Two polyurethanases PueA and PueB are major extracellular lipases partly secreted by the mediation of their cognate ABC exporter AprDEF in Pseudomonas protegens Pf-5.

Two polyurethanases PueA and PueB from Pseudomonas protegens Pf-5 have been reported to have hydrolytic activity against synthetic p-nitrophenyl palmitate of lipase substrate, and PueA may play a more effective role in this activity. However, it is still unknown whether PueA and PueB play similar parts in the lipase activity against natural acylglycerols and achieve the extracellular secretion via their cognate ABC exporter AprDEF. In this study, we investigated these questions through the construction of four markerless deletion mutants in Pf5139 (Δupp derivative of Pf-5), two heterologous co-expression strains and their three control strains in lipase-free Escherichia coli BL21(DE3), and detected their lipase activities by the tributyrin plate assay and the liquid culture assay. The results showed that PueA and PueB, classified as subfamily I.3 lipases, are major extracellular lipases involved in the uptake of oil in Pf-5, and PueA plays a leading role in extracellular lipase activity. In addition, the extracellular secretion of PueA and PueB can be partly mediated via AprDEF in Pf-5 and BL21(DE3). Finally, PueA and PueB are also able to achieve the extracellular secretion without the assistance of AprDEF in Pf-5 and BL21(DE3).

Gut microbiota and allergy/asthma: From pathogenesis to new therapeutic strategies.

Asthma and atopy, classically associated with hyper-activation of the T helper 2 (Th2) arm of adaptive immunity, are among the most common chronic illnesses worldwide. Emerging evidence relates atopy and asthma to the composition and function of gut microbiota composition. Moreover, certain gut microbial strains have been shown to inhibit or attenuate immune responses associated with chronic inflammation in experimental models. Although still a relatively nascent field of research, evidence to date suggests that the gut microbiome may represent fertile targets for prevention or management of allergic asthma and other diseases in which adaptive immune dysfunction is a prominent feature. The oral probiotics/prebiotic represents a possible therapeutic for improving asthma and allergic disease. Especially, recent technological developments that permit identification of microbes and their products using culture-independent molecular detection techniques. In this review, we literaturely summarise the aggravation or improvement of metabolic diseases by role of gut microbiota, probiotics/prebiotic treatment.

First Report of Branch Blight of Tree Peony Caused by Phoma glomerata in China.

Tree peony (Paeonia suffruticosa Andrews) is a perennial woody deciduous shrub native to China and famous for its beautiful flowers. Starting in early autumn 2010, blighted branches of tree peony were detected in the International Peony Garden in Luoyang. The disease incidence was greater than 10% and disease symptoms included bulb atrophy and twig and branch dieback. Pycnidia were embedded within the bark of diseased branches. They were small, black, ostiolate, and measured 145 to 275 × 140 to 251 µm. Pycnoconidia were single-celled, hyaline or sandy beige, rounded to ellipsoidal, and 3.9 to 10.3 × 2.3 to 7.0 µm. Pure cultures were obtained by plating the pycnoconidia on potato dextrose agar (PDA). In culture, the fungus produced a circular, white to pink colony with pyknotic and linter shaped aerial mycelium. Numerous pycnidia, initially brown and dark at maturity, were embedded in the mycelium, especially in the center of the colony, with a few of them scattered in the edge. The morphological characteristics were consistent with Phoma (2). The ITS1-5.8S-ITS2 region of three isolates were PCR amplified and sequenced with primers ITS1 and ITS4. Sequences (GenBank Accession No. JX885584) showed 99% identity with reference isolates of Peyronellaea glomerata (Corda) Goid (AB470906.1 and HQ380779.1) and Phoma glomerata (Corda) Wollenw. & Hochapfel (EU098115.1). These two species are synonyms (1). To test pathogenicity, nine healthy branches of 3-year-old potted tree peony plants were wound-inoculated with a PDA disk containing pycnidia from an actively growing colony of P. glomerata. Three control branches were inoculated with sterile PDA disks. Each inoculated branch was wrapped in a plastic bag and maintained in a greenhouse at 25 to 28°C. After 3 days, brown patches appeared on inoculated branches and extended by up to 1 cm. Pycnidia identical to those observed in the field and in storage appeared on all inoculated branches 7 days after inoculation. Control branches did not show symptoms. The pathogen was reisolated from inoculated branches, fulfilling Koch's postulates. P. glomerata was reported as the causal agent of withering of flowers and young shoots of grapevines in Yugoslavia (3). To our knowledge, P. glomerata and Botryosphaeria dothidea have always been reported together, causing branch wilting or dieback. To our knowledge, this is the first report of branch blight of tree peony caused by P. glomerata in China. References: (1) M. M. Aveskamp et al. Mycol. Soc. Am. 101:363, 2009. (2) G. H. Boerema et al. Studies in Mycology, 3, 1973. (3) A. Saric-Sabados et al. Atti Ist. bot. Univ. Pavia 18:101, 1960.

