Discrimination of alleles and copy numbers at the <Emphasis Type="Italic">Q</Emphasis> locus in hexaploid wheat using quantitative pyrosequencing

Speltoid spikes are characterized by pyramidal spike morphology featuring an elongated rachis and tenacious glumes. Speltoids are considered undesirable spike aberrants in wheat breeding leading to increased heterogeneity within a cultivar candidate. As a consequence, the presence of speltoids may result in rejection of a cultivar candidate during official field trials or denial of cultivar certification during seed multiplication. A reliable method is, thus, required to assess the occurrence of speltoids, early on in a wheat breeding program. The domestication gene Q located on the long arm of wheat chromosome 5A is known to suppress the speltoid phenotype in wheat. Here, a quantitative pyrosequencing assay was developed to distinguish between normal wheat plants, which possess two copies of the Q allele, and aberrants, which are either aneuploids lacking the correct number of chromosome 5A copies or plants which carry the primitive q allele. An accurate and reproducible determination of the Q gene copy number was achieved for different wheat genotypes based on homoeologous sequence quantification with two primer combinations at the Q locus. Single plants with one to four copies of the Q allele could be detected by quantitative pyrosequencing which corresponded to the occurrence of speltoid (1 Q allele), normal (2 Q alleles), and compact (more than 2 Q alleles) spikes. Q and q specific alleles could be differentiated at SNP position 2299 of the Q gene. This SNP is assumed to be related to the emergence of free-threshing wheat forms. To our knowledge this is the first report for detection of aneuploids and differentiation of Q alleles in bread wheat using pyrosequencing technology. In future, quantitative pyrosequencing assay can be applied in wheat breeding programs to carry out marker-assisted selection against the presence of speltoid spike aberrants.     

Probability of freedom from disease after the first detection and eradication of PRRS in Sweden: Scenario-tree modelling of the surveillance system

In July 2007, PRRS was detected for the first time in Sweden. A total of eight positive herds were identified and various measures were taken to eradicate the disease, including restrictions and slaughter of infected herds. Subsequently, both active and passive surveillance activities were undertaken. This study describes stochastic scenario-tree modelling of all the various surveillance system components, to estimate the current probability that Sweden is free from PRRS. The model includes all actions taken after the first positive herd was detected. The surveillance system components included in the model were as follows: investigations undertaken in association with the outbreak, a serological study based on samples collected at slaughter, samples collected in the national PRRS surveillance programme and passive clinical surveillance. The probability of freedom was estimated in time steps of 1 month, from July to December 2007. After each time step, the calculated posterior probability of freedom from the previous month, combined with the probability of introduction, was used as a prior probability for the next month.The result from the model showed a 99.8% probability that Sweden was free from PRRS at the end of December 2007. The estimated total sensitivity of the surveillance system varied between 81.2% and 94.3% and was highest during the first months after the outbreak. For sensitivity analysis purposes, the model was also applied using higher risks of introduction. However, this did not make considerable difference to the final estimates.     

Effects of dietary phytoestrogens on plasma testosterone and triiodothyronine (T3) levels in male goat kids

Background

Exposure to xenoestrogens in humans and animals has gained increasing attention due to the effects of these compounds on reproduction. The present study was undertaken to investigate the influence of low-dose dietary phytoestrogen exposure, i.e. a mixture of genistein, daidzein, biochanin A and formononetin, on the establishment of testosterone production during puberty in male goat kids.

Methods

Goat kids at the age of 3 months received either a standard diet or a diet supplemented with phytoestrogens (3 - 4 mg/kg/day) for ~3 months. Plasma testosterone and total and free triiodothyronine (T₃) concentrations were determined weekly. Testicular levels of testosterone and cAMP were measured at the end of the experiment. Repeated measurement analysis of variance using the MIXED procedure on the generated averages, according to the Statistical Analysis System program package (Release 6.12, 1996, SAS Institute Inc., Cary, NC, USA) was carried out.

