Optimization of a species-specific polymerase chain reaction assay for identification of Pentatrichomonas hominis in canine fecal specimens

OBJECTIVE: To determine the optimum reaction conditions and detection limits of PCR assay for identification of Pentatrichomonas hominis in DNA extracted from canine feces. SAMPLE POPULATION: DNA extracted from feces of 4 dogs with diarrhea from which trichomonads were observed, 81 dogs that had feces submitted to a diagnostic laboratory, and 19 dogs residing in a laboratory animal facility. PROCEDURES: Optimum reaction conditions and absolute and practical detection limits of 2 P hominis 18S species-specific primer pairs were determined by use of an in vitro cultivated canine isolate of P hominis in the presence and absence of canine feces. The optimized PCR assay was applied to amplification of P hominis 18S rRNA genes from DNA extracted from the feces of dogs. RESULTS: Under optimized conditions, a primer pair was identified as able to detect as few as 1 P hominis organism/180-mg fecal sample. The PCR assay identified P hominis in diarrheic feces of 4 dogs in which trichomonads were seen by light microscopy. The P hominis genes were not amplified from other fecal samples examined. CONCLUSIONS AND CLINICAL RELEVANCE: Molecular identification of P hominis in feces of 4 dogs with trichomonosis and diarrhea reported here validates the identity of this species in such infections. Sensitive and specific PCR amplification of P hominis 18S rRNA genes from DNA extracted from feces will directly facilitate studies examining pathogenicity of this trichomonad and enable differentiation of P hominis from other known or novel species of trichomonads that may infect the gastrointestinal tract of dogs.     

Hydrosedimentology of nested subtropical watersheds with native and eucalyptus forests

Purpose

Information on the effects of eucalyptus forests on hydrosedimentological processes is scarce, particularly at the catchment scale. Monitoring and mathematical modeling are efficient scientific tools used to address the lack of information for natural resource management and the representation and prediction of those processes. This study evaluates the effects of eucalyptus cultivation on hydrosedimentological processes in watersheds and to use the Limburg soil erosion model (LISEM) to represent and predict hydrological processes.

Material and methods

The study was conducted in two forested watersheds: the main watershed (94.46 ha) and a nested sub-watershed (38.86 ha), both cultivated with eucalyptus and residual riparian native forest, located in southern Brazil. Hydrosedimentalogical monitoring was conducted from 16th February 2011 to 31st December 2012, and LISEM model calibrations were performed on the bases of six storms events.

Results and discussion

The sediment yield for 2011 was 41.6 Mg km^?2 and 38.5 Mg km^?2 for the watershed and sub-watershed, respectively. An extreme event in 2012 provided greater sediment yield for the sub-watershed (99.8 Mg km^?2) than that for the watershed (51.7 Mg km^?2). Rainfall events with a greater maximum intensity generated rapid discharge and suspended sediment concentration responses in the sub-watershed due to the smaller drainage area and steeper landscape. In the main watershed, the accumulation of flood waves occurred for most events, with less steep hydrographs, and a later occurrence of the discharge peak after that of the sub-watershed. The LISEM adequately reproduced the peak discharge and runoff for the calibrated events; however, the peak time and the shape of the hydrograph were not adequately represented.

Conclusions

The hydrosedimentological patterns of the watershed and sub-watershed, both cultivated with eucalyptus, was characterized by sedimentographs preceding hydrographs during rainfall–runoff events where scale effects occur, with maximum discharge and specific sediment yield greater in the watershed than that in the sub-watershed. Empirical models based on hydrologic variables may be used for estimating the suspended sediment concentration and sediment yield. Therefore, LISEM may be used for the prediction of hydrological variables in these forested watersheds.     

