Application of duplex-PCR in rapid and reliable detection of toxigenic Vibrio cholerae in water samples in Thailand

Toxigenic Vibrio cholerae, the cause of cholera, is a native flora of the aquatic environment which is transmitted through drinking water and still remains the leading cause of morbidity and mortality in many developing countries including Thailand. The culture method (CM), which is routinely used for assessing water quality, has not proven as efficient as molecular methods because the notorious pathogen survives in water mostly in a non-culturable state. We employed duplex-polymerase chain reaction (duplex-PCR) for detection of tcpA and ctxA genes in toxigenic V. cholerae, and compared PCR detection with CM in various waters of Khon Kaen Municipality, Thailand. We also evaluated the effect of different pre-PCR conditions on the results of ctxA and tcpA detection including: 1) water filtered and enriched in alkaline peptone water (APW) for 3 h before PCR, 2) water filtered without enrichment before PCR, and 3) use of only enrichment in APW for 6 h before PCR. Of the 96 water samples (taken from waste-water, potable and waste-water from patients' houses, and from rivers) tested, 48 (50%) were positive for ctxA and tcpA by duplex-PCR, whereas only 29 (30%) were positive for V. cholerae by CM. Of the 29 V. cholerae isolated by CM, 2 (7%) were toxigenic V. cholerae belonging to serovar O1, while the rests were non-O1/ non-O139. Results revealed, therefore, that ctxA and tcpA-targeted duplex PCR is more sensitive than CM for detection of toxigenic V. cholerae from water samples because CM detected much less toxigenic V. cholerae than the non-toxigenic V. cholerae. Template DNA as low as 100 fg or 23 cells of V. cholerae in the water sample was detected in duplex PCR. Pre-PCR filtration followed by enrichment for 3 h significantly increase in the efficiency of duplex-PCR detection of toxigenic V. cholerae.     

抗虫玉米CM8101定性检测方法的建立

转基因抗虫玉米CM8101是经人工改造合成的转Bt抗虫基因Cry1Ab?Ma转基因玉米新品系,由我国自主研发,具有更优良的抗虫性,目前已进入生产性试验阶段,具有广阔的产业化应用前景。依据CM8101的5′端旁侧序列信息,设计并筛选出最佳引物探针组合,通过普通PCR和实时荧光PCR技术,建立了转基因抗虫玉米CM8101的两种特异性定性检测方法。结果显示,普通PCR检测方法检出限达0.1%,实时荧光PCR检测方法检出限达0.05%。这两种定性检测方法的建立为今后准确高效检测转基因抗虫玉米CM8101及其产品提供了新的参考方法。     

Cerebral malaria is frequently associated with latent parasitemia among the semi-immune population of eastern Sudan

The accurate diagnosis of malaria starts with clinical suspicion, confirmed by reliable laboratory results. A hospital-based study, described here, was carried out in a malaria mesoendemic area in eastern Sudan, where the inhabitants are semi-immune to malaria, and the fever threshold of parasitemia is not above the detection level of microscopy. Thus, we hypothesized that patients with symptoms highly suggestive of cerebral malaria (CM), but aparasitemic by microscopy, may have submicroscopic parasitemia. Patients in our malaria clinic were screened by microscopy, and 120 individuals were selected for the study, including febrile patients with and without microscopically detectable parasitemia, and apparently healthy individuals. In the two former groups there were patients with severe anemia and deep coma. Polymerase chain reaction (PCR) for parasite detection and ELISA tests for measuring serum antibody levels were carried out on all blood samples. A majority of the febrile patients who were parasite negative by microscopy showed the presence of a Plasmodium falciparum infection by PCR. The occurrence of P. falciparum infection with parasitemia below the detection level of microscopy was recognized more often in patients with CM symptoms than in those with severe malarial anemia (SMA), and in older rather than younger patients. Patients clinically suspected (CS) of having CM ((CS)CM) mostly were infected with a single clone, and a large proportion of them acquired antibodies (Abs) against merozoite surface protein (MSP) antigens (Ags). The therapeutic response to quinine treatment was comparable between patients with (CS)CM and CM. In conclusion, uniquely in this setting, CM can be associated with sub-patent parasitemia; thus, a diagnostic tool more sensitive than microscopy is needed.     

