[Effect of CD4-positive T lymphocyte expression rate on pain control and prognosis in stage â £ non-small cell lung cancer patients with cancerous pain].

Objective: To investigate the effect of CD4-positive T lymphocyte expression rate on the pain control and prognosis of stage Ⅳ non-small cell lung cancer (NSCLC) patients with cancerous pain. Methods: The clinical data of 128 stage Ⅳ NSCLC patients with cancerous pain who were admitted to the Affiliated Cancer Hospital of Zhengzhou University from January to December 2020 were retrospectively analyzed, including 92 males and 36 females, with a male-to-female ratio of 23∶9 and an average age of (56±21) years old. The expression rate of CD4-positive T lymphocytes in peripheral blood was routinely detected on admission, and the expression rate of CD4-positive T lymphocytes ≥45% was defined as the CD4 high expression group, and<45% was defined as the low expression group. The differences in the time required for pain control, the dosage of opioids and the incidence of adverse reactions between the two groups were compared and analyzed. Survival analysis was performed by Kaplan-Meier method, and the overall survival (OS) time and progression-free survival (PFS) time of the two groups were calculated. Cox regression model was used to analyze the influencing factors of patients' OS time and PFS time. Results: The median time required for pain control in the high CD4 expression group [M (Q1, Q3)] was 18.6 (4.6, 21.5) h, which was lower than that in the low CD4 expression group [28.2 (7.1, 38.9) h] (P=0.012). The dosage of morphine in the CD4 high expression group [M (Q1, Q3)] was 88.6 (42.5, 295.0) mg, which was lower than that in the low expression group [145.8 (82.5, 442.5) mg] (P=0.010). There was no significant difference in the incidence of adverse reactions such as nausea and vomiting, constipation, urinary retention, intestinal obstruction, and respiratory depression between the two groups (all P>0.05). The OS time and PFS time in the CD4 high expression group [M (Q1, Q3)] were 12.5 (8.1, 13.8) months and 8.5(3.1, 9.4) months, respectively, which were higher than those in the CD4 low expression group [8.6 (4.1, 12.9) months and 6.5 (2.1, 7.9) months, respectively] (all P<0.01). Cox multivariate analysis showed that high expression of CD4 was a protective factor affecting OS (HR=0.876, 95%CI: 1.224-6.641, P=0.004) and PFS (HR=0.675, 95%CI: 1.742-5.930, P=0.031) Conclusion: The stage Ⅳ NSCLC patients with cancerous pain and high expression of CD4-positive T lymphocytes have shorter pain control time, less morphine dosage, and longer OS and PFS time.

[Changes of coagulation factor activity and its clinical significance in tumor patients with abnormal coagulation function].

Objective: To investigate the changes and clinical significance of coagulation factor activity in tumor patients with abnormal coagulation function. Methods: The clinical data of cancer patients who were hospitalized in Henan Cancer Hospital from January 2020 to June 2021 was collected. Thromboelastography (TEG) was used to monitor the coagulation function of tumor patients. Accordingly, 196 tumor patients with abnormal coagulation function were in the test group, and 36 tumor patients with normal status were in the control group. According to the coagulation index (CI) value of the TEG test results, the test group was divided into two groups: hypercoagulability test group (n=104): CI value>3; hypocoagulation test group (n=92): CI value<-3. Meanwhile, each test group was divided into 3 subgroups according to the R value, K value, α angle and MA value of the TEG results, namely hypercoagulable group one (n=37), hypercoagulable group two (n=34), and hypercoagulable group three (n=33); hypocoagulation group one (n=33), hypocoagulation group two (n=30), and hypocoagulation group three (n=29). The activities of coagulation factors (F) Ⅱ, Ⅴ, Ⅶ, Ⅷ, Ⅸ, Ⅹ, Ⅺ, Ⅻ and von willebrand factor (vWF) were measured and compared for all patients in the test groups and the control group in the same period. Results: Compared with the normal control group, the hypercoagulable group one showed higher FⅡ, FⅤ, FⅦ, FⅧ, FⅪ and vWF activity, and the values were (1 105±281), (1 352±326), (1 628±397), (1 795±314), (1 389±288) and (1 908±486) U/L, respectively (P<0.01). The hypercoagulable group two showed higher FⅡ, FⅤ, FⅦ, FⅧ FⅩ and vWF activity, and the values were (1 068±189), (1 194±205), (1 529±394), (1 562±241), (1 150±196) and (1 722±415) U/L, respectively (P<0.05). Hypercoagulable group three showed higher FⅦ, FⅩ and vWF activity, and the values were (1 411±196), (1 212±327) and (1 713±457) U/L, respectively (P<0.01). In contrast, the hypocoagulation group one showed lower FⅤ, FⅧ, FⅨ, FⅫ and vWF activity, and the values were (732±96), (695±64), (1 216±191), (832±128) and (1 088±117) U/L, respectively (P<0.05). Hypocoagulation group two showed lower FⅤ, FⅦ, FⅧ and FⅫ activity, and the values were (714±102), (1 125±108), (783±95) and (912±111) U/L, respectively (P<0.01). Hypocoagulation group three had lower FⅪ, FⅫ and vWF activity, and the values were (812±92), (827±179) and (916±76) U/L, respectively (P<0.01). Conclusions: Only part of the coagulation factor activity changes significantly in the tumor patients with abnormal coagulation function. In tumor patients with hypercoagulable state, the high activity of FⅡ and FⅩ becomes an important factor in anticoagulant therapy, while high FⅤ, FⅧ activity can cause deep vein thrombosis. In the hypocoagulation state, the significant decreases of FⅤ and FⅧ activity often cause bleeding or oozing.

