Rapid methodology for antigenic profiling of FMDV field strains and for the control of identity, purity and viral integrity in commercial virus vaccines using monoclonal antibodies

Monoclonal antibodies (MAbs) developed against different foot-and-mouth disease virus (FMDV) vaccine strains were extensively used to study any possible antigenic variations during vaccine production in Argentine facilities. Additionally, a typing ELISA using strain specific MAbs was developed to detect potential cross contaminations among FMDV strains in master and working seeds with high specificity and sensitivity and to confirm strains identity in formulated vaccines. This assay was carried out for the South American strains currently in use in production facilities in Argentina (A24/Cruzeiro, A/Argentina/01, O1/Campos and C3/Indaial) and for the strain O/Taiwan, produced only for export to Asia. These non-cross reactive MAbs were also used to analyze the integrity of viral particles belonging to each one of the individual strains, following isolation of 140S virions by means of sucrose density gradients from the aqueous phase of commercial polyvalent vaccines. Antigenic profiles were defined for FMDV reference strains using panels of MAbs, and a coefficient of correlation of reactivity with these panels was calculated to establish consistent identity upon serial passages of master and production seeds. A comparison of vaccine and field strain antigenic profiles performed using coefficients of correlation allowed the rapid identification of two main groups of serotype A viruses collected during the last FMD epidemic in Argentina, whose reactivity matched closely to A/Argentina/2000 and A/Argentina/2001 strains.     

Effect of seasonality and agricultural practices on occurrence of entomopathogenic nematodes and soil characteristics in La Rioja (Northern Spain)

The effects of seasonality and land management on entomopathogenic nematodes (EPNs) distribution and on soil characteristics were studied in natural areas and agricultural fields under organic and conventional management in La Rioja (Northern Spain). The population density of the native EPNs as well as air temperature, rainfall, soil fertility, soil moisture, and soil content of heavy metals were seasonally recorded over 2 consecutive years. The highest occurrence of EPNs was observed in autumn, followed by spring and summer. Agricultural management influenced the amount of soil organic matter, nutrients and heavy metals, as well as soil volumetric moisture and temperature, leading to statistical differences mainly between natural and agricultural sites, but also in some cases between different types of agricultural management. The usefulness of soil organic matter, C, N, P and K content, as well as the C/N, C/P and N/P ratios as indicators for differentiating conventional and organic crop management as well as the role of agricultural practices such as tillage and fertilizing is discussed.     

Comparison of Three Methods for Monitoring Populations of Different Genotypes of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens in the Rhizosphere

ABSTRACT Pseudomonas fluorescens strains producing the antibiotic 2,4-diacetylphloroglucinol (DAPG) have biocontrol activity against a broad spectrum of root and seedling diseases. In this study, we determined the effect of genotype on the ability to isolate and quantify introduced 2,4-DAPG producers from the rhizosphere of wheat using three different methods: traditional dilution plating on selective media, colony hybridization followed by polymerase chain reaction (PCR), and phlD-specific PCR-based dilution endpoint assay. Regression analysis of the population densities of 10 2,4-DAPG-producing P. fluorescens, representing five genotypes, determined by the three different methods demonstrated that the relationship was linear (P < 0.001) and the techniques were very similar (i.e., slopes equal to 1.0). The phlD-specific PCR-based assay had a slightly lower limit of detection than the other two methods (log 3.3 versus log 4.0 CFU/g of fresh root weight). With the colony hybridization procedure, we observed that the phlD probe, derived from strain P. fluorescens Q8r1-96, hybridized more strongly to colonies of BOX-PCR genotypes D (strains W2-6, L5.1-96, Q8r1-96, and Q8r2-96) and K (strain F113) compared with strains of genotypes A (Pf-5 and CHA0), B (Q2-87), and L (1M1-96 and W4-4). Colony hybridization alone overestimated the actual densities of some strains, thus requiring an additional PCR step to obtain accurate estimates. In contrast, population densities estimated for three of the bacterial treatments (strains CHA0, W2-6, and Q8r2-96) with the PCR-based assay were significantly (P < 0.041) smaller by 7.6 to 9.2% and 6.4 to 9.4% than population densities detected by the dilution plating and colony hybridization techniques, respectively. In this paper, we discuss the relative advantages of the different methods for detecting 2,4-DAPG producers.     