Cryptococcus Neoformans Meningitis Cases Among China’s HIV-Infected Population may have been Severely Under-Reported

Cryptococcal meningitis (CM) is the leading fungal infection of the central nervous system. Globally, most CM cases have been reported from patients with compromised immunities, especially those infected with HIV. However, reports from China have shown that most CM infections were from HIV-negative, immunocompetent hosts. Here, we reviewed the published reports and found those studies were almost exclusively based on patients from hospitals associated with Chinese universities but not from specialized infectious diseases hospitals where most Chinese HIV-infected patients have been treated. Thus, we believe CM cases among China’s HIV-infected population may have been severely under-reported. Analyses of CM cases in specialized infectious diseases hospitals are needed to identify the true epidemiological pattern of CM in China.

     

Role of the CM2 protein in the influenza C virus replication cycle

CM2 is the second membrane protein of influenza C virus. Although its biochemical characteristics, coding strategy, and properties as an ion channel have been extensively studied, the role(s) of CM2 in the virus replication cycle remains to be clarified. In order to elucidate this role, in the present study we generated CM2-deficient influenza C virus-like particles (VLPs) and examined the VLP-producing 293T cells, VLPs, and VLP-infected HMV-II cells. Quantification of viral RNA (vRNA) in the VLPs by real-time PCR revealed that the CM2-deficient VLPs contain approximately one-third of the vRNA found in wild-type VLPs although no significant differences were detected in the expression levels of viral components in VLP-producing cells or in the number and morphology of the generated VLPs. This finding suggests that CM2 is involved in the genome packaging process into VLPs. Furthermore, HMV-II cells infected with CM2-deficient VLPs exhibited significantly reduced reporter gene expression. Although CM2-deficient VLPs could be internalized into HMV-II cells as efficiently as wild-type VLPs, a smaller amount of vRNA was detected in the nuclear fraction of CM2-deficient VLP-infected cells than in that of wild-type VLP-infected cells, suggesting that the uncoating process of the CM2-deficient VLPs in the infected cells did not proceed in an appropriate manner. Taken together, the data obtained in the present study indicate that CM2 has a potential role in the genome packaging and uncoating processes of the virus replication cycle.     

Altered expression of the DNA mismatch repair proteins hMLH1 and hMSH2 in cutaneous dysplastic nevi and malignant melanoma

Molecular alterations in the mismatch repair system suggest that this mechanism may be important in the evolution of cutaneous melanoma. Our current study evaluated the expression of two mismatch repair proteins, hMLH1 and hMSH2, in dysplastic nevi (DN) and cutaneous melanoma (CM). Immunohistochemical staining of these proteins was performed on 55 CM and 30 DN specimens. The staining results were divided into three groups: negative, partially positive and strongly positive. Normal adjacent skin cells served as an internal control for positive immunostaining. Altered immunoreactivity of one of the proteins was found in four (13.4%) DN and seven (12.7%) CM. Lack of staining for hMLH1 was observed in two (6.7%) cases of DN and five (9.1%) cases of CM; staining for hMSH2 was absent in two (6.7%) of the DN and two (3.6%) of the CM specimens. Partially positive staining was found in 33.3% and 53.3% for hMLH1 and hMSH2, respectively, in DN, and in 54.5% and 69.1%, respectively, in CMM. Our study shows that complete or partial loss of MMR protein expression occurs in a subset of both DN and CM and may represent a distinct pathway in the development of some DN and CM.     