[Comparison of effects of celiac plexus block alone or in combination with retroperitoneal metastatic lymph node injection for pancreatic cancer-related pain].

Objective: To analyze the analgesic effect of CT-guided celiac nerve plexus destruction or celiac plexus destruction combined with absolute ethanol injection on retroperitoneal enlarged lymph nodes in patients with pancreatic cancer with retroperitoneal lymph node metastasis (combined therapy). Methods: Retrospective analysis of clinical data of 187 patients with pancreatic cancer and retroperitoneal lymph node metastasis admitted to Zhengzhou University Cancer Hospital from January 2014 to December 2018 due to poor abdominal pain control. According to the treatment method, they were divided into 2 groups: Group A (n=48) , treated with CT-guided celiac plexus destruction; Group B (n=139) , treated with CT-guided combined therapy. The analgesic effect, morphine application dose, and adverse reactions were compared before surgery, 1 week, 1 month, and 3 months after surgery. Results: The oral morphine doses of patients in Group A before surgery and 1 day, 1 week, 1 month, and 3 months after surgery were (107±34) , (65±23) , (35±12) , (48±18) , (81±25) mg. The oral morphine doses of patients in Group B before surgery and 1 day, 1 week, 1 month, and 3 months after surgery were (112±37) , (53±17) , (27±14) , (42±16) , (63±20) mg. Compared with that before surgery, the oral morphine doses were significantly reduced at 1 day, 1 week, 1 month, and 3 months after surgery in both groups (P<0.05 or P<0.01) . The effective rate and excellent rate of pain treatment in Group A at 1 week after operation were 83.3% and 60.4%, in Group B were 95.7% and 75.5%, respectively. The effective rate and excellent rate of pain treatment in Group A at one month after operation were 71.7% and 45.6%, in Group B were 89.0% and 67.6%, respectively; The effective rate and excellent rate of pain treatment in Group A at three months after operation were 48.6% and 25.7%, respectively, in Group B were 72.6% and 47.0%; Compared with Group A, the effective rate and excellent rate of pain treatment in Group B were increased, and the differences were statistically significant (P<0.05 or P<0.01). There was no significant difference in the incidence of nausea and vomiting between the two groups of patients before and 1 day after surgery, but the incidence of nausea and vomiting at 1 week, 1 month, and 3 months after surgery in Group B was significantly reduced, and the differences were statistically significant (P<0.05 or P<0.01). Compared with that before surgery, the incidence of nausea and vomiting in Group A was significantly reduced at 1 week and 1 month after operation, and the difference was statistically significant (P<0.01); The incidence of nausea and vomiting in Group B was significantly reduced at 1 day, 1 week, 1 month, and 3 months after operation, and the differences were statistically significant (P<0.01). Compared with 1 day after surgery, the incidence of nausea and vomiting in Group A was significantly reduced at 1 week and 1 month after surgery (P<0.05 or P<0.01). The incidence of nausea and vomiting in Group B was significantly reduced at 1 week, 1 month, and 3 months after operation, and the differences were statistically significant (P<0.01). Compared with 1 week after surgery, the incidence of nausea and vomiting in the two groups increased at 3 months after surgery, and the differences were statistically significant (P<0.05 or P<0.01). Compared with 1 month after surgery, the incidence of nausea and vomiting in Group A increased at 3 months after surgery, and the difference was statistically significant (P<0.05). There was no significant difference in the incidence of transient hypotension after surgery in the two groups. The difference in the incidence of postoperative diarrhea was not statistically significant. The incidence was highest within 1 day after surgery and generally recovered within 7 days after surgery. Conclusions: The two schemes can effectively relieve pain in patients with pancreatic cancer with retroperitoneal lymph node metastasis, reduce morphine dose. The combination therapy has higher efficiency and excellent rate, lower morphine dosage after surgery, and lower incidence of nausea and vomiting.

[Effect of opioid-related gene polymorphisms on patients with high-dose opioid-tolerant cancer pain].

Objective: To investigate the relationship between single nucleotide polymorphisms of opioid-related genes and high-dose opioid tolerance in patients with cancer pain. Methods: Twenty patients (high-dose opioid tolerance group, group A) who were hospitalized in Henan Cancer Hospital from June 2016 to June 2018 and who received high-dose opioid for pain control for more than 1 week were selected as case groups (group A). Thirty patients with stage Ⅳ tumors who were hospitalized in Henan Cancer Hospital and did not have opioid tolerance were randomly selected as the control group (group B). The peripheral blood samples of two groups were taken for DNA extraction. Gene polymorphisms were detected in 15 single nucleotide polymorphisms (SNP) (rs1799971, rs754891060, rs200637194, rs1045642, rs7438135, rs7439366, rs2242480, rs1080985, rs529520, rs581111, rs2234918, rs4680, rs6276, rs3732765, rs9859538) of the nine opioid receptor-related genes (OPRM1,ABCB1,UGT2B7,CYP3A4,CYP2D6,OPRD1, COMT,DRD2,P2RY12) which most likely to affect high-dose opioid tolerance in patients with cancer pain. Results: The distribution of different genotypes of rs7438135 locus in UGT2B7 gene were statistically significant between the two groups (χ(2)=9.68, P=0.004). The difference in the distribution of the different genotypes of the rs3732765 locus of the P2RY12 gene in the two groups were at the significant edge (χ(2)=5.57, P=0.05). A correlation analysis between the relevant SNP locus and the risk of high-dose opioid tolerance in cancer patients indicated that individuals with rs7438135 GA genotype in cancer patients were at 6.19 times more likely to have high doses of opioid tolerance than individuals with AA genotypes. Conclusions: The rs7438135 locus gene polymorphism of UGT2B7 gene may be a risk predictor for high-dose opioid tolerance. The rs3732765 site of the P2RY12 gene may be a potential predictor of high-dose opioid tolerance.

