Assessing calves as carriers of Cryptosporidium and Giardia with zoonotic potential on dairy and beef farms within a water catchment area by mutation scanning

In the present study, we undertook a molecular epidemiological survey of Cryptosporidium and Giardia in calves on three dairy and two beef farms within an open drinking water catchment area (Melbourne, Australia). Faecal samples (n = 474) were collected from calves at two time points (5 months apart) and tested using a PCR‐based mutation scanning‐targeted sequencing phylogenetic approach, employing regions within the genes of small subunit (SSU) of ribosomal RNA (designated partial SSU), 60 kDa glycoprotein (pgp60) and triose phosphate isomerase (ptpi) as genetic markers. Using partial SSU, the C. bovis, C. parvum, C. ryanae and a new genotype of Cryptosporidium were characterised from totals of 74 (15.6%), 35 (7.3%), 37 (7.8%) and 9 (1.9%) samples, respectively. Using pgp60, C. parvum genotype IIa subgenotype A18G3R1 was detected in 29 samples. Using ptpi, G. duodenalis assemblages A and E were detected in totals of 10 (2.1%) and 130 (27.4%) samples, respectively. The present study showed that a considerable proportion of dairy and beef calves in this open water catchment region excreted Cryptosporidium (i.e. subgenotype IIaA18G3R1) and Giardia (e.g. assemblage A) that are consistent with those infecting humans, inferring that they are of zoonotic importance. Future work should focus on exploring, in a temporal and spatial way, whether these parasites occur in the environment and water of the catchment reservoir.     

Phytochemical investigation of Euphorbia trigona

Euphorbia species (Family: Euphorbeaceae) have wide applicability in traditional medicines and biofuel sector, and are also rich sources of secondary metabolites, especially terpenoids, attributed with various pharmacological properties. Though 82 Euphorbia species are reported in India, most of the species are uninvestigated for their constituents or potential utilities. Present study reports the isolation and characterization of chemical constituents, estimation of major triterpenoids using a validated HPTLC method and cytotoxicity of plant and latex extracts of Euphorbia trigona. As the secondary metabolites epi-friedelinyl acetate, 3β-friedelinol, taraxerol, rhoiptlenone, stigmasterol and stearic acid were isolated and characterized from E. trigona. HPTLC estimation showed 3β-friedelinol at 0.91 ?± ?0.11% and taraxerol at 1.45 ?± ?0.12% was present in E. trigona plant powder (dry weight). Plant and latex extracts were found non toxic towards normal cell line H9C2 (cardiac myoblasts) and showed insignificant cytotoxic effects towards HeLa (cervical cancer cell line) upto 100 ?μg/ml concentration.     

Cross-mixing study of a poisonous Cestrum species,Cestrum diurnum in herbal raw material by chemical fingerprinting using LC-ESI-QTOF-MS/MS

Poisonous plants are widely distributed and may have risk of phytotoxicity upon mixing with medicinal plants. Several species of Cestrum genus are poisonous and linked with many serious health issues. In the present study, cross-mixing of a toxic plant, Cestrum diurnum with morphologically resembling medicinal plant, Adhatoda vasica was studied using chemical fingerprinting approach. LC-ESI-MS/MS tool was used to develop the chemical fingerprints of three toxic species of Cestrum, including, C. diurnum, C. nocturnum and C. parqui. Total forty-three compounds were identified using high-resolution LC-ESI-MS/MS data comparison. Chemometric analyses were done to compare the distribution of identified compounds present in these Cestrum species. One of the identified compounds, nornicotine (a toxic compound) was also quantified using LC-IT-MS/MS. Adulteration study was conducted by mixing toxic C. diurnum in A. vasica with various ratios (w/w) and five differentiable compounds were identified to detect the adulteration. The method was able to detect up to the limit of 5% mixing of toxic C. diurnum. Moreover, cytotoxicity of the methanolic extracts of these three species were also studied on normal human PBMC (peripheral blood mononuclear cells) and all found to be toxic, while the C. nocturnum showed the highest level of toxicity with the IC₅₀ 12.5 μg/mL.     