Results

No significant difference in plasma testosterone concentration between the groups was detected during the first 7 weeks. However, at the age of 5 months (i.e. October 1, week 8) phytoestrogen-treated animals showed significantly higher testosterone concentrations than control animals (37.5 nmol/l vs 19.1 nmol/l). This elevation was preceded by a rise in plasma total T₃ that occurred on September 17 (week 6). A slightly higher concentration of free T₃ was detected in the phytoestrogen group at the same time point, but it was not until October 8 and 15 (week 9 and 10) that a significant difference was found between the groups. At the termination of the experiment, testicular cAMP levels were significantly lower in goats fed a phytoestrogen-supplemented diet. Phytoestrogen-fed animals also had lower plasma and testicular testosterone concentrations, but these differences were not statistically significant.

Conclusion

Our findings suggest that phytoestrogens can stimulate testosterone synthesis during puberty in male goats by increasing the secretion of T₃; a hormone known to stimulate Leydig cell steroidogenesis. It is possible that feedback signalling underlies the tendency towards decreased steroid production at the end of the experiment.     

Retinal degeneration in nine Swedish Jämthund dogs

The Jämthund is the fourth most common breed in Sweden with approximately 1600 pups registered each year. Although it has been known that some adult dogs go blind, so they cannot hunt, the Jämthund dog has historically not been screened for hereditary eye diseases. This report describes nine Swedish Jämthund dogs with retinal degeneration. These dogs represent all Jämthund dogs diagnosed with progressive retinal atrophy (PRA) by the Swedish Eye Panel and registered with the Swedish Kennel Club from January 1998 to September 2008. The dogs were examined with indirect opthalmoscopy and slitlamp biomicroscopy. Additionally, electroretinograms (ERGs) following ECVO guidelines were performed in two dogs (one affected and one normal) and the eyes from three affected dogs were examined by light‐microscopy postmortem. Typical findings were bilateral symmetric generalized retinal degeneration with tapetal hyper‐reflectivity, attenuation of blood vessels and pigment clumping in the nontapetal fundus. These retinal findings progressed with time in two dogs after re‐examination. Visual impairment, especially under dim light conditions, was observed in the affected dogs. ERG from one affected dog showed profoundly reduced rod responses, whereas cone responses were better preserved. Microscopic changes in the eyes from three dogs were characterized by a severe diffuse predominantly outer retinal degeneration and atrophy. Re‐sequencing of the prcd‐gene for eight of the nine investigated dogs revealed that none of the individuals carried disease allele that has been associated with prcd‐PRA in other breeds. In conclusion, ophthalmoscopic, electroretinographic, and light‐microscopic alterations observed in nine Jämthund dogs were compatible with PRA. The prcd mutation was excluded as a cause of this retinopathy.     

The yin and yang of corpus luteum-derived endothelial cells: balancing life and death

A dense network of capillaries irrigates the corpus luteum (CL) allowing an intricate cross talk between luteal steroiodgenic and endothelial cell (EC) types. Indeed, luteal endothelial cells (LEC) play pivotal roles throughout the entire CL life-span. Microvascular endothelial cells are locally specialized to accommodate the needs of individual tissues, therefore unraveling the characteristics of LEC is imperative in CL physiology. Numerous studies demonstrated that endothelium-derived endothelin-1 (ET-1) is upregulated by the luteolytic hormone-prostaglandin F2alpha (PGF2alpha) and functions as an important element of the luteolytic cascade. To have a better insight on its synthesis and action, members of ET system (ET-1, ET converting enzyme -ECE-1 and ET(A) and ET(B) receptors) were quantified in LEC. The characteristic phenotype of these cells, identified by high ET-1 receptor expression (both ET(A), ET(B)) and low ET-1 and ECE-1 levels, was gradually lost during culture suggesting that luteal microenvironment sustains the selective phenotype of its resident endothelial cells. Proper vascularization and endothelial cell activity per se are essential for normal CL function. Therefore, factors affecting vascular growth are expected to play major role in the regulation of luteal function. Concomitantly with the angiogenic process, luteal PGF2alpha and its receptors (PGFR) are induced and maintained during most of the CL life-span, suggesting a possible role of PGF2alpha in LEC proliferation and function. Dispersed LEC expressed PGFR and incubation with the prostaglandin stimulated mitogen-activated protein kinase (MAPK) signaling cascade. PGF2alpha activated p42/44 MAPK phosphorylation also in long-term cultured LEC. In this cell type, PGF2alpha increased cell number, 3H-Thymidine incorporation and cell survival. Additionally, PGF2alpha rapidly and transiently stimulated the expression of immediate-early response genes, i.e. c-fos and c-jun mRNA, further suggesting a mitogenic effect for this prostaglandin in LEC. These data imply that PGF2alpha may assume different and perhaps opposing roles depending on luteal microenvironment.     