Assignment of Opsonic Values to Pneumococcal Reference Serum 007sp for Use in Opsonophagocytic Assays for 13 Serotypes.

Opsonophagocytic assays (OPAs) are routinely used for assessing the immunogenicity of pneumococcal vaccines, with OPA data often being utilized for licensure of new vaccine formulations. However, no reference serum for pneumococcal OPAs is available, making evaluation of data among different laboratories difficult. This international collaboration was initiated to (i) assign consensus opsonic indexes (OIs) to FDA pneumococcal reference serum lot 007sp (here referred to as 007sp) and a panel of serum samples used for calibration of the OPA and (ii) determine if the normalization of the OPA results obtained with test samples to those obtained with 007sp decreases the variability in OPA results among laboratories. To meet these goals, six participating laboratories tested a panel of serum samples in five runs for 13 serotypes. For each serum sample, consensus OIs were obtained using a mixed-effects analysis of variance model. For the calibration serum samples, normalized consensus values were also determined on the basis of the results obtained with 007sp. For each serotype, the overall reduction in interlaboratory variability was calculated by comparing the coefficients of variation of the unadjusted and the normalized values. Normalization of the results substantially reduced the interlaboratory variability, ranging from a 15% reduction in variability for serotype 9V to a 64% reduction for serotype 7F. Normalization also increased the proportion of data within 2-fold of the consensus value from approximately 70% (average for all serotypes) to >90%. On the basis of the data obtained in this study, pneumococcal reference standard lot 007sp will likely be a useful reagent for the normalization of pneumococcal OPA results from different laboratories. The data also support the use of the 16 FDA serum samples used for calibration of the OPA as part of the initial evaluation of new assays or periodic assessment of established assays.

[Epidemiologic characteristics of noroviruses isolated in outpatients with acute gastroenteritis in Hangzhou area, from 2014 to 2015].

OBJECTIVE: To explore the epidemiologic characteristics of noroviruses isolated in patients with acute gastroenteritis in Hangzhou between March 2014 and April 2015. METHODS: Stool specimens and clinical data were collected from 1 109 patients with acute gastroenteritis. Specimens were detected for noroviruses with GⅠand GⅡsubtypes by one-step double real-time RT-PCR. Some of the positive specimens were then randomly selected and amplified by multiplex RT-PCR. Those positive PCR products were sequenced and analyzed phylogenetically for testing the partial capsids of noroviruses. RESULTS: Of the 1 109 stool specimens, positive rate of noroviruses was 26.87% (298/1 109). GⅡgenotype was the major viruses with the proportion as 25.52% (283/1 109), while 1.35% (15/1 109) belonged to GⅠgenotypes. There was no significant difference in the noroviruses detection rate of the different genders (P>0.05). However, in different age groups, GⅡgenotypes were predominant types of noroviruses, and the positive rates of GⅡgenotypes were 16.94% (<5 years-old), 19.45% (5-18 years-old) and 32.26% (≥18 years-old), respectively. In different seasons, noroviruses could be detected all year round, with positive rate as 29.67%-37.08% in the highly epidemic seasons (between December and March of the following year). The distribution trends were seen certain difference between noroviruses-GⅡand GⅠtypes. Additionally, results from the sequence analysis demonstrated that GⅡ-4 genotype was the prevalent strain of GⅡ genotypes, clustered into GⅡ-4/Sydney (46.43%, 13/28) and GⅡ-4/2006b (25.0%, 7/28), while GⅠstrains clustered into GⅠ-1. CONCLUSION: Noroviruses appeared one of the major pathogens, leading to acute gastroenteritis. G Ⅱgenotypes of noroviruses, especially the G Ⅱ-4/Sydney variant strains and GⅡ-4/2006b variant strains, were considered to be the prevalent strains prevailed in Hangzhou areas from 2014 to 2015.

[Monitoring and research on pathogen spectrum in patients with acute diarrhea from sentinel hospital of Zhejiang Province during 2009 to 2014].

