 1 and 2. In adding to the all features noted above, the author could also observe an interesting feature that the xylem elements in mid-vein vascular bundle tissues are considerably disorganized to show the unspecialized vessel elements, the irregularly arranged xylem elements. However, this kind of irregularities which occur in xylem under the virus infection has not been reported previously. The features noted on the internal structure of vascular bundle under the condition of infection by the shoot cluster disease on chinese date trees appear to be more or less closely similar to the symptoms of the bunchy-top of banana and the yellow dwarf disease of barley in respect to the fact that whether phloem necrosis takes place as a primary symptom or a secondary symptom. In all these disease, primary histological changes of hypoplasia and hypertrophy are preceeded by the necrosis of phloem.
A double-stranded RNA (dsRNA) mycovirus was detected in malformed fruiting bodies of Pleurotus ostreatus strain ASI2792, one of bottle cultivated commercial strains of the edible oyster mushroom. The partial RNA-dependent RNA polymerase (RdRp) gene of the P. ostreatus ASI2792 mycovirus (PoV-ASI2792) was cloned, and a cDNA sequences alignment revealed that the sequence was identical to the RdRp gene of a known PoSV found in the P. ostreatus strain. To investigate the symptoms of PoV-ASI2792 infection by comparing the isogenic virus-free P. ostreatus strains with a virus-infected strain, isogenic virus-cured P. ostreatus strains were obtained by the mycelial fragmentation method for virus curing. The absence of virus was verified with gel electrophoresis after dsRNA-specific virus purification and Northern blot analysis using a partial RdRp cDNA of PoV-ASI2792. The growth rate and mycelial dry weight of virus-infected P. ostreatus strain with PoV-ASI2792 mycovirus were compared to those of three virus-free isogenic strains on 10 different media. The virus-cured strains showed distinctly higher mycelial growth rates and dry weights on all kinds of experimental culture media, with at least a 2.2-fold higher mycelial growth rate on mushroom complete media (MCM) and Hamada media, and a 2.7-fold higher mycelial dry weight on MCM and yeast-malt-glucose agar media than those of the virus-infected strain. These results suggest that the infection of PoV mycovirus has a deleterious effect on the vegetative growth of P. ostreatus.
Soybean mosaic virus (SMV) is the predominant viral pathogen that affects the yield and quality of soybean. The natural host range for SMV is very narrow, and generally limited to Leguminosae. However, we found that SMV can naturally infect Pinellia ternata and Atractylodes macrocephala. In order to clarify the molecular mechanisms underlying the cross-family infection of SMV, we used double-stranded RNA extraction, rapid amplification of cDNA ends polymerase chain reaction and Gibson assembly techniques to carry out SMV full-length genome amplification from susceptible soybeans and constructed an infectious cDNA clone for SMV. The genome of the SMV Shanxi isolate (SMV-SX) consists of 9,587 nt and encodes a polyprotein consisting of 3,067 aa. SMV-SX and SMV-XFQ008 had the highest nucleotide and amino acid sequence identities of 97.03% and 98.50%, respectively. A phylogenetic tree indicated that SMV-SX and SMV-XFQ018 were clustered together, sharing the closest relationship. We then constructed a pSMV-SX infectious cDNA clone by Gibson assembly technology and used this clone to inoculate soybean and Ailanthus altissima; the symptoms of these hosts were similar to those caused by the virus isolated from natural infected plant tissue. This method of construction not only makes up for the time-consuming and laborious defect of traditional methods used to construct infectious cDNA clones, but also avoids the toxicity of the Potyvirus special sequence to Escherichia coli, thus providing a useful cloning strategy for the construction of infectious cDNA clones for other viruses and laying down a foundation for the further investigation of SMV cross-family infection mechanisms.
Pepper mild mottle virus (PMMoV), one of the most prevalent viruses in chili pepper (Capsicum annuum L.) is a non-enveloped, rod-shaped, single-stranded positive-sense RNA virus classified in the genus Tobamovirus. The supernatants of five bacterial cultures (Pseudomonas putida [PP], Bacillus licheniformis [BLI], P. fluorescens [PF], Serratia marcescens [SER], and B. amyloliquifaciens [BA]) were analyzed to find novel antiviral agents to PMMoV in chili pepper. Foliar spraying with supernatants (1:1, v/v) obtained from Luria-Bertani broth cultures of PP, BLI, PF, SER, and BA inhibited PMMoV infection of chili pepper if applied before the PMMoV inoculation. Double-antibody sandwich enzyme-linked immunosorbent assay showed that treatments of five supernatants resulted in 51-66% reductions in PMMoV accumulation in the treated chili pepper. To identify key compounds in supernatants of PP, BLI, PF, SER, and BA, the supernatants were subjected to gas chromatography-mass spectrometry. The 24 different types of compounds were identified from the supernatants of PP, BLI, PF, SER, and BA. The compounds vary from supernatants of one bacterial culture to another which includes simple compounds-alkanes, ketones, alcohols, and an aromatic ring containing compounds. The compounds triggered the inhibitory effect on PMMoV propagation in chili pepper plants. In conclusion, the cultures could be used to further conduct tissue culture and field trial experiments as potential bio-control agents.
This study describes the isolation of a Toxocara canis species-specific excretory-secretory(ES) antigen and the development of an enzyme-linked immunosorbent assay(ELISA) based on this antigen. Analysis of the ES antigens of T. canis, Toxocara vitulorum, Ascaris lumbricoides and Necator americanus larval antigen was performed by SDS-PAGE followed by western blotting. A 57 kDa T. canis-specific antibody fraction(TcES-57) was identified by western blotting and labelling with anti-Toxocara antibodies(from experimental rabbits and human patients) and tracing with anti-human or anti-rabbit peroxidase conjugate. No protein fraction of 57 kDa was detected in ES or larval antigens collected from T. canis, T. vitulorum, A. lumbricoides and N. americanus. Using TcES-57, a specific anti-serum was produced in rabbits and a double sandwich ELISA was developed. This test was validated using known seropositive sera from toxocariasis patients, sera from A. lumbricoides or N. americanus patients, and 50 serum samples from cats. These tests revealed that TcES-57 antigen is specific to T. canis infection and does not cross react with sera of other related infections. Thus, ELISA based on TcES-57 antigen was proven to be an effective tool in the diagnosis of toxocariasis and studies on the role of T. canis in the epidemiology of human toxocariasis.
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