Ultrasensitive detection of genetically modified maize DNA by capillary gel electrophoresis with laser-induced fluorescence using different fluorescent intercalating dyes

In this work, four different fluorescent intercalating dyes are compared for the ultrasensitive CGE-LIF detection of DNA from transgenic maize in flours. The fluorescent intercalating dyes compared are YOPRO-1, SYBR-Green-I, Ethidium bromide (EthBr), and EnhanCE. For all the four dyes optimum concentrations are established, and efficient separations of DNA fragments ranging in size from 80 to 1000 bp are obtained. The comparative study demonstrates that SYBR-Green-I and YOPRO-1 provide better limits of detection (LODs) than EnhanCE or EthBr (i.e., LODs are, respectively, 700, 1000, 11300, and 97400 zmol, calculated for a 200-bp DNA fragment). Separations using YOPRO-1 are faster than those using SYBR-Green-I (30 min vs 47 min for the analysis of the 80-1000 bp DNA fragments). Also, separations using YOPRO-1 are more efficient than those using SYBR-Green-I (e.g., 2.4 x 10(6) plates/m vs 1.6 x 10(6) plates/m, respectively, calculated for the 200-bp fragment). Also, buffer depletion and cost per analysis are worse with SYBR-Green-I than with YOPRO-1. Therefore, YOPRO-1 was selected as the preferred intercalating dye. Using this fluorescent compound, analysis time reproducibility for the CGE-LIF separation of the DNA fragments is determined to be better than 1.7% (% RSD, n = 10) within the same day, and better than 1.9% (% RSD, n = 30) for three different days. Moreover, the fluorescence signal obtained using this dye is shown to vary linearly with the DNA concentration in the range studied, i.e., 1-500 ng/microL. It is demonstrated that by using this method 0.01% of transgenic maize can be detected in flour by direct injection of the PCR-amplified sample.     

The growth curve of animals

Several analytical expressions have been proposed as models of weight versus time to describe growth but these have not been completely satisfactory. Some of them represent a good adjustment for only a part of the growth process; others have parameters of little biological significance or not enough practical interpretation. Some also involve troublesome numerical work, or do not have enough flexibility to accommodate exceptional cases.In this study, a simple flexible and analytical expression with biologically significant parameters is presented. In the authors' opinion this expression eliminates many of the deficiencies mentioned above.The proposed expression is validated with data available in the literature. Cases were selected and used to show the adequacy of the expression for adjusting quantitative variations between and within species.     

Correlation of Different Biochemical Parameters in Blood Sera of Healthy and Sick Cows

Metabolic diseases of cows represent the leading internal pathology in Lithuania in terms of incidence and economic impact. This paper summarizes the mineral metabolic state of milk cows, and details the influence of feeding on serum levels of calcium, nonorganic phosphorus, magnesium, potassium, sodium, glucose, total protein, and urea quantity, and correlationd among them, in healthy dry or post-calving cows as well as in cows with osteomalacia and milk fever after calving. There was less pronounced hypocalcaemia and there were only minor changes in phosphorus, magnesium, potassium and sodium in the serum of healthy dry and post-calving cows that had silage and mineral-vitamin supplements, compared to cows that did not have supplements and silage. There was a fall in calcium and phosphorus (to 1.89 +/- 0.12 mmol/L and 0.71 +/- 0.06 mmol/L, respectively) in the blood of cows sick with milk fever after calving, while levels of magnesium and potassium were increased. The correlation between calcium and phosphorus was r = 0.6993, p < 0.001 in the serum of sick cows. There was a fall in calcium, phosphorus and magnesium (to 1.86 +/- 0.46 mmol/L, 0.75 +/- 0.37 mmol/L, and 0.60 +/- 0.19 mmol/L, respectively) and an increase in sodium level (to 158.90 +/- 19.30 mmol/L) in the blood of cows with osteomalacia in comparison with healthy cows.     

