Franco Mutinelli

Clinical, Gross, and Microscopic Findings in Different Avian Species Naturally Infected During the H7N1 Low- and High-Pathogenicity Avian Influenza Epidemics in Italy During 1999 and 2000

Abstract From the end of March to the beginning of December 1999, an epidemic of low-pathogenicity avian influenza (LPAI), caused by a H7N1 type A influenza virus, affected the intensively reared poultry population of Northeastern Italy. A total of 199 flocks were diagnosed with influenza infection. The highest number affected flocks were in meat turkeys (164), with only a limited number of turkey breeder, chicken (breeders, broilers, and table egg layers), and guinea fowl flocks infected. Following the circulation of the LPAI virus in a susceptible population for several months, a high-pathogenicity avian influenza (HPAI) virus emerged. Over 13 million birds on 413 different premises were affected by the HPAI virus, including turkey, chicken, guinea fowl, pheasant, Japanese quail, ostrich, and waterfowl flocks. In the present paper we report on the clinical, gross, histopathological, and immunohistochemical investigations performed on different avian species naturally infected by the LPAI virus and the HPAI virus.

Screening of natural compounds for the control of nosema disease in honeybees (Apis mellifera)

The potential of some natural compounds (thymol, vetiver essential oil, lysozyme, resveratrol) for the control of nosema infection in honeybees was evaluated. A first trial aimed at screening substances, in candy preparations, on the basis of their toxicity to honeybees and bees’ dietary preferences. None of the tested substances showed an increased bee mortality or decreased bee preference, and were therefore considered suitable for further testing. In the second trial the effects of the natural compounds on nosema diseased honeybees were evaluated: bees were individually dosed with nosema spores and fed candies prepared with the screened substances. The results showed that bees fed with thymol and resveratrol candies had significantly lower infection rates, and bees supplied with resveratrol prepared candy also lived significantly longer. We suggest that thymol and resveratrol could be useful in alternative strategies for the control of nosema disease.ZusammenfassungEffektive Wirkstoffe zur Bekämpfung der Nosemose werden europaweit dringend benötigt, da es keine zugelassenen Präparate gibt und sich die Krankheit zunehmend ausbreitet. Die Nosemose ist ein weltweites Problem für die Bienenhaltung. Die Krankheit wird durch die Mikrosporidien Nosema apis Zander und Nosema ceranae Fries verursacht, wobei sie als obligate Parasiten des Darmepithels die adulten Bienen schädigen. Die Ansteckung erfolgt über Sporen, die in großer Menge im Kot von kranken Bienen nachweisbar sind. Sie sind länger als ein Jahr in Waben und Wasser infektiös und können zusätzlich über Trophallaxis verbreitet werden. Das einzige bisher wirksame Produkt zur Bekämpfung der Nosemose ist das Antibiotikum Fumagillin, das aber in vielen Ländern verboten ist. Das Ziel dieser Untersuchung war es, verschiedene Naturstoffe, die ohne das Risiko einer Rückstandsbildung in Bienenprodukten eingesetzt werden können, hinsichtlich ihrer Wirksamkeit gegen die Nosemose zu testen. Da von Thymol, Vetiveröl, Lysozym und Resveratrol bereits Berichte über inhibitorische Wirkungen auf Mikrosporidien vorlagen, wurden diese für die Tests ausgewählt und den Bienen über das Futter appliziert. In einem ersten Versuch wurden die Substanzen in zwei unterschiedlichen Konzentrationen hinsichtlich der toxischen Effekte und ihrer Akzeptanz als Futterzusatz geprüft. Jeweils 20 Bienen wurden in kleinen Holzkäfigen bei 33 °C und 70 % RF im Dunkeln gehalten und die Futterabnahme sowie die Anzahl toter Bienen bis zum elften Tag erfasst. Dabei wurden keine Unterschiede zwischen den unterschiedlich gefütterten Versuchsgruppen festgestellt (Abb. 1, 2). In einem zweiten Versuch wurden Nosema-freie Bienen einzeln mit 1 μL einer sporenhaltigen Zuckerlösung mit jeweils 18000 N. ceranae Sporen gefüttert. 30 Bienen pro Käfig wurden mit Zuckerteig, der jeweils die höchste getestete Konzentration der o. a. Substanzen enthielt, gefüttert. Aus jedem Käfig wurden 8, 13, 19 und 25 Tage nach Versuchsbeginn je 2 Bienen entnommen und deren Sporenbelastung bestimmt, um die Entwicklung der Nosemainfektion zu verfolgen. Zusätzlich wurde täglich die Anzahl toter Bienen registriert, um die Überlebensraten zu berechnen. Die mit Resveratrol gefütterten Bienen wiesen eine signifikant höhere mittlere Überlebenszeit auf (Abb. 3, Tab. II). Bei Bienen, die mit Resveratrol und Thymol gefüttert wurden, kam es am letzten Untersuchungszeitpunkt (Tag 25) zu einer Reduzierung des Nosema-Infektionsgrades, während in den anderen Gruppen die Anzahl an Sporen zunahm (Tab. III). Bei den mit Thymol gefütterten Bienen war die Sporenkonzentration zu allen Untersuchungszeitpunkten am geringsten. Es scheint, dass Thymol und Resveratrol geeignete Substanzen sind, um die Entwicklung des Parasiten zu hemmen. Thymol wird bereits als wirksames Akarizid gegen Varroa-Milben in der Imkerpraxis eingesetzt und Resveratrol ist bekannt für seine spezifischen antioxidativen Eigenschaften, die in anderen Versuchen bereits zur Verlängerung der Lebensdauer kleiner Invertebraten geführt haben.

