Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique

BACKGROUND: Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. OBJECTIVE: The objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. METHODS: CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. RESULTS: Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. CONCLUSION: The microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations.     

Malassezia spp. overgrowth in allergic cats

A series of 18 allergic cats with multifocal Malassezia spp. overgrowth is reported: atopic dermatitis was diagnosed in 16, an adverse food reaction in another and one was euthanized 2 months after diagnosis of Malassezia overgrowth. All the cats were otherwise healthy and those tested (16 out of 18) for feline leukaemia or feline immunodeficiency virus infections were all negative. At dermatological examination, multifocal alopecia, erythema, crusting and greasy adherent brownish scales were variably distributed on all cats. Cytological examination revealed Malassezia spp. overgrowth with/without bacterial infection in facial skin (n = 11), ventral neck (n = 6), abdomen (n = 6), ear canal (n = 4), chin (n = 2), ear pinnae (n = 2), interdigital (n = 1) and claw folds skin (n = 1). Moreover, in two cats Malassezia pachydermatis was isolated in fungal cultures from lesional skin. Azoles therapy alone was prescribed in seven, azoles and antibacterial therapy in eight and azoles with both antibacterial and anti-inflammatory therapy in three of the cats. After 3-4 weeks of treatment, substantial reduction of pruritus and skin lesions was observed in all 11 cats treated with a combined therapy and in five of seven treated solely with azoles. Malassezia spp. overgrowth may represent a secondary cutaneous problem in allergic cats particularly in those presented for dermatological examination displaying greasy adherent brownish scales. The favourable response to treatment with antifungal treatments alone suggests that, as in dogs, Malassezia spp. may be partly responsible for both pruritus and cutaneous lesions in allergic cats.     

Application of morphometric measures in estimation of body weight and discrimination of Astyanax lacustris and Astyanax fasciatus

The objective of this study was to verify the direct and indirect correlation between morphometric measures, ratios, body weight and yield in two lambari species Astyanax lacustris and Astyanax fasciatus and whether the discriminant analysis is capable of separating and allocating the species. We used 102 lambari yellow tail and 60 lambari red tail. The fish were weighed and submitted to the evaluation of the morphometric measurements. The direct and indirect effects were evaluated by the method of track analysis, considering weight at slaughter, weight of body parts and body yields as dependent variables and measures and morphometric ratios as explanatory variables. Astyanax lacustris presented higher height and body width, carcass yield and trunk, while A. fasciatus presented higher head yield and viscera weight. The discriminant analysis was able to classify 79.5% of the two species. Track analysis demonstrated that the morphometric measurements can be used for estimation of body and body components’ weight in A. lacustris and A. fasciatus. However, regarding the corporal yield; the morphometric measures were insufficient to explain the yield variations of the species.     

Identification,partial characterization,and use of grey mullet ( Mugil cephalus ) vitellogenins for the development of ELISA and biosensor immunoassays

Vitellogenin (Vtg) has proven to be a sensitive and simple biomarker in determining sex, sexual maturity, and xenoestrogenic effects in fish. Thus, our investigation has been focused on identification, partial characterization, and quantification of grey mullet (Mugil cephalus) Vtg through the use of a variety of biochemical and immunological analytical techniques. Mullet is considered both a promising aquaculture candidate and an important species for improving sediment quality in polyculture systems. In the first part of this work, grey mullet Vtg was purified from plasma of 17β-estradiol (E2)-induced male fish by a one-step chromatographic protocol, and partially characterized. Specific polyclonal antibodies were then raised against the mullet Vtg, and both an indirect ELISA and an optical immunosensor were set up and validated to quantify plasma Vtg. The indirect ELISA and the optical immunosensor assay developed showed linear measuring in the range 56.8–1047.1 ng mL−1 and 70–739 ng mL−1 Vtg concentrations in standard solutions, respectively. The results obtained suggest that the indirect ELISA allows Vtg detection over a wide dynamic range, thus resulting more suitable for rapid and sensitive sample screening. Therefore, we suggest that the direct immunosensor is a promising tool which needs more investigation to improve the sensitivity.     

