Specific Diagnosis of Two Root-Knot Nematodes, Meloidogyne chitwoodi and M. fallax, with Satellite DNA Probes

ABSTRACT Meloidogyne chitwoodi and M. fallax are serious pests of potato, and both species have been recently designated as quarantine organisms in the European Community and in Canada. The sympatric and less damaging species M. hapla is often found associated with both of them under temperate climates. Here, we describe the use of satellite DNA (satDNA) sequences previously isolated from these three root-knot nematode species for the development of specific diagnostic procedures. In dot-blot experiments, it was unambiguously possible to separate M. chitwoodi and M. fallax from M. hapla using satDNA monomers as probes. In squash-blot experiments, satDNAs allowed discrimination between single individuals of M. chitwoodi or M. fallax from M. hapla, even within root tissues, without the need for DNA purification. The same results were obtained with radioactive or digoxigenin-labeled probes with no loss of sensitivity in detection. M. fallax and M. chitwoodi could not be distinguished. From this study, it is concluded that such cloned satDNA sequences may constitute a powerful tool for the identification and management of Meloidogyne spp. populations in the field and for the implementation of quarantine regulations against these pests.     

Variation in resistance to the root-knot nematode Meloidogyne incognita in tomato genotypes bearing the Mi gene

Root-knot nematodes ( Meloidogyne spp.) are among the main pathogens of tomato ( Lycopersicon esculentum ) worldwide. Plant resistance is currently the method of choice for controlling these pests and all the commercially available resistant cultivars carry the dominant Mi gene, which confers resistance to the three main species Meloidogyne arenaria , M. incognita and M. javanica . However the emergence of virulent biotypes able to overcome the tomato resistance gene may constitute a severe limitation to such a control strategy. To date, little was known of the possible influence of the homozygous vs heterozygous allelic state of the Mi locus, or the tomato genetic background, on the expression of the resistance. In order to test both these factors, the resistance was evaluated of a large panel of L. esculentum genotypes (selected from the Vilmorin germplasm stock collection) to seven M. incognita lines avirulent or virulent against the Mi gene. Plant resistance was estimated by counting the egg masses on the root systems after inoculation with second-stage juveniles (J₂). Reproduction of the nematodes was similar or, more often, significantly higher on heterozygous tomato genotypes than on homozygous ones, suggesting a possible dosage effect of the Mi gene. Data also indicated that the tomato genetic background had a major effect on the variations observed in nematode reproduction, especially when tomato genotypes were heterozygous for the Mi gene. These results have important consequences in terms of breeding strategies and durability of the resistance conferred by the Mi gene.     

Cutaneous extraskeletal mesenchymal chondrosarcoma in a cat

A 4-year-old, male cat was presented with a fixed, subcutaneous mass in the lumbosacral region. A histopathological examination revealed a well-defined but nonencapsulated neoplasm characterized by a proliferation of predominantly spindle cells, with high mitotic activity. Interspersed between these cells were single cellular elements with chondroid differentiation. Large areas of cartilaginous tissue with foci of endochondral ossification, necrosis and myxoid tissue were also observed within the neoplastic parenchyma. A diagnosis of extraskeletal mesenchymal chondrosarcoma was made based on the histological pattern - characterized by the coexistence of cartilaginous islands and undifferentiated mesenchymal cells, results of Alcian blue staining at various pH, immunohistochemical reactivity against vimentin and S-100, and the absence of skeletal involvement or other primary tumour sites. Clinical history of the cat excluded traumas, vaccinations or other types of subcutaneous inoculation. Six months on from surgical treatment, neither recurrence nor metastases have been detected.     