Objective: To explore pathogen spectrum constitution of acute diarrhea in outpatient and emergency of Zhejiang Province, and provide basis for treatment, prevention and control of the disease. Methods: During January 2009 to December 2014, we selected seven sentinel hospitals in different regions of Zhejiang, monitored and researched on pathogen spectrum in patients with acute diarrhea from outpatient and emergency. We recorded patients' personal basic information, the main symptoms and signs, and collected stool samples (5 g). Eight kinds of bacteria (Vibrio cholerae, Shigella spp., Salmonella spp., Diarrheagenic E. coli, Vibrio parahaemolyticus, Aeromonas hydrophila, Yersinia enterocolitica and Plesiomonas shigelloides) and five kinds of viruses (Rotavirus, Norovirus, Sappovirus, Astrovirus and Adenovirus) were detected. Chi-square test and Fisher's exact probability method were used to compare different characteristics of patients with single bacterial infection, single virus infection and multiple infection (bacteria-bacteria, bacteria-viruses, virus-virus). Results: During 2009 to 2014, 9 364 fecal samples from acute diarrhea patients were collected and tested, among which 3 500 cases were tested positive, with total positive rate of 37.38%. Positive rates of bacteria and viruses were 13.14% (1 230 cases) and 20.75% (1 943 cases), respectively. Mixed infection positive rate of multiple pathogens was 3.49% (327 cases). Positive rate of Vibrio parahaemolyticus (5.96% , 558 cases) was the highest among bacterial pathogens, followed by pathogenic Escherichia coli (3.86%, 361 cases). Viruses were mainly Norovirus (10.73%, 1 005 cases) and rotavirus (8.35%, 782 cases). A big difference existed in diarrheogenic pathogen spectrum between patients less than 15 years old and patients equal or older than 15 years old. Pathogens for patients less than 15 years old were mainly virus, with the positive rate of 32.69% (1 014 cases). However, the positive rate of bacteria was 16.86% (1 056 cases) in patients equal or older than 15 years old. Single bacterial infection was highest in age group of 25-34 years old (18.62%, 302 cases) , single virus infection was highest in age group of 1-4 years old (41.12%, 435 cases) , and mixed infections of multiple pathogens were mainly existed in age group of 1-4 years old (7.37%, 78 cases) . Pathogen positive rate were increasing year by year. Pathogen positive rate of patients with acute diarrhea has obvious seasonality, with single bacterial infection being highest during July to September and single virus infection being highest during December to March. Pathogen spectrum of outpatient and emergency patients with acute diarrhea in Zhejiang Province changed a little from 2009 to 2014, mainly rotavirus (22.34% (782/3 500)), norovirus (28.71% (1 005/3 500)), vibrio parahaemolyticus (15.92% (558/3 500)) and Escherichia coli (10.31% (361/3 500)). However, pathogen spectrums in different years owned different features. Conclusion: Common pathogens in outpatient and emergency patients with acute diarrhea in Zhejiang Province were tested with significant seasonal epidemic law. The composition of pathogenic spectrum was variant in different age group. Constitutes of major pathogen spectrum in different years differed a little.

A consortium of rhizobacterial strains and biochemical growth elicitors improve cold and drought stress tolerance in rice (Oryza sativa L.).

In the present study, a consortium of two rhizobacteria Bacillus amyloliquefaciens Bk7 and Brevibacillus laterosporus B4, termed 'BB', biochemical elicitors salicylic acid and ß-aminobutyric acid (SB) and their mixture (BBSB) were investigated for cold and drought stress tolerance in rice plants. After withholding water for 16 days, rice plants treated with BBSB showed 100% survival, improved seedling height (35.4 cm), shoot number (6.12), and showed minimum symptoms of chlorosis (19%), wilting (4%), necrosis (6%) and rolling of leaves. Similarly, BB inoculation enhanced plant growth and reduced overall symptoms in rice seedlings subjected to 0 ± 5 °C for 24 h. Our results imply several mechanisms underlying BB- and BBSB-elicited stress tolerance. In contrast to the control, both treatments significantly decreased leaf monodehydroascorbate (MDA) content and electrolyte leakage, and increased leaf proline and cholorophyll content. Moreover, activities of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) increased 3.0- and 3.6-fold, respectively. Moreover, expression of OsMYB3R-2, OsDIL, OsDREB1A and OsCDPK13 genes was significantly up-regulated, suggesting that these genes play important roles in abiotic stress tolerance of rice. In addition, bacterial strains Bk7 and B4 were able to produce high amounts of IAA and siderophores, and colonise the plant roots, while only strain Bk7 exhibited the capability to form biofilms and solubilise inorganic phosphate. This study indicates that the BB and BBSB bio-formulations can be used to confer induced systematic tolerance and improve the health of rice plants subject to chilling and drought stress.

γ-Aminobutyric acid-mediated neurotransmission in cerebellar-hypothalamic circuit attenuates gastric mucosal injury induced by ischemia-reperfusion.

BACKGROUND: Excessive greater splanchnic nerve (GSN) activation contributes to the progression of gastric ischemia-reperfusion (GI-R) injury. This study was designed to investigate the protective mechanism of cerebellar fastigial nucleus (FN) stimulation against GI-R injury. METHODS: The GI-R injury model was induced in rats by clamping the celiac artery for 30 min, and then reperfusion for 30 min, 1, 3, 6, or 24 h, respectively. KEY RESULTS: Microinjection of L-Glu (3, 6, 12 µg) into the FN dose-dependently attenuated GI-R injury and GSN activity. In addition, there was an enhancement of gastric mucosal blood flow in GI-R rats. Pretreatment with the glutamic acid decarboxylase antagonist into the FN, the GABAA receptor antagonist into the lateral hypothalamic area or lesion of superior cerebellar peduncle all reversed the protective effects of the FN stimulation. Furthermore, the FN stimulation reduced the TUNEL-positive gastric mucosal cell and Bax-positive gastric mucosal cell in GI-R rats. CONCLUSIONS & INFERENCES: These results indicate that the protective effects of the FN stimulation against GI-R injury may be mediated by attenuation of the excessive GSN activation, gastric mucosal cell apoptosis, and Bax expression in GI-R rats.

Specific and Sensitive Detection of Enterobacter mori Using Reliable RT-PCR.

Enterobacter mori, the causal agent of bacterial wilt in mulberry, is becoming a serious disease in mulberry orchards in China. Because no effective control strategy has been devised for this disease, the reliable screening of mulberry material for latent infection became necessary. Hence, a fast polymerase chain reaction (PCR) assay for the detection of E. mori was developed in this study. The primers were designed within regions of the RNA polymerase ß-subunit (rpoB) gene. The method is fast and simple and showed 100% sensitivity (no false negatives) and 100% specificity (no false positives), which was tested with 4 representative E. mori strains, 9 Enterobacter type strains, 2 strains of the other major mulberry bacterial pathogens (Ralstonia solanacearum and Pseudomonas syringae pv. mori) in China, 7 strains of other plant-associated pathogens, and 50 unidentified epiphytic bacterial isolates from mulberry plants. The real-time PCR assays reliably detected the DNA at at least 10 fg/µl and the bacterial cells at 102 CFU/ml from mulberry shoots and roots suspension. The strong positive reaction in testing of all symptomatic plants (with 100% positive) and parts of asymptomatic latent infected plant samples (with 36.4% positive) provided proof that this method is reliable and sensitive and suitable for screening plant material with latent infections of E. mori.

