Efficacy of loop mediated isothermal amplification (LAMP) assay for the laboratory identification of Mycobacterium tuberculosis isolates in a resource limited setting

Current methods of TB diagnosis are time consuming and less suited for developing countries. The LAMP (loop mediated isothermal amplification) is a rapid method more suitable for diagnosis in resource limited settings and has been proposed as a viable test requiring further evaluation for use as a laboratory method as well. We evaluated two LAMP assays, using culture lysates of clinical sputum samples (from Southern India) and compared it to a proprietary multiplex PCR reverse-hybridization line probe assay (‘GenoType MTBC’ from HAIN Lifescience GmbH, Germany). The LAMP procedure was modified to suit the local conditions. The Mycobacterium tuberculosis specific LAMP assay (‘MTB LAMP’) showed sensitivity and specificity, of 44.7% and 94.4% respectively in a 60 min format, 85.7% and 93.9% respectively in a 90 min format and 91.7%, and 90.9% respectively in a 120 min format. The Mycobacteria universal LAMP assay (‘Muniv LAMP’) showed a sensitivity of 99.1%. The LAMP was shown to be a rapid and accessible assay for the laboratory identification of M. tuberculosis isolates. Initial denaturation of template was shown to be essential for amplification in unpurified/dilute samples and longer incubation was shown to increase the sensitivity. The need for modification of protocols to yield better efficacy in this scenario needs to be addressed in subsequent studies.     

Comparative growth kinetics and virulence of four different isolates of entomopathogenic fungi in the house fly (Muscadomestica L.)

Virulence (speed of kill) of a fungal entomopathogen against a particular host insect depends on biological properties of the specific isolate-host combination, together with factors such as fungal dose. How these intrinsic and extrinsic factors affect the actual pattern and extent of fungal growth invivo is poorly understood. In this study we exposed adult house flies (Muscadomestica L.) to surfaces treated with high and low doses of Beauveriabassiana (isolates BbGHA and Bb5344), Metarhiziumanisopliae (strain MaF52) and M.anisopliae var. acridum (isolate Ma189) and used quantitative real-time PCR with species-specific primers to examine the relationship between fungal growth kinetics and virulence. At the highest dose, all fungal isolates killed flies significantly faster than controls, with BbGHA, Bb5344 and MaF52 roughly equivalent in virulence (median survival time (±SE) = 5.0 ± 0.10, 5.0 ± 0.08 and 5.0 ± 0.12 days, respectively) and Ma189 killing more slowly (MST = 8.0 ± 0.20 days). At the lower dose, effective virulence was reduced and only flies exposed to isolates BbGHA and Bb5344 died significantly faster than controls (MST = 12 ± 1.36, 15 ± 0.64, 18 ± 0.86 and 21.0 ± 0.0 days for BbGHA, Bb5344, MaF52 and Ma189, respectively). Real-time PCR assays revealed that flies exposed to surfaces treated with the high dose of spores had greater spore pickup than flies exposed to the low dose for each isolate. After pickup, a general pattern emerged for all isolates in which there was a significant reduction of recovered fungal DNA 48 h after exposure followed by a brief recovery phase, a stable period of little net change in fungal sequence counts, and then a dramatic increase in sequence counts of up to three orders of magnitude around the time of host death. However, while the patterns of growth were similar, there were quantitative differences such that higher final sequence counts were recovered in insects infected with the most lethal isolates and with the higher dose. These results suggest that variation in virulence between isolates, species and doses is determined more by quantitative rather than qualitative differences in fungal growth kinetics.     

NF1 mRNA biogenesis: effect of the genomic milieu in splicing regulation of the NF1 exon 37 region

We have studied the splicing regulation of NF1 exons 36 and 37. We show that they not only require an intact exonic Splicing Enhancer (ESE) within exon 37, but also need the genomic region stretching from exons 31 to 38. Any nucleotide change in two exon 37 third codon positions disrupts the ESE. The extent of exons 36 and 37 skipping due to a mutated ESE depends on the genomic context. This is a unique example of what may be a more general phenomena involved in the tuning of pre-mRNA processing and gene expression modulation in the chromosomal setting.     