Characterization of the bacterial population of the genital tract of adult cats

OBJECTIVE: To characterize the bacteria of the genital tract in adult cats; assess the effect of estrus, mating, and administration of progestins on those microorganisms in females; and evaluate whether results of bacteriologic culture of vaginal swabs are affected by cleansing of the vulva prior to sampling or by repeated sampling. ANIMALS: 66 female and 29 male cats undergoing routine ovariohysterectomy or castration. PROCEDURE: Specimens were obtained from vaginal and uterine or preputial mucosae with swabs moistened with sterile saline (0.9% NaCl) solution. In 9 cats, vaginal specimens were obtained before and after cleansing of the vulva with ethanol; in 7 female cats, 2 vaginal speci mens were obtained in immediate succession. RESULTS: Aerobic bacteria were most commonly isolated from cats' vaginas and prepuces; anaerobic bacteria were isolated frequently from males (41%) but rarely from females (5%). Generally, culture results were not affected by cleansing of the vulva or repeated vaginal sampling. The bacterial population of the vaginas of cats was influenced by stage of the estrous cycle but not by mating or administration of progestins. Bacteria were not isolated from the uterus of any cat. CONCLUSIONS AND CLINICAL RELEVANCE: In cats, bacteria of the genital tract in females are predominantly aerobic; in males, aerobic and anaerobic bacteria are found. The bacterial population of the vagina is affected by stage of the estrous cycle. Pure growth of bacteria in culture of genital tract specimens is a normal finding; antimicrobials should only be administered if clinical signs of genital infection are present.     

Nitrogen best management practice for citrus trees: I. Fruit yield,quality, and leaf nutritional status

Elevated levels of nitrate-nitrogen (NO₃-N) in the surficial aquifer above the drinking water quality standard, i.e. maximum contaminant limit (MCL; 10 mg L⁻¹), have been reported in some part of central Florida citrus production regions. Soils in this region are very sandy (sand content >95%), hence are vulnerable to leaching of soluble nutrients and chemicals below the rooting depth of the trees. The objective of this research was to develop N and irrigation best management practices for citrus in sandy soils to maintain optimal crop yield and quality, and to minimize potential leaching of nitrate below the root zone. Six years of field experiment was conducted in a high productive (mean fruit yield > 80 Mg ha⁻¹yr⁻¹) >20-year-old ‘Hamlin’ orange trees [Citrus sinensis (L.) Osbeck] on ‘Cleopatra mandarin’ (Citrus reticulata Blanco) rootstock grown on a well drained Tavares fine sand (hyperthermic, uncoated, Typic Quartzipsamments) in Highland county, FL. Nitrogen rates ranged from 112 to 280 kg ha⁻¹ yr⁻¹ applied as fertigation (FRT), water soluble granular (WSG), 50:50 mix of FRT and WSG, and controlled-release fertilizer (CRF). Tensiometers were used to monitor the soil water content as a basis to schedule optimal irrigation. Fruit yield response over the entire range of N rates was greater for the FRT and WSG sources as compared to that for the WSG + FRT or CRF sources. Using the regression analysis of the fruit yield in relation to N rate, the optimum N rate appeared to be at 260 kg ha⁻¹ yr⁻¹. Based on fruit production response in this study, the N requirement for production of 1 Mg of fruit varied from 2.2 to 2.6 kg across four N sources. This study demonstrated an increased N uptake efficiency, as a result of best management of N and irrigation applications. The optimal N and K concentration in the 4–6-month-old spring flush leaves were 26–30, and 15–18 g kg⁻¹, respectively. However, fruit yield response showed no significant relationship with concentrations of P in the 4–6-month-old spring flush leaves over a range of 0.8–2.4 g kg⁻¹. The results of fate and transport of N in soil and in soil solution with application of different rates and sources of N, and components of citrus tree N budget, are reported in a companion paper.     