First Report of Leaf Spot Disease of Peony Caused by Seimatosporium botan in China.

Tree peony (Paeonia suffruticosa Andrews), a perennial ligneous deciduous shrub in the Paeoniaceae family, is known for its beautiful and charming flowers. It is regarded as the flower symbol of China and is cultivated throughout the country. In August 2008, a previously unknown leaf spot was observed on peony cultivated in the Mountain Peony Garden located in the Luoyang area of Henan Province, China. In 2009, the leaf spot disease was observed in some gardens in the city of Luoyang, China. Initial symptoms appeared as small, round or irregular, brown, necrotic lesions in the middle of leaves. These lesions gradually enlarged up to 1 cm in diameter and were circular or irregular, brown to dark brown, and brown on the margins. In a humid atmosphere, black, sessile, discoid acervuli developed on the lesions, and the lesions sometimes became waxy-like, eventually coalesced, and nearly covered the entire leaf. Conidia produced in acervuli had two morphologically different types. One type had a single basal appendage, ellipsoid to fusiform, transversely three septate, 16 to 20 × 5 to 7 µm, smooth, basal cell obconic with a truncate base, subhyaline, 3 to 5 µm long; two central cells subcylindrical to dolioform, brown to dark brown, 8 to 10 µm long, apical cell conical with rounded apex, concolorous with the central cells, 4 to 5 µm long, basal appendage filiform, unbranched, excentric, 4 to 8 µm long. The other type had a single appendage at both ends, fusiform to subcylindrical, transversely three septate, 16 to 20 × 4 to 5 µm, smooth; basal cell obconic with a truncate base, subhyaline, 4 to 5 µm long; two central cells subcylindrical to dolioform, pale brown, 8 to 11 µm long; apical cell conical with an acute apex, hyaline to subhyaline, 4 to 5 µm long; basal appendage filiform, unbranched, excentric, 4 to 8 µm long; apical appendage filiform, unbranched, 4 to 8 µm long. Single conidial isolates of both types of conidia yielded identical colonies, which produced both types of conidia on potato dextrose agar (PDA), thus showing that both types of conidia belonged to the same fungus. Colonies on PDA were slimy in appearance, yellow to villous with an irregular taupe margin; reverse brown to grayish brown. Cultural and conidial characteristics of the isolates were similar to those of Seimatosporium botan (1). The DNA sequence for the fungus showed internal transcribed spacer region (ITS1-5.8S-ITS2) sequences (GenBank Accession No. HM067840) with 93% sequence identity to S. discosioides (Accession Nos. EF600970.1 and EF600969.1). This is the first submission of a S. botan sequence to GenBank. To determine pathogenicity, 20 healthy leaves of P. suffruticosa were inoculated by spraying a conidial suspension of S. botan onto the foliage. Ten leaves were sprayed with sterile water and served as controls. Plants were covered with plastic for 24 h to maintain high relative humidity. After 15 days, the symptoms described above were observed on leaves in all inoculated plants, whereas symptoms did not develop on the control plants. The pathogen was reisolated from inoculated leaves, fulfilling Koch's postulates. On the basis of morphology and ITS region sequences, we conclude that S. botan is the causal agent of leaf spots of P. suffruticosa. There is a report of S. botan on P. suffruticosa stems in Japan (1), but to our knowledge, this is the first report of leaf spot disease of peony caused by S. botan in China. References: (1) S. Hatakeyama et al. Mycoscience 45:106, 2004.