Indirect effect of 6-azauracil on Pythium debaryanum in cucumber

Protection of cucumber seedlings against damping off, caused byPythium debaryanum, was obtained by soaking the seeds in a solution of 1 ppm 6-azauracil (AzU), but not by treatment with a 10 or 100 ppm AzU solution. After the 1 ppm AzU treatment of the seeds, the rhizosphere microflora of the developing seedlings was changed; an increase of the bacterial population and a decrease of the fungal population was observed. SinceP. debaryanum is rather insensitive to AzU in vitro, it is suggested that the control of damping off is obtained in an indirect way, possibly via the microflora of the roots.Samenvatting Bescherming van komkommerzaailingen tegen aantasting doorPythium debaryanum werd verkregen na weken der zaden in een oplossing van 1 ppm 6-azauracil (AzU); 10 en 100 ppm AzU waren niet werkzaam. Na behandeling der zaden met 1 ppm AzU traden veranderingen in de rhizosfeermicroflora der kiemplanten op; de bacteriepopulatie nam toe en de schimmelpopulatie nam af. AangezienP. debaryanum in vitro vrijwel ongevoelig is voor AzU, wordt verondersteld dat de bescherming tegen aantasting door deze schimmel op indirecte wijze verkregen wordt, mogelijk via de microflora van de wortels.     

Haptoglobin and pig-major acute protein are increased in pigs with postweaning multisystemic wasting syndrome (PMWS)

The objective of this study was to determine the serum concentration levels of selected acute phase proteins (APP), haptoglobin (HPT) and pig-major acute phase protein (pig-MAP), in postweaning multisystemic wasting syndrome (PMWS) affected pigs and PCV2-subclinically infected pigs. In a first study, a group of 15 eight-week-old conventional pigs from a PMWS affected farm were bled and a complete necropsy, histopathology and in situ hybridisation to detect PCV2 were performed. Based on the results, pigs were classified as suffering from PMWS (n = 10) or healthy animals (n = 5). In a second study, a group of 45 pigs from another PMWS affected farm were selected and bled at 3, 7, 12 and 28 weeks of age. The assessment of PCV2 infection status in these pigs was retrospectively done by PCV2 PCR in serum samples. Selected APP were measured in the serum of all studied pigs by means of radial immunodiffusion. Mean HPT and pig-MAP levels were significantly increased (p = 0.004 and p = 0.0006 respectively) in PMWS-affected pigs when compared to levels found in healthy pigs (2.52 +/- 0.88 mg/mL vs. 1.06 +/- 0.73 mg/mL for HPT and 3.81 +/- 1.53 mg/mL vs. 0.76 +/- 0.34 mg/mL for pig-MAP). In the second study, no significant difference in mean HPT and pig-MAP values were observed between PCV2 PCR positive and negative pigs of any age. However, both APP increased significantly with age in PCV2 PCR negative pigs. Altogether, the present results suggest that APP levels are significantly increased in pigs that develop PMWS, but not in animals with a PCV2 subclinical infection.     

Effect of formic acid on performance, digestibility, intestinal histomorphology and plasma metabolite levels of broiler chickens

1. The effect of dietary formic acid on performance, digestibility, intestinal histomorphology and plasma metabolite levels of broiler chickens was studied. 2. An experiment with 120 Ross male broiler chickens was conducted from 1 to 42 d of age at the laboratory. There were 4 treatment groups: control (C), 10 mg/kg feed avilamycin (AV) and formic acid at two concentrations, 5 and 10 g/kg feed (FA5 and FA10, respectively). 3. No differences in weight gain, feed intake or feed conversion ratio were observed in male broiler chickens fed on the different diets. 4. An effect of the additives on ileal dry matter (DM) digestibility at 42 d of age was detected with the finisher diets; AV and 10 g/kg of feed formic acid slightly improved ileal DM digestibility compared to the other treatment groups. 5. Jejunum pH was not affected when 5 or 10 g/kg formic acid was added, and the results do not clearly show a positive effect of formic acid on the intestinal histomorphology. 6. No differences were noticed for blood metabolites in the different diets, and the levels of calcium and phosphorus in the plasma were not altered by formic acid addition. 7. In conclusion, when using broiler chickens under conditions of good hygiene, dietary formic acid did not have a clear positive effect on performance, intestinal histomorphology or plasma metabolite levels; however, there was a slight positive effect on the ileal digestibility of nutrients.     