鼠衣原体毒力基因的筛选和基因差异型菌株的建立

【目的】探讨鼠衣原体(Chlamydia muridarum,Cm)标准株与减毒株全基因组序列中存在的差异,筛选毒力基因并建立不同基因型的单克隆菌株,为后续致病机制的研究奠定基础。【方法】将Cm标准株G0和减毒株G28以高通量测序法进行全基因组测序,通过全基因组序列比对分析并筛选潜在的毒力相关基因;经空斑形成实验(Plaque assay)在混合菌G0和G28中大量挑取空斑,以毒力靶基因的PCR测序鉴定从G0和G28中筛选含有不同毒力基因型的单克隆菌株。【结果】全基因测序结果显示Cm G0与G28的TC0412、TC0237和TC0668基因明显不同。通过挑取空斑初步筛选了111个空斑样品,通过3个毒力基因的PCR测序鉴定最终获得了G0和G28来源的56个单克隆菌株,并根据TC0412蛋白型分成B3、D1和E1三组,每组中含有TC0237和TC0668基因差异性菌株。【结论】Cm的致病能力可能与TC0412、TC0237和TC0668基因有关,筛选获得的单克隆菌株通过分组匹配后可用于后续的毒力基因功能研究。     

中国家蚕抗菌肽ABP-CM4在E.coli中的融合表达及活性分析

参照天然抗菌肽CM4(ABP-CM4)氨基酸序列和大肠杆菌偏爱密码子,采用rPCR法获得CM4基因后重组到表达载体pET32a上,在E.coli中融合表达。表达产物以可溶性存在,经Ni2 -NTA琼脂糖亲和层析获得融合蛋白,再经甲酸切割、亲和层析和阳离子交换层析,得到纯化的重组抗菌肽。琼脂糖扩散法和液相测定法证明了纯化的抗菌肽具有抗菌活性。     

Completeness of the disease recording systems for dairy cows in Denmark, Finland, Norway and Sweden with special reference to clinical mastitis

ABSTRACT: BACKGROUND: In the Nordic countries Denmark, Finland, Norway and Sweden, the majority of dairy herds are covered by disease recording systems, in general based on veterinary registration of diagnoses and treatments. Disease data are submitted to the national cattle databases where they are combined with, e.g., production data at cow level, and used for breeding programmes, advisory work and herd health management. Previous studies have raised questions about the quality of the disease data. The main aim of this study was to examine the country-specific completeness of the disease data, regarding clinical mastitis (CM) diagnosis, in each of the national cattle databases. A second aim was to estimate country-specific CM incidence rates (IRs). RESULTS: Over 4 months in 2008, farmers in the four Nordic countries recorded clinical diseases in their dairy cows. Their registrations were matched to registrations in the central cattle databases. The country-specific completeness of disease registrations was calculated as the proportion of farmer-recorded cases that could be found in the central database. The completeness (95% confidence interval) for veterinary-supervised cases of CM was 0.94 (0.92, 0.97), 0.56 (0.48, 0.64), 0.82 (0.75, 0.90) and 0.78 (0.70, 0.85) in Denmark, Finland, Norway and Sweden, respectively. The completeness of registration of all CM cases, which includes all cases noted by farmers, regardless of whether the cows were seen or treated by a veterinarian or not, was 0.90 (0.87, 0.93), 0.51 (0.43, 0.59), 0.75 (0.67, 0.83) and 0.67 (0.60, 0.75), respectively, in the same countries. The IRs, estimated by Poisson regression in cases per 100 cow-years, based on the farmers' recordings, were 46.9 (41.7, 52.7), 38.6 (34.2, 43.5), 31.3 (27.2, 35.9) and 26.2 (23.2, 26.9), respectively, which was between 20% (DK) and 100% (FI) higher than the IRs based on recordings in the central cattle databases. CONCLUSIONS: The completeness for veterinary-supervised cases of CM was considerably less than 100% in all four Nordic countries and differed between countries. Hence, the number of CM cases in dairy cows is underestimated. This has an impact on all areas where the disease data are used.     