Application of a novel sorting system for equine mesenchymal stem cells (MSCs)

The objective of this study was to validate non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs) into subpopulations, for use with MSCs derived from equine muscle tissue, periosteal tissue, bone marrow, and adipose tissue. Cells were collected from 6 young, adult horses, postmortem. Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and left supragluteal subcutaneous adipose tissue. Aliquots of 800 × 10³ MSCs from each tissue source were separated and injected into a ribbon-like capillary device by continuous flow (GrFFF proprietary system). Cells were sorted into 6 fractions and absorbencies [optical density (OD)] were read. Six fractions from each of the 6 aliquots were then combined to provide pooled fractions that had adequate cell numbers to seed at equal concentrations into assays. Equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells were consistently sorted into 6 fractions that remained viable for use in further assays. Fraction 1 had more cuboidal morphology in culture when compared to the other fractions. Statistical analysis of the fraction absorbencies (OD) revealed a P-value of < 0.05 when fractions 2 and 3 were compared to fractions 1, 4, 5, and 6. It was concluded that non-equilibrium GrFFF is a valid method for sorting equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells into subpopulations that remain viable, thus securing its potential for use in equine stem cell applications and veterinary medicine.     

Soil fertility differences across a land-use intensification gradient in the highlands of Chiapas,Mexico

In the highlands of Chiapas, southern Mexico, soil texture and soil chemical properties were measured in 70 agricultural fields covering a range of slope positions and managements. Fields represented four corn cropping systems: long fallow, short fallow, pasture–cultivation rotation, and annual continuous cultivation, in addition to fallow at rest (forest, shrubland, and pastures). Fields were located in four slope positions (upper, middle, and lower slopes, and doline floor) in a karst landscape developed on limestone with additions of acid volcanic ashes. Distribution of clays and sands were related to the toposequence and the percentage of clay fraction increased from upper slope to doline floor. Sand presented a reverse pattern. Some soil chemical properties also vary in a characteristic way along the toposequence. Exchangeable Ca²⁺ and Mg²⁺ effective cation exchange capacity (ECEC) and pH were higher in the doline floor than in the other slope positions. Soil organic matter and total N contents were 30.4 and 35.2% higher under long fallow than under annual continuous cultivation. Soil Olsen P was greater under cultivation than under fallow (irrespective of the cropping system), with the highest values under annual continuous cultivation (16.2±8.3 mg kg⁻¹) and the lowest in the forest (5.3±2.5 mg kg⁻¹). Exchangeable K⁺ was 74 and 51% higher in cultivated plots of the long and the short fallow than under forest and shrubland, respectively. Exchangeable Ca²⁺ and Mg²⁺ and ECEC did not vary significantly among the cropping systems. Inputs of basic cations through burning of tree and shrub vegetation increased the soil pH. These results indicate that land use intensification is leading to a decline in soil organic matter and total N, whereas Olsen P increased with intensification due to the continuous application of P fertilizers and sheep manure as soil conditioners in intensively cultivated fields.     

Replacement of fish meal with co-extruded wet tuna viscera and corn meal in diets for white shrimp (Litopenaeus vannamei Boone)

A feeding trial was conducted to evaluate the potential of replacing fish meal with co‐extruded wet tuna viscera and corn meal in diets for juvenile white shrimp Litopenaeus vannamei. Five practical diets were formulated to contain 30% crude protein and 16.6 kJ g^?1. The feeding experiment was carried out in a recirculating seawater system with fifteen 25‐L tanks with 15 shrimp per tank. Each dietary treatment was fed in triplicate in a completely randomized design for 41 days. Percent weight gain (WG%), survival (S%) and feed conversion ratio (FCR) were calculated, comparisons of these parameters showed no statistical differences (P>0.05) among treatments. The shrimp almost tripled their weight during the feeding trial. Under the experimental conditions S%, WG% and FCR were either improved or were not significantly influenced by the replacement of fish meal with double co‐extruded viscera+corn, except for a slight increase in feed consumption without affecting FCR. Hence, this product can be included by up to 40% in practical shrimp diets without any detrimental effects.     

Angiotensin converting enzyme inhibitory activity in commercial fermented products. Formation of peptides under simulated gastrointestinal digestion

The angiotensin converting enzyme (ACE)-inhibitory activity of several commercial fermented milks was evaluated. Most of these products showed moderate inhibitory activity, but a few exceptions were detected. The high ACE-inhibitory activity found in some cases could be related to the origin of the milk. Two of these products were subjected to an enzymatic hydrolysis process, which simulates physiological digestion, to study the influence of digestion on ACE-inhibitory activity. The activity did not significantly change or increase during simulated gastrointestinal digestion. The peptides generated from one selected product during simulated digestion were sequenced by tandem spectrometry. Most peptides found at the end of the simulated digestion were released after 30 min of incubation with the pancreatic extract. This suggests that physiological digestion promotes the formation of active peptides from the proteins present in these fermented products. The potential ACE-inhibitory activity of the identified peptides is discussed with regard to their amino acid sequences.