Increase in phosphorylase activity during cold-induced sugar accumulation in potato tubers

Summary The accumulation of reducing sugars, sucrose and hexose phosphates in cv. Bintje and genotype KW77-2916 during storage at 2, 4, or 8°C was studied in relation to several catalytic activities. Bintje tubers accumulated sugars during storage at 2 or 4°C, whereas KW77-2916 showed reduced cold-sweetening at 2°C. The increase in glucose 6-phosphate and sucrose occurred concurrently and preceded the increase in reducing sugar concentration. Phosphorylase activity showed a strong interaction with temperature, storage duration and sugar accumulation in both genotypes. Invertase activity increased in Bintje concomitantly with the increase in reducing sugars, but this effect was less obvious in KW77-2916. The activities of other glycolytic and Krebs cycle enzymes showed no obvious correlation with sugar accumulation. It is suggested that the increase in phosphorylase activity acts as a triggering event in the sweetening of potato tubers during cold storage.     

Barley roots are not constrained to large-sized biopores in the subsoil of a deep Haplic Luvisol

Using the profile wall method, we determined the root-length density (RLD) of barley roots growing in large-sized biopores (diameter >2 mm) and in the bulk soil of a Haplic Luvisol down to 200 cm of soil depth. The maximum bulk density in the soil profile (1.52 g?cm^?3) was recorded in the Bt horizon (41–115 cm of soil depth). The proportion of RLD in biopores over the total RLD increased with increasing soil depth down to the 45–75 or 75–105 cm of soil layer but then decreased again in deeper soil. In contrast to earlier investigations, the maximum percentage of RLD in biopores recorded in this study was only 25 %. Root sampling from individually dissected biopores confirmed that roots did not predominantly grow in biopores. Results suggest that roots can use biopores as preferred pathways for growth through rather compact soil layers, whereas they can possibly leave the biopore and re-enter the bulk soil in deeper, less compact layers.     

Species selection for reforestations: what happens with historic local extinctions and habitat protection zones? A case study in the Cantabrian Range

Species selection for reforestations can be challenging in habitat protection zones, especially in human transformed landscapes, where nativeness of some species is not easily determined and yet is crucial for knowing whether a species can be used or not. We try to determine whether Pinus uncinata Ramond ex DC was present in the Cantabrian Range (Spain) and whether it could have disappeared recently due to anthropogenic causes. Plant morphology and needle anatomy were characterized in Pinus sylvestris L. and P. uncinata 2-year-old seedlings. An analysis of variance was used for selecting morphological markers capable of discriminating both species. These markers permit the comparison of seedlings coming from the highest zone of Puebla de Lillo relict pinewood (Cantabrian Range) with reference material from Castillo de Vinuesa (Iberian Range). This comparison was firstly made with two multivariate methods, a principal component analysis and a multivariate discriminant analysis. Subsequently, this was corroborated with the detection of a species-specific chloroplast DNA marker. The differences found between both species reflect the better adaptation of P. uncinata to typical highland environment. Several Puebla de Lillo seedlings had P. uncinata type morphology and haplotype, suggesting the presence of this species in the Cantabrian Range until recently. This discovery changes the current interpretation of the zone’s vegetation, enhancing the natural presence of highland open pinewoods. However, human activities (fire and livestock grazing) have driven them to extinction and have favoured the contemporary expansion of heathlands. Based on this P. uncinata case, we discuss how local extinctions can affect management and conservation policies negatively.     

Dermatophytes isolated from domestic and feral animals

In work over a period of 8 years, dermatophytes were recovered from 12 animal species in the North Island of New Zealand. A total of 552 dermatophytes were isolated and belonged to the Microsporum (6 species) and Trichophyton (6 species) genera.

Some unusual isolations are reported: Microsporum canis and Trichophyton mentagrophytes var. mentagrophytes were recovered from calves; Microsporum distortum from a dog, which was the first known isolation from an animal in the North Island; four horses, from the same stable, yielded Microsporum equinum which has not previously been recorded in this country; Trichophyton erinacei was recovered from lesions on a cat which is the first report of the dermatophyte from an animal other than the dog, hedgehog or man; Trichophyton equinum var. autotrophicum was a rare isolation from a dog.