[Research advances on skin sweat gland regeneration induced by stem cells and tissue engineering].

As the largest organ in mammals, skin is the first protective barrier against external stimuli. Sweat glands are one of the important cutaneous appendages and play an important role in maintaining electrolyte balance and regulating body temperature. Patients with extensive deep burns usually suffer from damage to deep dermis or the entire skin layer. The damaged sweat glands are difficult to repair and even unable to regenerate, which seriously affects patients' sweating and thermo-regulation function, reduces patients' quality of lives. How to achieve the functionalization of sweat glands has become one of the important researches in regenerative medicine of damaged skin. This review summarizes the translational and application technology of sweat gland regeneration and repair using stem cells and tissue engineering, in order to provide a theoretical basis for the research of sweat gland regeneration.

First Report of Pilidium concavum on Paeonia suffruticosa in China.

Paeonia suffruticosa Andrews, a deciduous perennial shrub, is known for its beautiful and charming flowers. It is regarded as the flower symbol of China and cultivated throughout the country. Since 2006, large, brown necrotic spots have been observed on numerous P. suffruticosa plants in gardens in Luoyang, China. Spots appeared each year and were observed on more than 50% of the plants, sometimes affecting more than half of the leaf. Initial symptoms appeared as small, round, water-soaked lesions in the middle or on the margin of leaves. These areas enlarged up to 1 to 3 cm in diameter and were circular or irregular, brown to dark brown, and pale brown on the margins. In a humid atmosphere, black, sessile, discoid conidiomata developed on the spots and exuded a pink spore mass that turned brown with age. Conidiophores were hyaline, unicellular, cylindrical, and fusiform and 5.0 to 8.0 µm long and 1.4 to 2.0 µm wide. Pure cultures were obtained by plating the spores on potato dextrose agar (PDA) medium. In culture, the fungus produced a gray-to-brown colony with whitish aerial mycelium. The morphology and size of conidia were comparable with previous descriptions of Pilidium concavum (Desm.) Höhn. (1). The ITS1-5.8S-ITS2 region of the isolate was amplified by PCR with primers ITS1 and ITS4 and sequenced. The 472-nt sequence was 100% identical to that of the Pilidium concavum specimen voucher BPI 1107275 (GenBank Accession No. AY487094). To validate Koch's postulates, pathogenicity was tested by inoculating 10 leaves of P. suffruticosa with mycelia plugs from a colony growing on PDA; leaves inoculated with the plugs of PDA medium only served as the control. Leaves were covered with plastic for 24 h to maintain high relative humidity. After 7 days, 100% of the mycelium-inoculated leaves showed symptoms identical to those observed on P. suffruticosa leaves affected in the field, whereas all leaves inoculated with PDA medium only remained free of symptoms. Reisolation of the fungus from leaf lesions confirmed that the causal agent was Pilidium concavum. Thus, we concluded that Pilidium concavum is the causal agent of leaf spots of P. suffruticosa. This disease has been reported to be frequently occurring on P. suffruticosa stems imported from Japan (1), but to our knowledge, this is the first report of Pilidium concavum on P. suffruticosa in China. References: (1) M. E. Palm. Mycologia 83:787, 1991.

Quantitation of porcine reproductive and respiratory syndrome virus RNA in semen by single-tube reverse transcription-nested polymerase chain reaction.