Immunohistochemical expression of heat shock proteins,p63 and androgen receptor in benign prostatic hyperplasia and prostatic carcinoma in the dog

This study compared heat shock proteins Hsp60, Hsp72 and Hsp73, along with p63 and androgen receptor (AR) immunoexpression between 16 cases of benign prostatic hyperplasia (BPH) and 11 prostatic carcinomas (PCa) in dogs. The proportion of Hsp60‐positive cells was higher in PCa compared with BPH (P = 0.033), whereas the frequency and intensity of Hsp73 immunostaining did not differ significantly between the two groups. Hsp72‐immunostained nuclei formed a discontinuous layer along the basement membrane in BPH, whereas cells in this layer in PCa were negative or weakly positive. Hsp72 nuclear score showed significant positive associations with both p63 (P = 0.016) and AR (P = 0.009) scores. Double immunofluorescence revealed Hsp72‐p63 and Hsp72‐AR co‐expressions in basal cell nuclei. Aberrant cytoplasmic p63 immunolabelling was observed in 3 of 11 PCa cases. These results suggest a role of the combined expression of Hsp72, p63 and AR in basal epithelial cells in canine BPH and PCa.     

Economics of Variable Rate Lime in Indiana

In Indiana, variable rate application (VRA) of lime is often considered a good place to start site-specific management (SSM). This is because soil pH is one of the most variable of manageable soil characteristics in the state, the availability of essential nutrients is closely related to soil pH, and because spreaders can be retrofitted relatively inexpensively to do VRA. The objective of this study is to evaluate the profitability of VRA for lime as a stand-alone activity. The methodology involves a spreadsheet model using corn and soybean pH response functions estimated with small plot data. The overall results indicate increased annual returns to corn and soybean production with site-specific pH management strategies. On average, SSM with agronomic recommendations provides an increased annual return of $7.24 per hectare (ha) (+1.78%). SSM with the economic decision rule provides an average increase in annual return of $19.55 ha^–1 (+4.82%). Information strategy, which uses site-specific information to determine the economically optimal uniform rate of lime, provides an average increase in annual return of $14.38 ha^–1 (+3.54%).     

Tumour‐infiltrating lymphocytes in canine melanocytic tumours: An investigation on the prognostic role of CD3+ and CD20+ lymphocytic populations

The study of the immune response in several types of tumours has been rapidly increasing in recent years with the dual aim of understanding the interactions between neoplastic and immune cells and their importance in cancer pathogenesis and progression, as well as identifying targets for cancer immunotherapy. Despite being considered one of the most immunogenic tumour types, melanoma can progress in the presence of abundant lymphocytic infiltration, therefore suggesting that the immune response is not able to efficiently control tumour growth. The purpose of this study was to investigate whether the density, distribution and grade of tumour‐infiltrating lymphocytes (TILs) in 97 canine melanocytic tumours is associated with histologic indicators of malignancy and can be considered a prognostic factor in the dog. As a further step in the characterization of the immune response in melanocytic tumours, an immunohistochemical investigation was performed to evaluate the two main populations of TILs, T‐lymphocytes (CD3⁺) and B‐lymphocytes (CD20⁺). The results of our study show that TILs are present in a large proportion of canine melanocytic tumours, especially in oral melanomas, and that the infiltrate is usually mild. The quantity of CD20⁺ TILs was significantly associated with some histologic prognostic factors, such as the mitotic count, the cellular pleomorphism and the percentage of pigmented cells. Remarkably, a high infiltration of CD20⁺ TILs was associated with tumour‐related death, presence of metastasis/recurrence, shorter overall and disease‐free survival, increased hazard of death and of developing recurrence/metastasis, hence representing a potential new negative prognostic factor in canine melanocytic tumours.     

PDGFs and PDGFRs in canine osteosarcoma: New targets for innovative therapeutic strategies in comparative oncology

Platelet derived growth factor receptor (PDGFR)α and PDGFRβ are tyrosine kinase receptors that are overexpressed in 70–80% of human osteosarcomas (OSAs) and may be suitable therapeutic targets for specific kinase inhibitors (TKIs). Canine OSA shows histopathological and clinical features similar to human OSA, and is considered an excellent model in comparative oncology. This study investigated PDGF-A, PDGF-B, PDGFRα and PDGFRβ expression in 33 canine OSA samples by immunohistochemistry and in seven primary canine OSA cell lines by Western blot and quantitative PCR analysis.Immunohistochemical data showed that PDGF-A and PDGF-B are expressed in 42% and 60% of the OSAs analysed, respectively, while PDGFRα and PDGFRβ were expressed in 78% and 81% of cases, respectively. Quantitative PCR data showed that all canine OSA cell lines overexpressed PDGFRα, while 6/7 overexpressed PDGFRβ and PDGF-A relative to a normal osteoblastic cell line. Moreover, in vitro treatment with a specific PDGFR inhibitor, AG1296, caused a dose- and time-dependent decrease in AKT phosphorylation. Collectively, these data show that PDGFRs/PDGFs are co-expressed in canine osteosarcomas, which suggests that an autocrine and/or paracrine loop is involved and that they play an important role in the aetiology of OSA. PDGFRs may be suitable targets for the treatment of canine OSA with a specific TKI.     