Cryptococcal Meningitis in Patients with Autoimmune Hemolytic Anemia

To summarize the epidemiology, clinical features, treatment, and outcome of cryptococcal meningitis (CM) in autoimmune hemolytic anemia (AIHA) patients and to provide a reference for the prevention and control of AIHA complicated with CM, we evaluated five cases of CM in patients with AIHA treated in our hospital from 2003 to 2013 and eight related foreign cases. All of the clinical isolates were Cryptococcus neoformans var. grubii and grouped into the VNI genotype and serotype A. The clinical features exhibit significant features. Headache, nausea, and fever are common symptoms of AIHA complicated with CM. The early clinical manifestations lack specificity, which may lead to delayed diagnosis and treatment. Long-term use of prednisone (≥15 mg day^?1), poor control of anemia, and splenectomy are risk factors for AIHA complicated with cryptococcal infection. The combination of intravenous amphotericin B and oral 5-fluorocytosine remains the preferred treatment for AIHA complicated with CM.     

Diagnostic Alternatives Used by Veterinary Surgeons for the Recording of Udder Diseases in the Norwegian Health Card System for Cattle

A questionnaire, in which 7 cases of udder disease were described, was distributed to 890 veterinarians in Norway. They were requested to classify the cases according to the diagnostic alternatives listed in the Norwegian Health Card System for Cattle (NHCSC). The NHCSC recordings are used for progeny testing of bulls, for disease monitoring, and for research purposes. The aim of this study was to evaluate the quality of the recordings for udder diseases. The questionnaire was answered by 633 veterinary surgeons. Four cases of clinical mastitis (CM) with abnormal secretion as well as other clinical signs of inflammation were correctly classified as CM by almost 100% of the veterinary surgeons. A cow in the final stage of lactation, showing no clinical signs except for clots in the milk, was considered not to be a clinical case by more than 25% of the veterinary surgeons. A typical case of subclinical mastitis (SM) was reported as SM by 83% of the veterinary surgeons, and as CM by 16%. A subclinical case with a recent history of clots in the secretion was classified as SM by 66% of the veterinary surgeons, but almost 40% either reported CM as their sole diagnosis or considered the case to be CM in combination with SM. Of the clinical cases, those exhibiting marked local signs of inflammation and a systemic reaction were correctly classified as acute clinical mastitis (ACM) by 96%–98% of the veterinary surgeons. In the NHCSC, the diagnostic alternatives for cases of CM are ACM and chronic clinical mastitis (CCM). One case, for which the diagnosis subacute clinical mastitis was appropriate according to standard definitions, was classified as CCM by 66%), and as ACM by 6%. Based on the information given in the questionnaire, the diagnosis for 2 of the clinical cases could have been either ACM or CCM, and for both cases each of these 2 alternatives was reported by more than 43% of the veterinary surgeons. A teat lesion, which was present together with ACM in one cow, was reported by 91% of the veterinary surgeons.     

Chromosomal mosaicism detected by karyotyping and chromosomal microarray analysis in prenatal diagnosis

To investigate the incidence and clinical significance of chromosomal mosaicism (CM) in prenatal diagnosis by G-banding karyotyping and chromosomal microarray analysis (CMA). This is a single-centre retrospective study of invasive prenatal diagnosis for CM. From 5758 karyotyping results and 6066 CMA results, 104 foetal cases with CM were selected and analysed further. In total, 50% (52/104) of foetal cases with CM were affected by ultrasound-detectable phenotypes. Regardless of whether they were singleton or twin pregnancies, isolated structural defects in one system (51.35%, 19/37 in singletons; 86.67%, 13/15 in twins) and a single soft marker (18.92%, 7/37 in singletons; 13.33%, 2/15 in twins) were the most common ultrasound anomalies. Mosaic autosomal trisomy (19.23%, 20/104) was the most frequent type, and its rate was higher in phenotypic foetuses (28.85%, 15/52) than in non-phenotypic foetuses (9.62%, 5/52). There was no difference in mosaic fractions between phenotypic and non-phenotypic foetuses based on specimen sources or overall classification. Discordant mosaic results were observed in 16 cases (15.38%, 16/104) from different specimens or different testing methods. Genetic counselling and clinical management regarding CM in prenatal diagnosis remain challenging due to the variable phenotypes and unclear significance. Greater caution should be used in prenatal counselling, and more comprehensive assays involving serial ultrasound examinations, different specimens or testing methods verifications and follow-up should be applied.     