Grain Discoloration of Rice Caused by Pantoea ananatis (synonym Erwinia uredovora) in China.

In the autumn of 2008, a new bacterial disease of rice was noted in paddy fields near Hangzhou, Zhejiang Province, China. The disease caused severe discoloration of rice grains on cv. Zhong-zhe-you 1 (Oryza sativa L.). It often occurred at early flowering of hybrid rice. Initially, light, rusty, water-soaked lesions appeared on the lemma or palea and then turned brown. More immature and lighter grains were observed on panicles at harvest. No bacterial ooze was observed. Ten bacterial isolates were recovered from eight samples of discolored rice grains (1). Six isolates were selected for identification. They were similar to those of the reference strain of Pantoea ananatis (Serrano, synonym Erwinia uredovora) LMG 2665T (ATCC 33244) from Belgium in phenotypic tests based on the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA), pathogenicity tests, gas chromatographic analysis of fatty acid methyl esters (FAME) using the Microbial Identification System (MIDI Inc, Newark, DE) with the aerobic bacterial library (TAB 5.0), and electron microscopy (TEM,KYKY-1000B, Japan). All isolates were facultatively anaerobic, gram-negative rods that measured 1.6 to 2.5 × 0.5 to 0.7 µm and had three to six peritrichous flagella. Colonies on nutrient agar were yellow and raised with smooth margins. A hypersensitive reaction was observed on tobacco (Nicotiana tobacum cv. Benshi) 24 h after inoculation. All isolates were identified as P. ananatis with Biolog similarity indices of 0.716 to 0.852 and FAME similarity indices of 0.783 to 0.903. Further identification as P. ananatis was done by 16S rDNA sequence analysis. Amplicons were produced from three strains using the universal primers (3) fD2: 5'-AGA GTT TGA TCA TGG CTC AG-3' forward primer and rP1: 5'-ACG GTT ACC TTG TTA CGA CTT-3' reverse primer and then sequenced (GenBank Accession Nos. GU324769, GU324770, and GU338399). A BlastN search of GenBank revealed that they had 97 to 98% nt identity with P. ananatis strain 3Pe76 (GenBank Accession No. EF178449). Koch's postulates were completed by spray inoculating panicles of rice cv. Zhong-zhe-you 1 at booting stage, grown in pots, with cell suspensions containing 108 CFU/ml of the six strains at 25 to 29°C. Three plants were inoculated with each strain, controls were sprayed with water, and the experiment was repeated once. Three weeks after inoculation, all strains produced symptoms on panicles similar to those observed in the field. Yellow pigmented bacteria were reisolated from symptomatic panicles and their identity was confirmed by FAMEs. These results indicate that the pathogen is P. ananatis (2), which also causes leaf blight and bulb decay of onion. To our knowledge, this is the first report of rice grain discoloration caused by P. ananatis in China. The disease cycle on rice and the control strategies in the regions are being further studied. References: (1) J. Y. Luo et al. Plant Dis. 91:1363, 2007. (2) H. G. Truper and L. de Clari. Int. J. Syst. Bacteriol. 47:908, 1997. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

First Report of Bacterial Head Rot of Broccoli Caused by Pseudomonas fluorescens in China.

During warm and humid periods in the winters from 2005 to 2008, head rot symptoms on broccoli (cv. Sijilv) (Brassica oleracea L. var italica Planch) were observed in commercial fields in Ningbo, Zhejiang Province, China. In agreement with the report of Cui and Harling (1), water-soaked lesions developed on the buds and then progressed into a brown-black soft rot. Longitudinal sections of the symptomatic inflorescences showed brown discoloration and rotting of the internal tissues. Broccoli production is hampered by the disease, with disease incidence ranging from 65 to 81%. Bacteria were isolated by streaking on nutrient agar (3) and individual colonies formed after 2 to 3 days of incubation at 28°C. Fifteen of thirty isolates induced hypersensitive reactions (HR) on tobacco leaves (Nicotiana tabacum cv. Samsun) within 48 h. All the HR-positive strains were fluorescent on King's medium B and the colonies were smooth, convex, entire, and round. Classical bacteriological tests indicated that the fluorescent strains were gram negative, obligate aerobes, arginine dihydrolase positive, and oxidase positive. Also, the fluorescent strains were positive for the production of levan from sucrose. Five representative strains were further characterized by the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA) and gas chromatography of fatty acid methyl esters (FAME) using the Microbial Identification System (MIDI Inc., Newark, DE) with the aerobic bacterial library (TSBA50). The five strains were identified as Pseudomonas fluorescens with Biolog and FAME similarity indexes of 0.61 to 0.68 and 0.52 to 0.58, respectively. The 16S rRNA gene sequence of broccoli strain PFB-01 (GenBank Accession No. GQ352649) was determined according to Li et al. (2). A subsequent GenBank search showed that this sequence had 98% nucleotide identity with the type strain of P. fluorescens (ATCC 17386T, GenBank Accession No. AF094726). Koch's postulates were completed by the inoculation of broccoli heads (cv. Sijilv) with cell suspensions (107 CFU/ml) of the above five strains by spraying on the surface of subcorymbs. Each treatment had five replicates. All strains induced head rot symptoms similar to those observed in natural infections. No symptoms were noted on the control plants inoculated with sterile water. Bacteria were successfully reisolated from symptomatic heads and confirmed by the cellular fatty acid composition. To our knowledge, this is the first report in China that P. fluorescens is the causal pathogen of bacterial head rot of broccoli. References: (1) X. Cui and R. Harling. Phytopathology 96:408, 2006. (2) B. Li et al. J. Phytopathol. 154:711, 2006. (3) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society. St. Paul, MN, 2001.

