Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4)

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.     

Veterinary pathology in the United Kingdom: past, present, and future

This article presents a historical perspective on veterinary anatomic pathology in the United Kingdom from the late nineteenth century to the present. Prior to World War II, the specialty was a rather general one that also included bacteriology and parasitology and was only slightly affected by strong Germanic developments in cell and tissue pathology. The few notable figures of this era include John McFadyean, Sidney Gaiger, and J.R.M Innes. The specialty developed strongly in the second half of the twentieth century, led by a small number of individuals, and was greatly aided by the development of specialist colleges and residency training. Key individuals of this era include W.F. Blakemore, Ernest Cotchin, R.J.M. Franklin, W.F.H. Jarrett, A.R. Jennings, and A.C. Palmer. A remarkable feature of this period has been the increased employment of veterinary pathologists in biomedical industry and in private diagnostic laboratories. While standards of pathology practice have benefited from the college initiatives, there are major financial constraints on the availability of funded training posts in the United Kingdom, and there remain considerable shortages in the supply of pathologists trained to contemporary standards. The acknowledged professional and scientific importance of veterinary pathology needs to be translated into effective financial support for the training that underpins competence in this specialty. Further developments seem likely to be dominated by advances in the technology of tissue handling, applications of molecular biology to pathology, and greater use of telepathology in teaching, in quality assurance, and in continuing professional development.     

The second-generation curcumin analogue RL71 elicits G2/M cell cycle arrest and apoptosis in canine osteosarcoma cells

Canine osteosarcoma is an aggressive cancer, comprising 85% of canine bone neoplasms. Current treatment practices of surgery and chemotherapy increase 1-year survival by only 45%. The curcumin analogue RL71, has demonstrated potent in vitro and in vivo efficacy in several models of human breast cancer through increased apoptosis and cell cycle arrest. Thus, the present study aimed to investigate efficacy of curcumin analogues in two canine osteosarcoma cell lines. Osteosarcoma cell viability was assessed using the sulforhodamine B assay and mechanisms of action were determined by analysing the levels of cell cycle and apoptotic regulatory proteins via Western blotting. Further evidence was obtained using flow cytometry to detect cell cycle distribution and the number of apoptotic cells. RL71 was the most potent curcumin analogue with EC₅₀ values of 0.64 ± 0.04 and 0.38 ± 0.009 μM (n = 3) in D-17 (commercial) and Gracie canine osteosarcoma cells, respectively. RL71 significantly increased the ratio of cleaved-caspase 3 to pro-caspase 3 and the level of apoptotic cells at the 2× and 5× EC₅₀ concentration (p < 0.001, n = 3). Furthermore, at the same concentration, RL71 significantly increased the number of cells in the G2/M phase. In conclusion, RL71 has potent cytotoxic activity in canine osteosarcoma cells triggering G2/M arrest and apoptosis at concentrations achievable in vivo. Future research should further investigate molecular mechanisms for these changes in other canine osteosarcoma cell lines prior to in vivo investigation.     

Use of intralesional cisplatin to successfully treat distal limb haemangiosarcoma in a foal

A 4-month old, 200 kg, grey warmblood colt presented for a firm, non painful mass on the distal medial aspect of the left third metatarsus. Excisional biopsy revealed a diagnosis of haemangiosarcoma. Equine haemangiosarcoma is uncommon and only limited reports of successful treatment are available. The prognosis for survival is therefore considered to be poor. After two separate incidences of recurrence with incomplete excision of the tumour, intralesional treatment with cisplatin without excision or debulking was performed on three separate occasions. Intralesional cisplatin injection was performed at monthly intervals for three treatments. Four years post treatment with cisplatin, the horse remained in remission. This case report describes the diagnostic and treatment challenges for successful treatment of a primary haemangiosarcoma on the distal limb of a warmblood foal using intralesional cisplatin chemotherapy.     

Determining crop-water production functions using yield–irrigation gradient algorithms

Crop-water production functions (CWPFs) are a useful tool for irrigation planning, but derivation of CWPFs by field experimentation is expensive, and traditional analytical techniques are not well suited to derivation of CWPFs. Physiologically based crop models provide a useful tool for simulation of agricultural experiments, but they have not been extensively applied to the task of CWPF determination. A new algorithm type based upon differential crop yield response to irrigation (“yield–irrigation gradients” [YIG]) is presented that uses these crop models to determine planning-level irrigation schedules and CWPFs. Three specific algorithms are developed within this type, varying in complexity, performance, and computational costs. Performance of the YIG methods is compared against a standard reference evapotranspiration method. In particular, the randomized iterative YIG (RIYIG) algorithm provides near-optimal results but at the highest computational costs of all the methods specified. All of the techniques presented have general applicability and are not limited to any one crop or location.     