Mastocytosis in children: clinicopathological study based on 35 cases

Immunohistochemical staining is useful in the diagnosis of bone marrow infiltration in systemic mastocytosis. However, it is not clear if antibody staining may be helpful in the diagnosis of cutaneous mastocytosis (CM). We studied the histological appearance of CM in 35 pediatric patients. Cases were assigned to three basic clinical groups: I--Urticaria pigmentosa (UP, n=29); II--Mastocytomas (n=4); and III--Diffuse Cutaneous Mastocytosis (DCM, n=2). The analysis of clinical information revealed an association between the presence of diarrhea and a higher number of cells/field. Nine doubtful cases, all of them macules, were selected based on the scarcity of mast cells (MC) and the absence or rarity of other inflammatory cells. We compared the number of cells identified in Giemsa and immunohistochemical stains in definite and doubtful cases. The intraclass correlation statistic tested the concordance between each staining method. All 9 dubious cases according to the Giemsa stain had their CM diagnosis confirmed by the immunohistochemistry analysis. The intraclass correlation between Giemsa and c-kit was good (0.7) when the number of MC was high. However, there was no correlation between the mast cells counts in the two different stains in the dubious cases. The immunohistochemistry with c-kit might make CM diagnosis easier, especially in the macular cases, when there is a lower number of MC.     

Pancreatic cancer stimulates pancreatic stellate cell proliferation and TIMP-1 production through the MAP kinase pathway

Pancreatic adenocarcinoma is characterized by an intense desmoplastic reaction that surrounds the tumor. Pancreatic stellate cells (PSCs) are thought to be responsible for production of this extracellular matrix. When activated, PSCs have a myofibroblast phenotype and produce not only components of the extracellular matrix including collagen, fibronectin, and laminin, but also matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). Since PSCs are found in the stroma surrounding human pancreatic adenocarcinoma, we postulate that pancreatic cancer could impact PSC proliferation and TIMP-1 production. Rat PSCs were isolated and cultured. Isolated PSCs were exposed to PANC-1 conditioned medium (CM) and proliferation, activation of the mitogen-activated protein (MAP) kinase pathway, and TIMP-1 gene induction were determined. Exposure to PANC-1 CM increased PSC DNA synthesis, cell number, and TIMP-1 mRNA (real-time PCR) as well as activating the extracellular-regulated kinase (ERK) 1/2. Inhibition of ERK 1/2 phosphorylation (U0126) prevented the increases in growth and TIMP-1 expression. PANC-1 CM stimulates PSC proliferation and TIMP-1 through the MAP kinase (ERK 1/2) pathway.     

大肠杆菌苯丙氨酸生物合成关键酶的串联表达

ａｒｏＧ基因编码的 ３－脱氧－２－阿拉伯庚酮糖－７－磷酸合成酶（ＤＡＨＰ　Ｓｙｎｔｈｅｔａｓｅ　ＤＳ）和 ｐｈｅＡ基因编码的分支酸变位酶／预苯酸脱水酶（Ｃｈｏｒｉｍａｔｅ ｍｕｔａｓｅ／ Ｐｒｅｐｈｅｎａｔｅ　ｄｅｈｙｄｒａｔａｓｅ,ＣＷ／ＰＤ）都是本丙氨酸合成途径中的关键酶,为了通过基因工程手段来增加本丙氨酸生物的产量,在利用高效的原核表达载体ｐＢＶ２２ ０对ｐｈｅＡ基因编码的ＣＭ／ ＰＤ 酶进行了表达的基础上,采用ＰＣＲ方法扩增了抗反馈抑制的ａｒｃＧ基因,进行克隆表达,并与ｐｈｅＡ基因串联,以ＰＲＰＬ－ａｒｏＧ－ＰＬ－ｐｈｅＡ的形式,实现了２种酶基因在大肠杆菌中的表达, ＳＤＳＰＡＧＥ 图谱显示了新增的４３ｋｕ及３５ｋｕ蛋白带,经酶活性测定ＤＳ、ＣＭ／ＰＤ酶的比活分别提高了 ４．６７倍、８０５／１０．７１倍。     