Genetic differentiation and subspecies development of the giant panda as revealed by DNA fingerprinting

Over the last 100 years giant pandas (Ailuropoda melanoleuca) have been separated into six completely isolated mountain ranges. DNA fingerprinting revealed different differentiation patterns in giant pandas, including early-stage, late-stage, recent divergence and recent genetic depression. A separation around 10 000 years ago resulted in highly significant differences in DNA fingerprints and morphological characteristics between Qinling and Sichuan populations. Supported by morphological differences, the genetic data were used to classify the Qinling population as a new subspecies, A. m. qinlingensis, while the Sichuan populations were classified into the original subspecies, A. m. melanoleuca. Thus, the Qinling population deserves management as a separate unit. In the Sichuan populations, two management units were defined, including Qionglai-Minshan and Daxiangling-Xiaoxiangling-Liangshan. Our data suggest urgent measures are needed to establish green corridors between subpopulations in each mountain range to increase gene flow and genetic variation to ensure long-term survival.     

Highly sensitive non‐isotopic restriction endonuclease fingerprinting of nucleotide variability in the gp60 gene within Cryptosporidium species,genotypes and subgenotypes infective to humans,and its implications

The high‐resolution analysis of genetic variation has major implications for the identification of parasites and micro‐organisms to species and subspecies as well as for population genetic and epidemiological studies. In this study, we critically assessed the effectiveness of a PCR‐based restriction endonuclease fingerprinting (REF) method for the detection of mutations in the 60 kDa glycoprotein gene (gp60) of Cryptosporidium, a genus of parasitic protists of major human and animal health importance globally. This gene displays substantial intraspecific variability in sequence, particularly in a TCA (perfect and imperfect) microsatellite region, is present as a single copy in the nuclear genome and is used widely as a marker in molecular epidemiological studies of Cryptosporidium hominis and C. parvum, the two predominant species that infect humans. The results of this study demonstrated an exquisite capacity of REF to detect nucleotide variability in the gp60 gene within each of the two species. The differentiation of genotypes/subgenotypes based on REF analysis was supported by targeted sequencing, allowing the detection of levels of variation as low as a single‐nucleotide transversion for amplicons of ∼1 kb in size. The high‐throughput potential and relatively low‐cost of REF make it a particularly useful tool for large‐scale genetic analyses of C. hominis and C. parvum. REF could also be utilized for comparative surveys of genetic variability across large nuclear genomic regions. Such analyses of Cryptosporidium in clinical and environmental samples by REF have important implications for identifying sources of infection, modes of transmission and/or possible infectivity to humans, thus assisting in the surveillance and control of cryptosporidiosis. Given its excellent mutation detection capacity, REF should find broad applicability to various single‐copy genes as well as a wide range of other protozoan and metazoan parasites. (The nucleotide sequences reported in this article are available in the GenBank database under accession numbers GU214343–GU214371).     

Quantitation of trace levels of phenyltin compounds using HPTLC

Mixtures of tetraphenyltin, triphenyltin chloride, diphenyltin dichloride, and monophenyltin trichloride were resolved using high performance thin layer chromatography on silica gel with retention factor values of 0.80, 0.35, 0.20 and 0.01, respectively. Inorganic tin impurities were strongly adsorbed and did not migrate from the origin. Diphenyltin dichloride, monophenyltin trichloride, and inorganic tin components reacted with morin to produce fluorescent complexes. Postdevelopment exposure of the plate to ultraviolet light photodegraded the organic components which, after morin treatment, exhibited greater fluorescence than the organotins. This photolysis technique permitted the visualization of the otherwise non-fluorescent tetraphenyltin and weakly fluorescent triphenyltin spots.The components were quantitated using scanning densitometry. The working range varied from a maximum of 222 ng to a minimum of 1 ng, depending on the component and the excitation wavelength chosen. Thirty standards each containing five components were spotted, developed, derivatized, and scanned three times to produce 480 pieces of data within four hours. Calibration curves showed an instrumental error of 1.5% relative standard deviation units, and a spotter and intraplate variation of 9.0% relative standard deviation units. The inherent multiplicity of high performance thin layer chromatography allows for multiple sampling and analysis, thereby yielding significantly increased precision and high sample throughput. The chromatography and detection of butyltins and cyclohexyltins were also examined.     