Parasitism of Ascogregarina taiwanensis and Ascogregarina culicis (Apicomplexa: Lecudinidae) in larvae of Aedes albopictus and Aedes aegypti (Diptera: Culicidae) from Manaus, Amazon region, Brazil

The aim of the study was to determine the existence of Ascogregarina spp. in larvae of Aedes albopictus and Aedes aegypti collected in urban and suburban areas of Manaus, Amazon region, Brazil. Between May 2004 and July 2005, the mid-gut of 3rd and 4th instar larvae, collected in tire traps in six neighborhoods of Manaus, was examined for the presence of trophozoites of Ascogregarina. Coexistence of Ae. albopictus larvae infected by A. taiwanensis, and Ae. aegypti larvae by A. culicis, was detected in traps in the field. The percentage of Ae. albopictus larvae infected by A. taiwanensis ranged from 21% to 93.5% and of Ae. aegypti larvae infected by A. culicis from 22% to 95%. The mean infection intensity was similar in both species of Aedes. In traps located in Mauazinho, the replacement of Ae. aegypti by Ae. albopictus larvae was observed. In Manaus, Ae. albopictus larvae were parasitized by A. taiwanensis, and Ae. aegypti larvae by A. culicis. Infection rates were high when the species of Aedes were found separately.     

Comparative virulence phenotypes and molecular genotypes of Puccinia striiformis f. sp. tritici, the wheat stripe rust pathogen in China and the United States

Stripe rust (yellow rust) of wheat, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases in both China and the United States. The Chinese and US populations of the stripe rust fungus were compared for their virulence phenotypes on wheat cultivars used to differentiate races of the pathogen in China and the US and molecular genotypes using simple sequence repeat (SSR) markers. From 86 Chinese isolates, 54 races were identified based on reactions on the 17 Chinese differentials and 52 races were identified based on the 20 US differentials. The selected 51 US isolates, representing 50 races based on the US differentials, were identified as 41 races using the Chinese differentials. A total of 132 virulence phenotypes were identified from the 137 isolates based on reactions on both Chinese and US differentials. None of the isolates from the two countries had identical virulence phenotypes on both sets of differentials. From the 137 isolates, SSR markers identified 102 genotypes, of which 71 from China and 31 from the US. The virulence data clustered the 137 isolates into 20 virulence groups (VGs) and the marker data clustered the isolates into seven molecular groups (MGs). Virulence and SSR data had a low (r = 0.34), but significant (P = 0.01) correlation. Principal component analyses using either the virulence data or the SSR data separated the isolates into three groups: group a consisting of only Chinese isolates, group b consisting of both Chinese and US isolates and group c consisting of mostly US isolates. A neighbour-joining tree generated using the molecular data suggested that the P. striiformis f. sp. tritici populations of China and the US in general evolved independently.     

Eurytrema pancreaticum: the in vitro effect of praziquantel and triclabendazole on the adult fluke

The efficacy and tolerance of the 80 microg/ml praziquantel (PZQ) and 40 microg/ml triclabendazole (TCZ) against adult stage Eurytrema pancreaticum in vitro were investigated at 3, 12, and 15 h incubation. Motility of the flukes and histopathological changes were studied. Sudden paralysis and death were observed after exposed to PZQ as early as 3h incubation. In contrast, the TCZ treated flukes showed active mobility at all intervals. By light microscopic examination, severe damages in various organs such as tegument, muscle, and testes were observed early at 12h incubation of these drugs. PZQ caused more severe damage to flukes than TCZ. There were vigorous contraction of musculature, progressive shrinkage of circular and longitudinal muscles, vacuolization and disintegration of the tegument disrupting the worms' outer surface including detachment of spines in the PZQ treatment. The cells in testes were slightly increased in size and followed by degeneration leaving several hollow spaces. The uterus and vitelline glands remained unaffected. The direct observation of the fluke motility and light microscopic study highly suggested that PZQ was more effective than TCZ treatment for the eurytremiasis infection.     

Cryptosporidium parvum: the course of Cryptosporidium parvum infection in C57BL/6 mice co-infected with the nematode Heligmosomoides bakeri

The effects of Heligmosomoides bakeri infection on the course of a concurrent Cryptosporidium parvum infection were studied in C57BL/6 mice. Mice were initially infected with 80 L₃ of H. bakeri and then challenged with 10⁴ oocysts of C. parvum, administered during the patent period of the nematode infection (28 day post H. bakeri infection). The number of C. parvum oocysts excreted in the feces and the number of adult H. bakeri in the small intestine were monitored during the experiment. Concurrent H. bakeri infection resulted in a prolonged course of infection with C. parvum. The intensities of both parasite infections were higher in co-infections. We also investigated the cellular immune response at 14 and 42 days post infection C. parvum. During infection with C. parvum there was an increase in production of IFN-γ and IL-12 but co-infection with H. bakeri inhibited IFN-γ secretion. The present study is the first to demonstrate that infection with H. bakeri markedly exacerbates the intensity of a concurrent C. parvum infection in laboratory mice and also affects immune effectors mechanisms in co-infection with H. bakeri.     