Changes in antimicrobial susceptibility of Actinobacillus pleuropneumoniae isolated from pigs in Spain during the last decade

A total of 229 Spanish Actinobacillus pleuropneumoniae isolates recovered from diseased pigs with pleuropneumonia from 1997 to 2004 was tested for their susceptibility to 11 antimicrobials in a broth microdilution method. All the isolates were susceptible to florfenicol and most of them to cephalothin; however, a high rate of resistance was observed to tetracycline. A bimodal or multimodal distribution of isolates over the MIC range were observed for penicillins, tetracycline, trimethoprim, sulfisoxazole and nalidixic acid, suggesting the development of acquired resistance. Eight resistance patterns were established, and 21.1% of the isolates were resistant to at least two antimicrobials. In addition, a considerable increase in the resistance to tetracyclines was observed during the last decade in Spain, when compared with other A. pleuropneumoniae strains isolated during 1987-1988 (Gutiérrez, C.B., Píriz, S., Vadillo, S., Rodríguez Ferri, E.F., 1993. In vitro susceptibility of Actinobacillus pleuropneumoniae strains to 42 antimicrobial agents. Am. J. Vet. Res. 54, 546-550); this finding was also observed for gentamicin in minor percentage.     

Molecular characterization of Haemophilus parasuis ferric hydroxamate uptake (fhu) genes and constitutive expression of the FhuA receptor

Bacteria have evolved a set of highly specialized proteins to capture iron in iron-depleted environments. The acquisition and uptake of iron present in the extracellular milieu of eukaryotic organisms is indispensable for the growth and survival of microbial pathogens in the course of infection. Haemophilus parasuis is the causative agent of Gl?sser disease, which is responsible for considerable financial losses in pig-rearing worldwide. To gain insight into the mechanisms involved in siderophore-mediated iron uptake in H. parasuis, genes in the H. parasuis ferric hydroxamate uptake (Fhu) region were amplified in the work being reported here. As has been described in A. pleuropneumoniae, an Fhu genomic region was also present in H. parasuis, being composed of four potential consecutive open reading frames (ORF) designated as fhuC, fhuD, fhuB, and fhuA, respectively. By immunoblotting, using a cross-reactive polyclonal antibody raised against Actinobacillus pleuropneumoniae FhuA protein, it was demonstrated that this protein was constitutively expressed in H. parasuis and its level of expression was not modified under conditions of restricted iron availability. This is the first report describing the presence of the fhu genes in H. parasuis. Our results indicate that FhuA protein expression is not affected under iron-restricted conditions, however, it is one of the targets of the humoral immune response.     

The growing prevalence of Nosema ceranae in honey bees in Spain, an emerging problem for the last decade

Microsporidiosis caused by infection with Nosema apis or Nosema ceranae has become one of the most widespread diseases of honey bees and can cause important economic losses for beekeepers. Honey can be contaminated by spores of both species and it has been reported as a suitable matrix to study the field prevalence of other honey bee sporulated pathogens. Historical honey sample collections from the CAR laboratory (Centro Apícola Regional) were analyzed by PCR to identify the earliest instance of emergence, and to determine whether the presence of Nosema spp. in honey was linked to the spread of these microsporidia in honey bee apiaries. A total of 240 frozen honey samples were analyzed by PCR and the results compared with rates of Nosema spp. infection in worker bee samples from different years and geographical areas. The presence of Nosema spp. in hive-stored honey from naturally infected honey bee colonies (from an experimental apiary) was also monitored, and although collected honey bees resulted in a more suitable sample to study the presence of microsporidian parasites in the colonies, a high probability of finding Nosema spp. in their hive-stored honey was observed. The first honey sample in which N. ceranae was detected dates back to the year 2000. In subsequent years, the number of samples containing N. ceranae tended to increase, as did the detection of Nosema spp. in adult worker bees. The presence of N. ceranae as early as 2000, long before generalized bee depopulation and colony losses in 2004 may be consistent with a long incubation period for nosemosis type C or related with other unknown factors. The current prevalence of nosemosis, primarily due to N. ceranae, has reached epidemic levels in Spain as confirmed by the analysis of worker honey bees and commercial honey.