Characterization of Antioxidant Activity of Momordica Charantia Fruit by Infrared-Based Fingerprinting

Momordica charantia is widely consumed edible fruit. The food and pharmaceutical industries use it as a natural antioxidant. However, the quality control of M. charantia-based medicinal products is questionable due to the complexity of metabolites in this fruit. Hence, this study has developed a statistical model in predicting the antioxidant value through the 2, 2-diphenyl-1 picrylhydrazyl radical scavenging activity and ferric reducing antioxidant power based on infrared spectroscopy with attenuated total reflectance. This technique was reliably used for quality control. Six ethanol extracts (0, 20, 40, 60, 80, and 100% in water) of this plant’s fruit were prepared. The radical scavenging and ferric reducing antioxidant power activities were measured and the chemical profiling of the extracts was fingerprinted by infrared spectroscopy between 4,000 and 600?cm^?1 at a resolution of 4?cm^?1. Statistical analysis was developed by correlating the bioactivity and infrared spectra of each extract using orthogonal partial least square discriminant analysis. The C–N, C?O, C–O, C–H, and OH infrared signals were positively correlated with biological activity. The antioxidant activity of the fruit of M. charantia may be due to the presence of several antioxidants that work synergistically.     

Metabonomic study on women of reproductive age treated with nutritional intervention: screening potential biomarkers related to neural tube defects occurrence

Nutritional intervention is effective in reducing the risk of neural tube defects (NTDs). To determine the effects of nutritional supplementation on human metabolism, a metabonomic study was carried out on 96 women of reproductive age. Subjects with nutritional intervention were given fortified wheat flour (containing folic acid, vitamin B₁, vitamin B₂, ferric sodium edetate and zinc oxide) for 8 months. Serum metabolic fingerprinting was detected via ultraperformance liquid chromatography in tandem with time‐of‐flight mass spectrometry (UPLC‐TOF MS), and data acquired was processed by multivariate statistical analysis. The result revealed a significant difference between the control and intervention group. Twenty potential biomarkers, including fructose 6‐phosphate, sphingosine 1‐phosphate, docosahexaenoic acid and hexadecanoic acid, were located and identified by the accurate mass measurement of TOF MS. These compounds are believed to be functionally related to anti‐oxidative competence in vivo. In conclusion, metabonomics study is a valuable approach in exploring the effect mechanism of nutritional intervention on NTD prevention. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of an HPTLC‐based diagnostic method for invasive aspergillosis

A rapid, sensitive and specific high‐performance thin‐layer chromatographic (HPTLC) method was developed and validated for determination of gliotoxin in Aspergillus infected immunocompromised patients with invasive aspergillosis (IA). Densitometric analysis of gliotoxin was carried out in the absorbance mode at 254 nm after single‐step extraction with chloroform. The method uses TLC aluminum plates pre‐coated with silica gel 60F‐254 as a stationary phase and toluene–isoamyl alcohol–methanol (10:0.5:0.5, v/v/v) as mobile phase, which gives compact spot of gliotoxin (R_f = 0.51). The calibration curve was linear (r² ≥ 0.994) between peak area and concentration in the tested range of 100–1000 ng spot^?1 with minimum detectable range 0.025 ng μ^?1 of serum sample. The mean ± SD value of slope and intercept of the standard chromatogram of gliotoxin were found to be 523.2 ± 1.555635 and 915.8 ± 30.68843, respectively. The developed method is simple, rapid, precise and less costly than earlier diagnostic methods, and different serum samples can be run on a single TLC plate for comparative analysis. The proposed method can be used to analyze gliotoxin in patient serum for easy, rapid and cost‐effective diagnosis of IA. Copyright © 2009 John Wiley & Sons, Ltd.     

Stability‐indicating HPTLC method for quantitative estimation of silybin in bulk drug and pharmaceutical dosage form

In the present study a novel stability‐indicating high‐performance thin‐layer chromatography (HPTLC) method for quantitative determination of silybin in bulk drug and nanoemulsion formulation has been developed and validated on silica using solvent chloroform–acetone–formic acid (9 : 2 : 1 v/v/v) (R_f of silybin 0.46 ± 0.05) in the absorbance mode at 296 nm. The method showed a good linear relationship (r² ± 0.999) in the concentration range 25–1500 ng per spot. It was found to be linear, accurate, precise, specific, robust and stability‐indicating and can be applied for quality control and standardization of several multi‐component hepatoprotective formulations as well as for stability testing of different dosage forms. The method proposed was also used to investigate the kinetics of acidic and alkaline degradation processes by quantification of drug at different temperature to calculate the activation energy and half‐life for silymarin degradation. Copyright © 2009 John Wiley & Sons, Ltd