Nitrogen Release from Slow-Release Fertilizers in Soils with Different Microbial Activities

Soil microbial activity is recognized as an important factor affecting nitrogen (N) release from slow-release fertilizers. However,studies on the effect of size and activity of soil microflora on fertilizer degradation have provided contrasting results. To date, no clear relationships exist between soil microbial activity and the release of N from slow-release fertilizers. Hence, the aim of this study was to better understand such relationships by determining the release of N from three slow-release fertilizers in soils with different microbial activities. Soils were amended with urea-formaldehyde (UF), isobutylidene diurea (IBDU), and crotonylidene diurea (CDU). Urea, a soluble fertilizer, was used as the control. Fertilized soil samples were placed in a leaching system, and the release of N was determined by measuring ammonium-N and nitrate-N concentrations in leachates during 90 d of incubation. Non-linear regression was used to fit N leaching rate to a first-order model. In all the treated soils, N was released in the order: urea (89%–100%) IBDU (59%–94%) UF (46%–73%) CDU (44%–56%). At the end of incubation, N released from CDU did not differ (P 0.05) among soils. On the contrary, UF and IBDU released significantly lower (P 0.05) amounts of N in the soil with higher microbial activity and lower pH.The rate constant (K_0) for UF was lower (P 0.05) in the soil with lower pH. Taken together, our results indicated that soil microbial size and microbial activity had a marginal effect on fertilizer mineralization.     

Soil microbial activity and crop sustainability in a long-term experiment with three soil-tillage and two crop-rotation systems

Reduction in soil disturbance can stimulate soil microbial biomass and improve its metabolic efficiency, resulting in better soil quality, which in turn, can increase crop productivity. In this study we evaluated microbial biomass of C (MB-C) by the fumigation-extraction (FE) or fumigation-incubation (FI) method; microbial biomass of N (MB-N); basal respiration (BR) induced or not with sucrose; metabolic quotient (obtained by the ratio BR/MB-C) induced (qCO₂(S)), or not with sucrose (qCO₂); and crop productivity in a 14-year experiment in the state of Paraná, southern Brazil. The experiment consisted of three soil-tillage systems [no-tillage (NT), conventional tillage (CT) and no-tillage using a field cultivator every 3 years (FC)] and two cropping systems [a soybean–wheat-crop sequence (CS), and a soybean–wheat–white lupin–maize–black oat–radish crop rotation (CR)]. There were six samplings in the 14th year, starting at the end of the winter crop (wheat in the CS and lupin in the CR plots) and finishing at full flowering of the summer crop (soybean in the CS and maize in the CR). Differences in microbiological parameters were greater than those detected in the total C (TCS) and total N (TNS) contents of the soil organic matter (SOM). Major differences were attributed to tillage, and on average NT was higher than the CT in the following parameters: TCS (19%), TNS (21%), MB-C evaluated by FE (74%) and FI (107%), and MB-N (142%). The sensibility of the microbial community and processes to soil disturbance in the tropics was highlighted, as even a moderate soil disturbance every 3 years (FC) affected microbial parameters but not SOM. The BR was the parameter that most promptly responded to soil disturbance, and strong differences were perceived by the ratio of qCO₂ evaluated with samples induced and non-induced with sucrose. At plowing, the qCO₂(S):qCO₂ was five times higher under CT, indicating a C-starving low-effective microbial population in the C-usage. In general, crop rotation had no effect on microbial parameters or SOM. Grain yield was affected by tillage and N was identified as a limiting nutrient. Linear regressions between grain yields and microbial parameters showed that soybean was benefited from improvements in the microbial biomass and metabolic efficiency, but with no significant effects observed for the maize crop. The results also indicate that the turnover of C and N in microbial communities in tropical soils is rapid, reinforcing the need to minimize soil disturbance and to balance inputs of N and C.     

Gene expression study of two widely used pig intestinal epithelial cell lines: IPEC-J2 and IPI-2I

The intestinal epithelial cells (IEC) play an important role in the immune system of swine, protecting against infectious and non-infectious environmental insults. The IEC participate in the innate immune response of the intestine through different mechanisms such as barrier function, mucus secretion, antibacterial peptide synthesis and participation in the cytokine/chemokine networks.Most of the current knowledge of intestinal cell functions has come from studies conducted on cell cultures generated from human cancers or from classical animal models. However, because the molecular and cellular elements of the immune system have been selected over evolutionary time in response to the species-specific environment, models of immune function based on mouse and human need to be applied cautiously in pig. Few models of swine small intestine epithelium exist and these are poorly characterised. In the present study we characterised the basal expression of epithelial and immune-related genes of two pig small intestine cell lines, IPEC-J2 and IPI-2I, under different culture conditions. These data represent essential background information for future studies on pig-intestinal pathogen interactions.