Species affinity was demonstrated with the dermatophytes M. canis; M. nanum; M. equinum; T. equinum; and T. verrucosum. These zoophilic species appear to be passed between individuals of the same species, with occasional infection in man and other animal species. T. mentagrophytes var. mentagrophytes had a wider distribution, being isolated from dogs, cats, guinea-pigs and rats. The infection in rats was subclinical.

The geophilic species M. cookei, T. ajelloiand and T. terrestre were recorded but not regarded as being pathogenic. M. gypseum was significant in cases involving dogs, horses and a cat, as arthrospores were seen invading the affected hairs.     

Radiation up-regulates the expression of VEGF in a canine oral melanoma cell line

To evaluate radiosensitivity and the effects of radiation on the expression of vascular endothelial growth factor (VEGF) and VEGF receptors in the canine oral melanoma cell line, TLM 1, cells were irradiated with doses of 0, 2, 4, 6, 8 and 10 Gray (Gy). Survival rates were then determined by a MTT assay, while vascular endothelial growth factor receptor (VEGFR)-1 and -2 expression was measured by flow cytometry and apoptotic cell death rates were investigated using an Annexin assay. Additionally, a commercially available canine VEGF ELISA kit was used to measure VEGF. Radiosensitivity was detected in TLM 1 cells, and mitotic and apoptotic cell death was found to occur in a radiation dose dependent manner. VEGF was secreted constitutively and significant up-regulation was observed in the 8 and 10 Gy irradiated cells. In addition, a minor portion of TLM 1 cells expressed vascular endothelial growth factor receptor (VEGFR)-1 intracellularly. VEGFR-2 was detected in the cytoplasm and was down-regulated following radiation with increasing dosages. In TLM 1 cells, apoptosis plays an important role in radiation induced cell death. It has also been suggested that the significantly higher VEGF production in the 8 and 10 Gy group could lead to tumour resistance.     

Development of a multianalyte enzyme-linked immunosorbent assay for permethrin and aroclors and its implementation for analysis of soil/sediment and house dust extracts

Development of a multianalyte enzyme-linked immunosorbent assay (ELISA) for detection of permethrin and aroclors 1248 or 1254 and implementation of the assay for analysis of soil/sediment samples are described. The feasibility of using the multianalyte ELISA to monitor aroclors 1254 and permethrin simultaneously was tested with permethrin and aroclor standards and with aroclor- and permethrin-containing soil/sediment and house dust samples. Comparison of the I?? and I?? values of the multianalyte with those of a single-analyte assay revealed similar results, and multianalyte ELISA determination of analyte amounts in soil/sediment dust samples yielded similar results to those of a single-analyte assay. A single-analyte assay of permethrin content in permethrin-containing dust samples showed that the ELISA can determine the analyte accurately in samples with dust matrix contents ranging from 6.25 to 100 mg as indicated by the good correlation between the results of the immunoassay and those of the gas chromatography analysis.     

Effects of the Consumption of Caffeinated and Decaffeinated Instant Coffee Beverages on Oxidative Stress Induced by Strenuous Exercise in Rats

Many authors attribute the antioxidant activity of brewed coffee to its caffeine content. In addition, caffeine intake has been associated with increased performance during physical exercise. This study analyzed the in vivo effects of drinking caffeinated and decaffeinated instant coffee (8%, w/v) on oxidative stress and antioxidant enzyme activity in the anterior tibialis muscles of rats subjected to intense exercise. It was observed that exercise induced lipid peroxidation (estimated using malondialdehyde) and protein oxidation (evaluated by determining the formation of carbonyl groups) in the muscle (P < 0.05). Decaffeinated instant coffee and caffeine solution did not exhibit antioxidant activity in vivo. Caffeinated instant coffee beverage intake did not induce changes in superoxide dismutase and glutathione peroxidase activities but was able to diminish lipid and protein oxidation in the anterior tibialis muscles of rats after exercise (P < 0.05), contributing to a reduction in the oxidative stress triggered by exercise.