Leukocyte endothelial cell interactions in pig to human organ xenograft rejection

In cases where antibody- and complement-mediated hyperacute rejection (HAR) of vascularized organ xenografts has been prevented, acute vascular rejection (AVR) and acute T cell-mediated rejection (ACR) cause graft destruction. Infiltration of leukocytes (innate and graft-primed T cells) into the graft execute the latter two rejection modalities. The leukocyte extravasation process, which is a prerequisite for graft infiltration, is governed by adhesion molecules, including the selectin, integrin and immunoglobulin protein families, and the chemokine protein family. The compatibility between porcine endothelial cell and human leukocyte adhesion molecules was investigated in dynamic adhesion and static transendothelial migration assays. The effect of human anti-pig antibodies on human leukocyte adhesion to, and transendothelial migration across, porcine endothelium was assessed under dynamic and static conditions, respectively. In contrast to previously published results, no difference in the ability of neutrophils to adhere to pig and human endothelium was observed. Furthermore, no evident quantitative or qualitative differences in the capacity of human and porcine endothelium to support transendothelial migration of human leukocytes (T, B and natural killer (NK) cells, monocytes, and neutrophils) could be detected. The presence of human anti-pig antibodies (Abs) modulated the migration of leukocytes across porcine endothelium, as well as neutrophil adhesion to porcine endothelium under conditions of flow. Antibodies specific for pig endothelial adhesion molecules can potentially be used as species (graft)-specific immunosuppressive reagents in order to prevent cellular organ xenograft rejection.     

Effects of management, feeding, and treatment on clinical and biochemical variables in cattle with displaced abomasum

OBJECTIVE: To determine effects of management, feeding, and treatment on clinical and biochemical variables in cows with displaced abomasum (DA). ANIMALS: 374 cows that received 470 treatments for DA. PROCEDURE: Blood and milk samples were obtained from 139 affected cows for analysis; for all cows, clinical data, management, feeding, and treatments were evaluated. RESULTS: Multiparous cows were more predisposed to DA than primiparous cows were, and Swedish Friesians were more predisposed than Swedish Red and Whites were. Eighty percent of cows had left-sided DA, and 20% had right-sided DA. In > 50% of affected cows, clinical signs appeared just before calving to 2 weeks after calving. Incidence of twin calves and periparturient diseases was significantly higher in affected cows than in the overall Swedish cow population. Content of neutral detergent fiber in the silage was low in herds with DA. Feeding a total mixed ration was a risk factor for DA. Treatment by surgical methods gave a significantly higher recovery rate than nonsurgical methods. CONCLUSIONS AND CLINICAL RELEVANCE: Displaced abomasum is a periparturient nutritional disease. Feeding roughage with low neutral detergent content is a more important causative factor than the amount of concentrates fed at the time of calving. The basic principle for prevention of DA is to maintain good ruminal filling before and at calving. The amount of high-quality roughage fed before and at calving should be kept to a minimum. By changing routines for periparturient feeding, it should be possible to reduce the incidence of DA.     

Photocatalytic degradation of pesticides and bio-molecules in water

Two approaches are suggested for the acceleration of the photocatalytic oxidation of organic contaminants of water: acceleration by oxidants and photo-enhancement by dyes. These processes were examined with several substances: two widely applied herbicides, bromacil (a uracil) and metribuzin (a triazine), and three proteins, studied as models of biocontaminated waters. The effects of oxygen and hydrogen peroxide indicated two different reaction patterns of photo-oxidation of the herbicides. With metribuzin, oxygen had a pronounced effect on the rate of photo-oxidation, while the influence of hydrogen peroxide was quite moderate; with bromacil, oxygen had a limited effect on the rate of photo-oxidation, which however was considerably enhanced by hydrogen peroxide. Acceleration of the photo-catalytic oxidation of colourless refractory contaminants by photo-excited dye was observed. Both UV and visible light were required for the enhanced decomposition. The mechanism of the reaction seems to involve a combination of oxidation by hydroxyl radicals, via the hole-electron semiconductor route, with subsequent oxidation of photo-intermediates by singlet oxygen formed by dye sensitization. The TiO2-photocatalyzed oxidation of proteins (albumin, ovalbumin and gamma-globulin) showed the susceptibility of proteins to photocleavage and of the amino acids to photocatalytic degradation. Tyrosine was the most sensitive, while the degradation of the aliphatic amino acids Gly and Asp was slow.