Porcine reproductive and respiratory syndrome virus (PRRSV) is in boar semen for extended periods of time as determined by reverse transcription-nested polymerase chain reaction (RT-nPCR) assay. The concentration of PRRSV RNA in semen and the biological significance of the detection level, however, remain to be resolved. In order to determine the concentration of PRRSV VR-2332 (a prototypic strain of North American isolates) in semen following infection, we established a 'standard curve'-quantitative competitive (SC-QC)-RT-nPCR assay as well as an equimolar QC-RT-nPCR assay. A deletion-type competitor RNA derived from the Lelystad virus, a European strain of PRRSV, ORF-7 gene standard which shares the nested sets of primer recognition sequences with the VR-2332 ORF-7 gene was used as an internal standard. The equimolar QC-RT-nPCR assay results revealed that the number of copies of PRRSV RNA in 1 TCID50/ml of virus derived from CL-2621 cell culture supernatants varied depending upon the type of samples in which virus was added; 143 +/- 24.0 and 266.5 +/- 48.5 copies in serum and semen samples spiked with PRRSV VR-2332, respectively. For the establishment of SC-QC-RT-nPCR assay, a standard curve was generated from band intensity ratios versus a series of known initial numbers of wild-type RNA copies which were quantified by the equimolar QC-RT-nPCR assay. Various initial numbers of copies of wild-type PRRSV RNA and each band intensity ratio with 1000 copies of competitor RNA were well correlated within the range of 100 to 200,000 copies (R2 = 0.947). A 'standard curve' quantitation assay using competitive single-tube RT-nPCR will offer a rapid and reliable way to quantify low concentrations of PRRSV RNA in semen.

Rapid diagnosis of porcine epidemic diarrhea virus infection by polymerase chain reaction.

The diagnosis of porcine epidemic diarrhea virus (PEDV) infection in the laboratory is rather fastidious because of difficulties in virus propagation. The feasibility of virus propagation in vivo is also limited by the handling of a number of samples at the same time. In this study, the detection of PEDV by reverse transcription polymerase chain reaction (RT-PCR) is described. The RT-PCR could detect up to 10(4) TCID50/ml of PEDV and did not show any cross reaction with transmissible gastroenteritis virus or porcine rotavirus. Using this method, the detection of PEDV in experimentally inoculated piglets was possible as early as one day after inoculation. These results suggest that the RT-PCR could be applicable for a rapid diagnosis of PEDV infection.

Bovine herpes virus expressing envelope protein (E2) of bovine viral diarrhea virus as a vaccine candidate.

The gene encoding the envelope protein (E2) of bovine viral diarrhea virus (BVDV) was expressed under the thymidine kinase (TK) promoter of Korean bovine herpesvirus 1 (BHV-1) isolate. Thymidine kinase negative (TK-) BHV-1 recombinants expressing E2 of BVDV were constructed and the expression of E2 was identified by immunofluorescence and Western blotting. Compared to wild type BHV-1, the recombinant BHV-1 had a delayed cytopathogenic effect in cells. The immunogenicity of the recombinant BHV-1 was examined in guinea pigs and cattle. Although an increase in body temperature was detected for a few days, the inoculated cattle returned to normal temperature with the development of neutralizing antibodies to BVDV.

Influence of hydration state and homologue composition of magnesium stearate on the physical chemical properties of liquid paraffin lipogels.

Lipogels were prepared by dispersing mixed (60:40 C(16)-C(18)) and pure (C(18)) homologue magnesium stearate (MgSt) in liquid paraffin, using three methods of preparation, i.e. addition of water at 95 °C during cooling cycle (method 1), homogenisation upon cooling (method 2) or cooling without addition of water or homogenisation (method 3). The systems were characterised by physical inspection, polarised, hot stage and scanning electron microscopy (SEM), differential scanning calorimetry (DSC), rheology, and X-ray diffraction (XRD). Systems formed stable semisolid lipogels (no syneresis), unstable solids showing syneresis or structured fluids, depending on the type of magnesium stearate used and the preparation technique. The stable semisolid lipogels containing mixed homologue MgSt (commercial-as received, anhydrous or dihydrate) prepared by methods 1 (∼ 1-2% water) and 2 contained α-crystalline lamellar structure. These were not present in the unstable solids formed with method 3 or in systems prepared from pure homologue MgSt which were generally structured fluids rather than semisolids. In addition, semisolid lipogels of pure homologue trihydrate MgSt prepared by method 3 showed plate-like crystals, implying pressure sensitivity. There is significantly more amorphous MgSt in the unstable solids compared to the stable semisolid lipogels, which are mainly crystalline (confirmed by XRD).

Evaluation for detection of Cryptosporidium oocysts in diarrheal feces of calves.