Changes in forest soil organic matter pools after a decade of elevated CO2 and O3

The impact of rising atmospheric carbon dioxide (CO₂) may be mitigated, in part, by enhanced rates of net primary production and greater C storage in plant biomass and soil organic matter (SOM). However, C sequestration in forest soils may be offset by other environmental changes such as increasing tropospheric ozone (O₃) or vary based on species-specific growth responses to elevated CO₂. To understand how projected increases in atmospheric CO₂ and O₃ alter SOM formation, we used physical fractionation to characterize soil C and N at the Rhinelander Free Air CO₂-O₃ Enrichment (FACE) experiment. Tracer amounts of ¹⁵NH₄⁺ were applied to the forest floor of Populus tremuloides, P. tremuloides-Betula papyrifera and P. tremuloides-Acer saccharum communities exposed to factorial CO₂ and O₃ treatments. The ¹⁵N tracer and strongly depleted ¹³C-CO₂ were traced into SOM fractions over four years. Over time, C and N increased in coarse particulate organic matter (cPOM) and decreased in mineral-associated organic matter (MAOM) under elevated CO₂ relative to ambient CO₂. As main effects, neither CO₂ nor O₃ significantly altered ¹⁵N recovery in SOM. Elevated CO₂ significantly increased new C in all SOM fractions, and significantly decreased old C in fine POM (fPOM) and MAOM over the duration of our study. Overall, our observations indicate that elevated CO₂ has altered SOM cycling at this site to favor C and N accumulation in less stable pools, with more rapid turnover. Elevated O₃ had the opposite effect, significantly reducing cPOM N by 15% and significantly increasing the C:N ratio by 7%. Our results demonstrate that CO₂ can enhance SOM turnover, potentially limiting long-term C sequestration in terrestrial ecosystems; plant community composition is an important determinant of the magnitude of this response.     

Genetics and molecular mapping of the naked grains in hexaploid oat

Avena sativa L. subsp. nudisativa has the ability to produce naked grains. Genetic studies on the naked trait of oat began over a century ago, but the genetic and molecular factors associated with the expression of this trait have not been fully clarified. The objectives of this study were to evaluate the naked trait in two oat populations of recombinant inbred lines (RILs), to determine the number of genes, to estimate the heritability, and to map genomic regions associated with the naked trait in hexaploid oat. Parental lines and RILs of each population were screened for the naked trait from plants grown in the field over a 2 year period. Based on the phenotypic data, the oat RILs were classed as naked, partially naked, partially hulled and hulled. In both populations and years, a great number of RILs showed variable expressivity for the naked trait. The genetic analysis indicated the action of a major gene (N1) with the action of modifying genes controlling the formation of naked grains. The results of the estimate of heritability show that environmental conditions do not have a great influence in determining the naked trait. The quantitative trait loci analysis detected a genomic region with a large effect on the naked trait that explained more than 50% of the phenotypic variation. Further studies are needed to validate the use of these molecular markers to assist breeding programs to select high quality and stable naked oat cultivars.     

The value of linking paleoecological and neoecological perspectives to understand spatially-explicit ecosystem resilience

Context

Predicting ecosystem resilience is a challenge, especially as climate change alters disturbance regimes and conditions for recovery. Recent research has highlighted the importance of spatially-explicit disturbance and resilience processes to long-term ecosystem dynamics. “Neoecological” approaches characterize resilience mechanisms at relatively fine spatio-temporal resolutions, but results are difficult to extrapolate across broad temporal scales or climatic ranges. Paleoecological methodologies can consider the effects of climates that differ from today. However, they are often limited to coarse-grained spatio-temporal resolutions.

Methods

In this synthesis, we describe implicit and explicit examples of studies that incorporate both neo- and paleoecological approaches. We propose ways to build on the strengths of both approaches in an explicit and proactive fashion.

Results

Linking the two approaches is a powerful way to surpass their respective limitations. Aligning spatial scales is critical: Paleoecological sampling design should incorporate knowledge of the spatial characteristics of the disturbance process, and neoecological studies benefit from a longer-term context to their conclusions. In some cases, modeling can incorporate non-spatial data from paleoecological records or emerging spatial paleo-data networks with mechanistic disturbance/recovery processes that operate at fine spatiotemporal scales.

Conclusions

Linking these two complementary approaches is a powerful way to build a complete understanding of ecosystem disturbance and resilience.