Bacterial Stem Rot of Oncidium Orchid Caused by a Dickeya sp. (ex Pectobacterium chrysanthemi) in Mainland China.

In December 2007, stem rot symptoms on orchids (Oncidium Gower Ramsey) were observed at a flower nursery in the Zhejiang Province of China. Initial symptoms were water-soaked lesions starting at the base of the stem. As these lesions expanded and elongated, the stem and leaf tissues became soft and watery. When examined with a microscope, cut edges of symptomatic stem and leaf tissues consistently exhibited bacterial streaming. The bacteria were isolated by streaking on nutrient agar (3). All isolates were gram-negative, facultative, anaerobic rods with peritrichous flagella. Infiltration of tobacco leaves (Nicotiana tabacum cv. Samsun) with the bacterial suspension of 108 CFU/ml resulted in typical hypersensitivity reactions within 24 h. Five representative isolates were further characterized by the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA) and gas chromatography of fatty acid methyl esters (FAME) by the Microbial Identification System (MIDI Inc., Newark, DE) with aerobic bacterial library (TSBA50). The five isolates were identified as Erwinia chrysanthemi (Pectobacterium chrysanthemi) with a Biolog and FAME similarity index of 0.81 to 0.88 and 0.62 to 0.75, respectively. The transfer of P. chrysamthemi to a novel genus, Dickeya gen. nov., was recently proposed (2). The almost complete 16S rDNA sequence from Oncidium isolate SCH-01 (1,604 bp; EMBL Accession No. FM946179) was determined according to the method of Li et al. (1). A subsequent GenBank search showed that this isolate is 98% identical to that of type strain CFBP 1269T of Dickeya dadantii (EMBL Accession No. AF520707) and CFBP 1200T of Dickeya dianthicola (EMBL Accession No. AF520708). Nevertheless, species identification within genus Dickeya is still difficult since only a limited number of strains of each species have been characterized fully. Koch's postulates were completed with the inoculation of Oncidium seedlings with cell suspensions (108 CFU/ml) by a pinprick at the base of the stem. All five representative isolates induced stem rot similar to that observed in natural infections. No symptoms were noted on the control plants inoculated with sterilized distilled water by the same method. The bacterium was reisolated from symptomatic stems of Oncidium plants. To our knowledge, this is the first report of stem rot on Oncidium orchid in Mainland China caused by the bacterium formerly referred to as P. chrysanthemi, now proposed as Dickeya sp. References: (1) B. Li et al. J. Phytopathol. 154:711, 2006. (2) R. Samson et al. Int. J. Syst. Evol. Microbiol. 55:1415, 2005. (3) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society. St. Paul, MN, 2001.

Isolation and characterization of antagonistic bacteria against bacterial leaf spot of Euphorbia pulcherrima.

AIMS: To investigate the inhibition potential of leaf-associated bacteria against the pathogen of bacterial leaf spot of Euphorbia pulcherrima. METHODS AND RESULTS: Seven out of 200 bacterial strains were effective antagonists by in vitro screening and the two strains PAB241 and PAB242 significantly reduced the disease incidence and severity as foliar treatments of E. pulcherrima. The two effective strains, PAB241 and PAB242, were both identified as Bacillus amyloliquefaciens by a polyphasic approach including phenotypic feature, carbon source utilization profile, fatty acid methyl esters and analysis of 16S rRNA gene sequence. In addition, the suspensions of B. amyloliquefaciens PAB241 and PAB242 showed antibacterial activities against the pathogen of bacterial leaf spot of E. pulcherrima under different treatments. CONCLUSIONS: The leaf-associated bacteria, B. amyloliquefaciens PAB241 and PAB242, markedly inhibited the growth of X. axonopodis pv. poinsettiicola under different treatments and protected E. pulcherrima from pathogen infection in growth chamber conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that showed B. amyloliquefaciens from plant leaves was a potential bactericide against bacterial leaf spot of E. pulcherrima.

Bacterial Stem Rot of Poinsettia Caused by a Dickeya sp. (Pectobacterium chrysanthemi) in China.