Culture of primary lung tumors using medium conditioned by a lung carcinoma cell line

Medium conditioned by the established lung tumor cell line A549 was used as a supplement to culture cells from primary solid lung tumors. Of 36 cases placed into culture, primary cells were obtained in 33 (91.7%). Of 29 cases in which subcultures were attempted, 18 (62.1%) were successful. Nine cell lines have been established by this technique to date. In growth assays, conditioned medium (CM) was found to stimulate both monolayer colony formation and growth in semi-solid medium of cells cultured from primary solid tumors. CM has been found to contain factors with the properties of both transforming growth factor alpha (TGF alpha) and insulin-like growth factor-I (IGF-I). The addition of a combination of these factors as purified peptides to basal medium at levels found in CM (0.1-0.5 ng/ml) stimulated colony formation of lung tumor cells by up to fourfold. These results indicate that secretion of growth factors may be important in tumor growth in vivo, and that use of CM may be a valuable tool for obtaining cultures from primary solid tumors.     

Estimating the impact of clinical mastitis in dairy cows on greenhouse gas emissions using a dynamic stochastic simulation model: a case study

The increasing attention for global warming is likely to contribute to the introduction of policies or other incentives to reduce greenhouse gas (GHG) emissions related to livestock production, including dairy. The dairy sector is an important contributor to GHG emissions. Clinical mastitis (CM), an intramammary infection, results in reduced milk production and fertility, increases culling and mortality of cows and, therefore, has a negative impact on the efficiency (output/input) of milk production. This may increase GHG emissions per unit of product. Our objective was to estimate the impact of CM in dairy cows on GHG emissions of milk production for the Dutch situation. A dynamic stochastic simulation model was developed to simulate the dynamics and losses of CM for individual lactations. Cows receive a parity (1 to 5+), a milk production and a calving interval (CI). Based on the parity, cows have a risk of CM, with a maximum of three cases in a lactation. Pathogens causing CM were classified as gram-positive bacteria, gram-negative bacteria, or other. Based on the parity and pathogen combinations, cows had a reduced milk production, discarded milk, prolonged CI and a risk of removal (culling and mortality) that reduce productivity of dairy cows and therefore increase GHG emissions per unit of product. Using life cycle assessment, emissions of GHGs were estimated from cradle to farm gate for processes along the milk production chain that are affected by CM. Processes included were feed production, enteric fermentation, and manure management. Emissions of GHGs were expressed as kg CO₂ equivalents per ton of fat-and-protein-corrected milk (kg CO₂e/t FPCM). Emissions of cows with CM increased on average by 57.5 (6.2%) kg CO₂e/t FPCM compared with cows without CM. This increase was caused by removal (39%), discarded milk (38%), reduced milk production (17%) and prolonged CI (6%). The GHG emissions increased by 48 kg CO₂e/t FPCM for cows with one case of CM, by 69 kg CO₂e/t FPCM for cows with two cases of CM and by 92 kg CO₂e/t FPCM for cows with three cases of CM compared with cows without CM. Preventing CM can be an effective strategy for farmers to reduce GHG emissions and can contribute to sustainable development of the dairy sector, because this also can improve the income of farmers and the welfare of cows. The impact of CM on GHG emissions, however, will vary between farms due to environmental conditions and management practices.