Epithelial‐to‐mesenchymal transition: immunohistochemical investigation of related molecules in canine cutaneous epithelial tumours

Background –  Epithelial‐to‐mesenchymal transition (EMT) is a multistep process, important in tumour invasion and metastasis, characterized by loss of epithelial markers, redistribution of β‐catenin and gain of mesenchymal markers. Hyposthesis/Objectives –  Our aim was to investigate the immunohistochemical aberrant expression of cytokeratin, vimentin, survivin and heat shock protein 72 (Hsp72) in canine cutaneous epithelial tumours, to understand the association of expression of these molecules with features of malignancy and their role in the EMT phenotype. Methods –  Ten canine squamous cell carcinomas (SCCs; one with lymph node metastasis), 30 canine hair follicle tumours (six pilomatricomas, eight infundibular keratinizing acanthomas, six trichoepitheliomas and 10 trichoblastomas) and five normal skin samples were investigated by immunohistochemistry using specific anti‐vimentin, ‐cytokeratin, ‐survivin and ‐Hsp72 antibodies. A semi‐quantitative method was used to analyse the results, as follows: 0 to <5%; ≥5 to <10%; ≥10 to <25%; and ≥25% of positive cells. Immunofluorescence was performed to investigate survivin–vimentin and survivin–Hsp72 colocalization in selected SCCs. Results –  In malignant hair follicle tumours and SCCs, a reduced intensity of cytokeratin and increased survivin and Hsp72 expression were observed. In SCCs, loss of cytokeratin expression and vimentin immunolabelling, suggestive of the EMT phenotype, were evident in <5% of neoplastic cells in the front of tumour invasion. In the same areas, strong nuclear survivin and cytoplasmic Hsp72 staining was evident, often colocalizing. Only a few neoplastic cells in the front of tumour invasion showed vimentin–survivin colocalization. Conclusions and clinical importance –  A possible simultaneous involvement of survivin and Hsp72 in tumour invasion and the multistep process of EMT of cutaneous epithelial tumours of dogs is suggested.     

Use of Lorenz curves and Gini coefficients to assess yield inequality within paddocks

The opportunity for site-specific management of crops depends on both the magnitude and spatial structure of yield variation. This study explored the applicability of Lorenz curves and Gini coefficients (G) to characterise the magnitude of the variation in grain yield. Maize crops were grown in farmers fields in a semi-arid region of central Argentina. Major sources of yield variation between and within paddocks included season, soil type and topography, rate of nitrogen fertiliser (nil to 132 kg N ha⁻¹), and the interactions among these factors. Nitrogen treatments were applied in a complete block strip trial (strip size700 m×9.8 m) with three replicates. Data were collected with an AgLeader™ yield monitor, and GIS software was used to create 9.8 m×9.8 m grids over the observations. Average yield in 0.7–2.8 ha field sections ranged from 1.6 to 7.0 t ha⁻¹. Gini coefficient ranged from 0.027 to 0.191 whereas its theoretical limits are 0 for a perfectly uniform population, and 1 for a theoretical population of infinite size where all units but one yield 0. Conditions conducive to high yield, e.g. adequate availability of nitrogen and water, reduced crop yield inequality, as quantified with G. The agronomic relevance of G was summarised in an inverse relationship with yield. Lorenz curves seemed particularly apt to present crop heterogeneity in terms of inequality, and to highlight the relative contribution of low- and high-yielding sections of the field to total paddock yield. Lorenz curves and Gini coefficients provide a potentially useful extension tool, a complement to yield maps and other statistical indices of yield variation, and further contact points between site-specific management, economics and ecology.