In December 2006, a rot symptom of unknown etiology was observed on stems of plants (Euphorbia pulcherrima cv. Fu-xing) at a flower nursery in the Zhejiang Province of China where we had previously reported leaf spot of poinsettia caused by Xanthomonas campestris (2). Chlorotic spots anywhere along the stem and purplish black petioles were the first noticeable symptoms. The spots rapidly coalesced, forming large irregular chlorotic areas. Petioles turned black and shriveled and affected leaves wilted. Infected tissues were soft and water soaked. Ten bacterial strains were isolated from the diseased samples and five were selected for identification. They were similar to those of the standard reference strains of Pectobacterium chrysanthemi (Dickeya sp.), LMG 2804 from Belgium and ZUPB20056 from China, in phenotypic tests based on the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA), pathogenicity tests, gas chromatography of fatty acid methyl esters (FAME) using the Microbial Identification System (MIDI Inc, Newark, DE) with aerobic bacterial library (TABA50), and transmission electron microscopy (TEM,KYKY-1000B, Japan). All strains tested were gram-negative facultative anaerobic rods measuring 1.5 to 3.6 × 0.6 to 1.1 µm, with peritrichous flagella. Colonies were gray-white and slightly raised with smooth margins on nutrient agar. They were negative for trehalose and positive for phosphatase production and reducing substances from sucrose. A hypersensitive reaction was observed on tobacco cv. Benshi, 24 h after inoculation. All five isolates, LMG 2804, and ZUPB20056 were identified as P. chrysanthemi (Dickeya sp.) with a Biolog similarity index of 0.58 to 0.83, 0.68, and 0.72 and a FAME similarity index of 0.52 to 0.80, 0.59, and 0.70, respectively. Identification as P. chrysanthemi (Dickeya sp.) was confirmed by PCR with specific primers used by Nassar et al (3). Koch's postulates were completed with the inoculation of 12 4-month-old intact poinsettia plants of cv. Fu-xing with cell suspensions containing 108 CFU/ml by a pinprick at the base of the stem. All five strains induced stem infection similar to those observed in natural infections. No symptoms were noted on the two control plants inoculated with sterilized distilled water by the same method. The bacterium was reisolated from symptomatic stems of poinsettia plants. P. chrysanthemi (Dickeya sp.) was first reported in United States as the cause of bacterial stem rot of poinsettia in 1972 (1). To our knowledge, this is the first report of poinsettia stem rot caused by P. chrysanthemi (Dickeya sp.) in China. The disease cycle and the control strategies of the bacterial stem rot of poinsettia in the regions are being further studied. References: (1) H. A. J. Hoitink et al. Plant Dis. Rep. 56:480, 1972. (2) B. Li et al. Plant Pathol. 55:293, 2006. (3) A. A. Nassar et al. Appl. Environ. Microbiol. 62:2228, 1996.

First Report of Pear Blossom Blast Caused by Pseudomonas syringae pv. syringae in China.

In the spring of 2006, a new bacterial disease was noted in pear orchards near Hangzhou, Zhejiang Province, China. The disease caused severe blossom blast on pears (Pyrus pyrifolia; cv. Cuiguan). Early symptoms of the disease included blackening of the calyx end of developing fruit, blackening of blossom clusters while leaves of affected blossom clusters appeared normal, or death of clusters consisting of both blossoms and leaves. Later, tips of twigs turned dark brown and died. No bacterial ooze was observed. Twelve bacterial isolates were recovered from ten samples of buds and blossoms. Six isolates were selected for identification. They were similar to those of the reference strains of Pseudomonas syringae pv. syringae LMG5570 and LMG 2230 from Belgium in phenotypic tests on the basis of the Biolog Microbial Identification System (version 4.2; Biolog Inc., Hayward, CA), pathogenicity tests, gas chromatographic analysis of fatty acid methyl esters (FAMEs) using the Microbial Identification System (MIDI Inc., Newark, DE) with aerobic bacterial library (TABA50), and electron microscopy (TEM, KYKY-1000B, Japan). All isolates tested were gram-negative, aerobic rods measuring 1.5 to 2.4 × 0.5 to 0.6 µm with 2 to 4 polar flagella. Fluorescent green diffusible pigment was produced on King's Medium B. Colonies were gray-white and slightly raised with smooth margins on nutrient agar. They produced levan on sucrose nutrient agar. A hypersensitive reaction was observed on tobacco cv. Benshi 24 h after inoculation. All isolates were identified as P. syringae pv. syringae with Biolog similarity index of 0.57 to 0.86 and FAME similarity index of 0.58 to 0.81. Identification as P. syringae pv. syringae was confirmed using 16S rDNA universal primers (2,3): 5'-AGA GTT TGA TCA TGG CTC AG-3' forward primer, 5'-ACG GTT ACC TTG TTA CGA CTT-3' reverse primer. The PCR fragments of the three isolates were sequenced and compared with sequences in GenBank. They had 99% similiarity with P. syringae pv. syringae 16S rRNA gene strain NCPPB 3869. Koch's postulates were conducted on buds of the original pear cultivar growing in pots and detached pear blossoms in flasks by spray inoculation with cell suspensions containing 108 CFU/ml of the six isolates at 18 to 22°C with two replications. The bacteria induced symptoms on buds and blossoms similar to those observed in the field. The bacterium was reisolated from symptomatic pear buds and internal ovary tissues. P. syringae pv. syringae was first reported in England as the cause of pear blossom blast in 1914 (1). After searching all the Chinese agricultural databases and major journals (National Knowledge Infrastructure database, Vip Chinese periodical database, Chinese wanfang database, China InfoBank, Scientia Agricultura Sinica, Acta Phytopathologica Sinica, Acta Phytophylacica Sinica, and Journal of Fruit Science), to our knowledge, this is the first report of pear blossom blast caused by P. syringae pv. syringae in China. The disease cycle on pear trees and the control strategies in the regions are being further studied. References: (1) B. P. Barker et al. Ann. Appl. Biol. 1:85, 1914. (2) U. Edward et al. Nucleic Acids Res. 17:7843,1989. (3) B. Li et al. J. Phytopathol. 34:141, 2006.

Bacterial Wilt of Mulberry (Morus alba) Caused by Enterobacter cloacae in China.