秦岭火地塘林区油松和华山松林乔木层净生产力与气候因子的关系

以秦岭火地塘林区油松(Pinus tabulaeformis)和华山松(Pinus armandi)林为研究对象,以其生物量及树高-胸径模型为基础,运用树木年轮宽度方法推算出1973年至2011年生物量和生产力年际动态,并通过相关分析和多元逐步回归分析探讨了油松和华山松林乔木层净生产力与温度、降水之间的关系。结果显示,该林区油松林和华山松林乔木层生物量39a间增长迅速,分别从1973年的15.32 t/hm2和7.53 t/hm2增长到2011年的175.97 t/hm2和130.98 t/hm2,平均年净生产力分别为4.18 t hm-2a-1和3.20 t hm-2a-1,油松林乔木层生物量和生产力均高于华山松林;气候分析表明年净生产力与降水关系不明显,与温度关系较为密切,随气温升高呈波动上升趋势:单月气候因子中上年7月温度、当年7月温度与油松林乔木层净生产力显著正相关,上年7月温度与华山松林乔木层净生产力显著正相关;油松林乔木层净生产力动态变化主要受1—7月平均温度影响,华山松林主要受5—7月平均温度影响;油松林生产力与温度因子的相关性高于华山松林。两种林型的生物量和生产力差异是由油松和华山松生物学特性所致。     

Change of color and components in sepals of chameleon hydrangea during maturation and senescence

The sepal color of a chameleon hydrangea, Hydrangea macrophylla cv. Hovaria™ ‘Homigo’ changes in four stages, from colorless to blue, then to green, and finally to red, during maturation and the senescence periods. To clarify the chemical mechanism of the color change, we analyzed the components of the sepals at each stage. Blue-colored sepals contained 3-O-sambubiosyl- and 3-O-glucosyldelphinidin along with three co-pigments, 5-O-p-coumaroyl-, 5-O-caffeoyl- and 3-O-caffeoylquinic acids. The contents of glycosyldelphinidins decreased toward the green-colored stage, with a coincident increase in the number of chloroplasts. During the last red colored stage, the two species of 3-O-glycosyldelphinidin almost disappeared, and another two anthocyanins, 3-O-sambubiosyl- and 3-O-glucosylcyanidin, increased in amounts. Mixing of 3-O-glycosylcyanidins, co-pigments, and Al³⁺ in a buffered solution at pH 3.0-3.5 gave not a blue, but a red, colored solution that was the same as that of the sepal color of the 4th stage. Sepals of hydrangea grown in an highland area also turned red in autumn, and contained the same cyanidin glycosides. The red coloration of the hydrangea during senescence was due to a change in anthocyanin biosynthesis.     

The effect of temperature and duration of exposure of potato tuber moth (Lepidoptera: Gelechiidae) in infested tubers to the biofumigant fungus Muscodor albus

The endophytic fungus, Muscodor albus produces several volatile compounds (alcohols, esters, ketones, acids and lipids) that are biocidal for a range of organisms including plant pathogenic bacteria and fungi, nematodes and insects. We studied the effects of these volatiles on 3-day-old potato tuber moth larvae within infested tubers inside sealed chambers. The length of exposure to M. albus significantly affected mortality of larvae, calculated as percentage of larvae failing to survive to the adult stage. Exposure durations of 3, 7, or 14 days at 24 degrees C followed by incubation in fresh air at 27 degrees C until emergence resulted in mortalities of 84.2, 95.5 and 99.6%, respectively. However, the longer exposures also resulted in increased levels of carbon dioxide (CO(2)) that are unacceptable for tuber storage. Effects of M. albus on larval survival was also monitored at 10, 15 and 24 degrees C, using an exposure duration of 7 days followed by incubation in clean air at 27 degrees C until emergence. Mortality of larvae was sharply reduced at the lower temperatures resulting in 50.8, 76.8, and 95.4% mortality, respectively. Tuber storage conditions, especially cooling rates, are discussed with respect to using M. albus as a fumigant without simultaneously producing unacceptable (for tuber storage) levels of CO(2).