Rapid screening of toxigenic vibrio cholerae O1 strains from south Iran by PCR-ELISA

BACKGROUND: The ability to sensitively detect Vibrio cholera with PCR-ELISA method represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen. The aim of this research is to evaluate the suitability of a PCR-enzyme-linked immunosorbent assay for sensitive and rapid detection of V. cholera O1. METHODS: The 398-bp sequence of a gene that codes for the cholera toxin B subunit was amplified by PCR. The digoxigenin-labeled amplified products were coated on microplates and detected by ELISA. The PCR product was also hybridized with biotin labelled probe and detected by ELISA using streptavidin. RESULTS AND CONCLUSION: The specificity of the PCR was determined using 10 bacterial strains and 50 samples from south Iran. The detection limit was 0.5 pg of the genomic DNA and five bacterial cells. Adaptation of PCR into PCR-ELISA assay format facilitates specific and sensitive detection and diagnosis of human cholera disease. We conclude that this PCR-ELISA is a diagnostic method that specifically detects toxin genes in V. cholera O1 strains. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.     

Prevalence and intensity of <Emphasis Type="Italic">Oestrus ovis</Emphasis> in sheep of Shiraz,southern Iran

Two thousand and two heads obtained from slaughtered sheep at the Fars abattoirs (Shiraz, Southern Iran) between April 2006 and April 2007 were examined for the presence of Oestrus ovis larvae. Of the total heads, 995 (49.7%) were infested with O. ovis larvae. O. ovis larvae were observed in both sexes and all age groups in each season of the year. A total of 6264 larvae were collected. The overall larval intensity for the infested sheep was 6.3, with 3.9 in spring, 5.3 in summer, 5.9 in autumn and 7.8 in winter. Prevalence ranged from 23.3% in spring to 80% in winter. Increased infestation was observed in older animals.     

Chromosome 1D as a possible location of a gene (s) controlling variation between wheat (Triticum aestivum L.) varieties for carbon isotope discrimination (Δ) under water-stress conditions

Cytoplasmic male-sterility (CMS) in pigeonpea has been reported when some wild relatives of pigeonpea were crossed as the female parent with cultivated types as the male parent. In this paper we report a new source of CMS developed by using the cultivated pigeonpea as the female parent and one of its wild relative Cajanus acutifolius as the pollen donor. This is the first report in pigeonpea where CMS has been developed using the cytoplasm of cultivated pigeonpea. Several pure line cultivars of pigeonpea restored pollen fertility whereas cv. HPL 24 partially maintained male-sterility. The wild species C. acutifolius used as one of the parents, maintained complete sterility. Cytological analysis revealed that both in male-sterile as well as the fertile floral buds, meiosis proceeded normally till the tetrad stage. However in the male-sterile genotypes during the formation of tetrads, the pollen mother cell (PMC) wall did not dissolve to release the tetrads unlike in the fertile genotypes and this major event was found to be responsible for male-sterility.     

Essential oils as reducing agents of cabbage peroxidase

Based on the antioxidant properties of essential oils, the effect of thyme, coriander, and rosemary was evaluated on the reduction of peroxidase in white cabbage “Brassica oleraceae var.capitata f. alba” and red cabbage “B. oleraceae var. capitata f. rubra”. To do this, pure, 50, 75, 100 and 200 μl/100 ml concentrations of essential oils were applied in vivo and in vitro of cabbages. Antioxidant activity of the essential oils was compared with that of ascorbic acid. Results revealed that the highest percentage of antioxidant activity was achieved in vitro using a pure concentration of coriander (52.65%) and rosemary (54.64%), and in vivo applying thyme (60.07%) of white cabbage. Applying in vitro pure concentration of coriander (46.39%) and 50 μl/100 ml of rosemary (41.64%), and in vivo 75 μl/100 ml of thyme (42.64%) in red cabbage showed high antioxidant activity. A high reduction of peroxidase was obtained in vitro using of essential oils in red and white cabbages.     

Quantitative real-time PCR and culture examination of Mycobacterium avium subsp. paratuberculosis at farm level

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in ruminants and may contribute to Crohn's disease in humans. The aim of this study was to determine the occurrence and quantity of MAP in cattle feces and milk in the Iranian context. In addition, we evaluated the effect of cattle age as well as farming system as risk factors contributing to MAP load. For this, a total sample of 373 consisting of 150 cattle feces (CF), 150 individual cow's milk (ICM), as well as 73 bulk-tank milk (BTM) was collected randomly and regardless of the cattle health status. The samples were assayed using F57 quantitative real-time PCR (qPCR) and culture method. According to the results of qPCR which was found ∼10 times more sensitive than culture assay, MAP was detected in 68.66% (103/150) of the CF, 12% (18/150) of the ICM and 52.05% (38/73) of the BTM samples. In contrast to the previous reports, the quantity of MAP in the BTM (2.03–5.97 log cfu/50 ml) was statistically (p < 0.01) higher than the ICM (0.90–1.97 log cfu/50 ml). Data suggested a direct relation (p < 0.01) between the cattle age and the quantity of MAP in the CF samples, while the relation was not statistically significant (p > 0.05) for the ICM. In addition, MAP load in the BTM samples obtained from traditional farms was significantly (p < 0.01) higher than that of the industrial ones, while the differences in CF and ICM was not significant (p > 0.05).     

Localization of herpes simplex virus type 1 DNA in latently infected BALB/c mice neurons using in situ polymerase chain reaction

Background: Herpes simplex virus type-1 (HSV-1) establishes a lifelong latent infection in neurons following primary infection. The existence of latent HSV-1 DNA in the trigeminal ganglia of infected BALB/c mice was examined using a direct in situ PCR technique, based on Digoxigenin-11-dUTP detection system with anti-digoxigenin-peroxidase and 3,3''-diaminobenzidine (DAB) substrate. Methods: Eight-week-old male BALB/c mice were inoculated via the eye by 10⁴ plaque forming unit of wild type Iranian isolates of HSV-1. After establishment of latency, trigeminal ganglia were removed and examined using in situ PCR to detect HSV-1 genome. Finally, the results of in situ PCR were verified by a two-round PCR method, using amplification cocktail of in situ reaction, as a template for a conventional gel base PCR. Results and Conclusion: The results suggest that a direct in situ PCR method using a peroxidase and DAB detection system is a useful means for detection of latent HSV-1 DNA in the latently infected ganglia. Key Words: Herpes simplex virus-1, Latency, In situ PCR, two-round PCR, Trigeminal ganglia     

Exogenous Spermidine Affected Leaf Characteristics and Growth of Common Bean Under Water Deficit Conditions

In order to investigate the effects of exogenous spermidine on water limitation-induced damage on common bean (Phaseolus vulgaris L.) genotypes, a field experiment was conducted over two growth seasons. The experiment was arranged as a split-split plot design and three replications. Treatments were three water stress levels (non-stress, moderate and severe stress), three bean genotypes (Akhtar, Pak and COS12) and two spermidine levels (control and foliar application). The results showed that water stress reduced markedly leaf relative water content, SPAD values, specific leaf area, leaf area index, plant height, relative growth rate, shoot dry weight and grain yield; however, increased leaf density, leaf relative electrolyte leakage, stomatal density, leaf angle and leaf temperature of bean genotypes. Spermidine application increased relative water content, SPAD values, leaf area, leaf area index, shoot dry weight and grain yield. Overall, exogenous spermidine usage as a free radical scavenger counteracted deleterious water deficit effects.