In August of 2006, a new bacterial disease was noted in Hangzhou mulberry orchards of Zhejiang Province, China where bacterial wilt of mulberry caused by Ralstonia solanacearum was previously reported (3). In the summer, the disease caused severe wilt, especially on 1- or 2-year-old mulberry plants, that resulted in premature plant death. Leaf wilt symptoms generally started on older leaves at the bottom of the plant and spread to the younger leaves. The leaves of infected plants became withered and dry, turned dark brown, and eventually the plants became defoliated. The root xylem of infected plants was moist and discolored with brown stripes. The phloem was asymptomatic, however, in severe infections, the phloem was decayed. The observation of wilting proceeding from the bottom of the plant to the top distinguishes this disease from bacterial wilt caused by R. solanacearum. Five bacterial strains isolated from infected mulberry plants showed characteristics similar to those of the standard reference strain of Enterobacter cloacae subsp. cloacae IBJ0611from China, but differed from R. solanacearum IBJ35, E. cancerogenus LMG2693T, and E. cloacae subsp. dissolvens LMG2683T from the University of Gent, Belgium in phenotypic tests, including the Biolog Identification System version 4.2 (Biolog Inc., Hayward CA), pathogenicity tests, transmission electron microscopy (TEM,KYKY-1000B, Japan) observation, and gas chromatographic analysis of fatty acid methyl esters (FAMEs) using the Microbial Identification System (MIDI Company, Newark, DE) with the aerobic bacterial library (TABA50). Isolates were gram negative, facultative anaerobic, rod shaped, 0.3 to 1.0 × 1.0 to 3.0 µm with peritrichous flagella. Colonies on nutrient agar were light yellow, smooth, circular, entire, and convex with no green fluorescent diffusible pigment on King's medium B (3). Weak hypersensitive reaction was observed on tobacco 3 days after inoculation. All five strains were identified as E. cloacae with Biolog similarity of 0.662 to 0.863 and FAMEs similarity of 0.632 to 0.701. Inoculation of 10 6-month-old intact mulberry plants of cv Husang with cell suspensions containing 109 CFU/ml by pinprick at the base of the stem reproduced symptoms observed in natural infections. No symptoms were noted on the two control plants inoculated by the same method but with sterilized distilled water. The bacterium was reisolated from the symptomatic mulberry plants. E. cloacae has been reported from the United States as the cause of internal yellowing of papaya fruits (1) and rhizome rot of edible ginger (2). To our knowledge, this is the first report of mulberry wilt caused by E. cloacae in China. References: (1) K. Nishijima et al. Plant Dis. 71:1029, 1987. (2) K. Nishijima et al. Plant Dis. 88:1318, 2004. (3) L. Xu et al. Acta Phytophylacica. Sin. 34:141, 2007.

Occurrence of Rust Caused by Puccinia carthami on Snow Lotus in China.

Snow lotus (Saussurea involucrata (Kar. & Kir.) Sch. Bip.) is an economically important medicinal herb increasingly grown in China in recent years. In August 2005, a rust disease on snow lotus plants commercially grown was found in the Tianshan mountain area of Xinjiang at 2,100 m above sea level. Disease incidence was approximately 15% of the plants observed in a commercial field in 2006. At the initial stage, tiny (1 to 2 mm long), orange brown pustules are formed on leaves. Later in the season, pustules turned chestnut brown to form bigger rust patches. Severely attacked leaves may die prematurely. During the growing season, rust pustules broke open to release reddish brown spores that cause secondary infection. The infected snow lotus plants were sampled, and the urediniospores and teliospores were observed for identification with a light microscope (4). Urediniospores were globose or broadly ellipsoid, 22 to 28 × 22 to 25 µm; teliospores were slightly or not constricted at the septum, 31.5 to 41.5 × 21.5 to 26.5 µm, wall was sienna to fulvous, occasionally chestnut colored, and pedicels were basal or oblique, verrucose, hyaline, and fragile. The pathogenicity test of the fungus was done by burying five leaves bearing telia around the roots of healthy safflower seedlings grown in a greenhouse under controlled conditions (25°C and 70% relative humidity) and healthy snow lotus seedlings grown under natural conditions with five replications. Symptoms were evaluated 60 days after inoculation. Similar rust symptoms were observed on both the snow lotus and safflower seedlings (1). The pycnidial and aecial stages of this autoecious rust were not observed in nature or the pathogenicity tests. The teliospores were reisolated and deposited at the Key Oasis Eco-Agriculture Laboratory of Xinjiang Production and Construction Group, Xinjiang and the Institute of Biotechnology, Zhejiang University. The causal organism of rust of snow lotus was identified as Puccinia carthami Corda on the basis of the morphology and pathogenicity test. To our knowledge, this is the first report of this rust on snow lotus (S. involucrata) (1-4). References: (1) W. L. Bruckart. Plant Dis. 83:181, 1999. (2) M. L. Deadman et al. Plant Dis. 89:208, 2005. (3) S. J. Kolte. Diseases of annual edible oilseeds crops. In: Rapeseed-Mustard and Sesame Diseases. Vol. II. CRC Press, Boca Raton, FL, 1985. (4) G. F. Laundon. N. Z. J. Bot. 8:310, 1970.

HLA and HPA typing in idiopathic thrombocytopenic purpura patients treated with Kami-kihi-to.

We performed human leukocyte antigen (HLA) and human platelet antigen (HPA) in patients with Kami-kihi-to-responsive idiopathic thrombocytopenic purpura. The HLA-A2, A61 and Cw1 were significantly increased in responders compared with nonresponders, as were HLA DRB1 *0901, DRB1 *1502, and DPB1 *0501. In contrast, HLA DPB1 *0201 and DPB1 *0901 were significantly decreased in responders. The a/b genotype of HPA-2 and a/a genotype of HPA-3 were markedly increased in nonresponders, and anti-GPIb antibody was also increased. These results suggest that HLA, HPA, and anti-GP antibody studies may predict the response of idiopathic thrombocytopenic purpura to Kami-kihi-to.