For the detection of Cryptosporidium oocysts, fecal samples were collected from 201 calves which showed diarrhea. Among the 201 samples, 29 samples (14.4%) were positive for Cryptosporidium spp. by the DMSO-modified acid-fast stain (MAFS), 23 samples (11.4%) were positive by commercial kit (Meridian Diagnostics, Cincinnati, Ohio) and 23 by the indirect immunofluorescence antibody (IFA) assay employing the monoclonal antibody (mAb C6). When tested by both IFA and MAFS, 20 fecal samples were positive for Cryptosporidium oocysts whereas 169 fecal samples were negative. If the MAFS is considered a standard method for oocyst detection, the IFA showed 69% of sensitivity and 98% of specificity. When tested by both IFA and commercial kit, 22 fecal samples were positive for Cryptosporidium oocysts while 177 samples were negative. One sample tested by IFA was found to be false negative, when compared with the results by commercial kit. The sensitivity of IFA was calculated as high as 96%; the specificity as 99% and the predictive value was also 99%. In the present study, IFA employing the mAb C6 revealed that 23 samples (11.4%) were positive among the 201 calves showing diarrhea. Of 23 IFA positive samples, 4 samples (5%) showed cryptosporidial oocysts more than 10(5) OPG. Therefore, it is concluded that the calves showing cryptosporidial oocysts more than 10(5) OPG in the feces were highly associated with clinical cryptosporidiosis.

[Experimental Cryptosporidium parvum infection in a Korean native calf isolated from a Korean mouse].

This study was performed to investigate experimental transmission of Cryptosporidium parvum in a calf. A 25-day-old Korean native calf was inoculated per os with 1 x 10(6) C. parvum oocysts isolated from a Korean mouse. The calf commenced oocyst discharge in feces on post-inoculation day 4, and continued until the day 11. The number of discharged oocysts peaked (4.9 x 10(5)) on post-inoculation day 6. However, the calf did not show signs of diarrhea. The present results indicate that C. parvum is cross-transmissible between the calf and the mouse.

Setaria marshalli infection in neonatal calves.

A total of 50 filariid worms of Setaria spp. was recovered from the peritoneal cavity of three neonatal calves infected with the Akabane virus. The parasites were identified as S. marshalli by their morphological characteristics. Males were 41-52 mm long and females 68-98 mm. Most of them were fully matured, indicating that the calves were infected prenatally. This is the first report of prenatal infection in calves by S. marshalli in Korea.

Enzyme-linked immunosorbent assay for detection of Trichinella spiralis antibodies and the surveillance of selected pig breeding farms in the Republic of Korea.

Trichinellosis is a parasitic zoonosis of public health importance. It is caused by Trichinella spiralis which has a wide host range including humans. In the present communication, the ELISA technique was employed on a total of 803 blood samples from 7 selected pig breeding farms in 1996 for diagnosis and surveillance of trichinellosis. Out of the entire 803 samples, nine were found to be suspected while one was positive by ELISA. But western blot analyses employed for further confirmation have shown that all of 10 samples did not react to larval excretory-secretory product antigens. These results indicate that pig breeding farms included in the present study are free from trichinellosis. However, it does not mean Korea is free from trichinellosis since human trichinellosis has recently been reported. The necessity of continued surveillance for trichinellosis in both pigs and wild animals was discussed.

Derivation of attenuated porcine epidemic diarrhea virus (PEDV) as vaccine candidate.

The field isolate of porcine epidemic diarrhea virus (PEDV) was serially passaged in Vero cells. The cell passaged PEDV, designated KPEDV-9, was tested for its pathogenicity in the neonatal pigs, immunogenicity and safety in the pregnant sows. The result indicated that KPEDV-9 at the 93rd passage revealed reduced pathogenicity in the neonatal pigs. Pregnant sows inoculated with the attenuated virus showed increased immune responses by ELISA. In addition, delivered piglets were protected from challenge of wild type PEDV. The safety test in pregnant sows indicated that all inoculated animals farrowed the average numbers of litters of piglets. The results of this study supported that the attenuated virus derived from serial passage could be applied as vaccine for protecting suckling piglets against PEDV infection.