Liposarcoma at the site of an implanted microchip in a dog.

An 11-year-old male, mixed breed dog was examined by the referring veterinarian for a subcutaneous mass, located in the lateral region of the neck, craniodorsally to the top of the left shoulder blade. The dog was regularly vaccinated and identified by a microchip (Indexel, Merial), which had been implanted in April 2000. The owner reported that he had first noticed the development of a small nodule at the site of the microchip implantation, in November 2001. At time of referral the mass was 10 6 cm, firm and painless. Using an image intensifier, the microchip was located in the subcutaneous fascia at the base of the mass (Fig. 1). This mass appeared to have developed in the subcutis, around the microchip itself, causing bulging of the skin contour. A complete physical examination excluded any further changes. Results of a complete pre-operative blood count and serum biochemistry analysis were within normal ranges. Thoracic radiographs, lateral and dorsoventral views, taken to exclude pulmonary metastasis, were normal. Following the veterinarian s advice, the owner decided to have the mass excised. On April 2003, surgery was performed under general anaesthesia. An elliptical excision with wide margins was made around the mass, which was well delimited and easily separated from the surrounding tissues. After surgery, the dog was discharged with routine antibiotic treatment for two weeks. Despite some post-operative exudation, treated by drainage, the surgical wound was completely healed within one month. Macroscopically, the mass appeared dark reddish due to haemorrhages and abundant vascularisation; it had a bumpy surface and was friable on sectioning. The intact

Expression of Ki67, BCL-2, and COX-2 in Canine Cutaneous Mast Cell Tumors Association With Grading and Prognosis

The expression of Ki67, BCL-2, and COX-2 was investigated in 53 canine cutaneous mast cell tumors (MCTs) by immunohistochemistry and quantitative real time polymerase chain reaction (qPCR) to evaluate their prognostic significance and the association with the histologic grading and the mitotic index (MI). MCTs were graded according to the Patnaik grading system and the novel 2-tier grading system proposed by Kiupel. The numbers of mitotic figures/10 high-power fields (MI) were counted. Both grading systems were significantly associated with prognosis. The Patnaik grading was of limited prognostic value for grade 2 MCTs, with 23% being associated with mortality. The concordance among pathologists was strongly improved by the application of the 2-tier grading system, and 71% of high-grade MCTs were associated with a high mortality rate. MI and Ki67 protein expression were significantly associated with grading and survival. No significant association between BCL-2 protein expression and either grading system or health status was observed. BCL-2 mRNA expression was significantly higher in grade 2 than in grade 1 MCTs, while no statistically significant differences were detected between low- and high-grade MCTs. The increased BCL-2 mRNA level was significantly associated with increased mortality rate. The COX-2 protein expression was detected in 78% of the MCTs investigated. However, neither association with the tumor grade nor with the health status was observed. COX-2 mRNA was significantly up-regulated in MCTs compared to surgical margins and control skin tissue, but it was neither associated with tumor grade nor with survival.

Expression of Matrix Metalloproteinases, Tissue Inhibitors of Metalloproteinases and Vascular Endothelial Growth Factor in Canine Mast Cell Tumours.

Degradation of the extracellular matrix and angiogenesis are associated with tumour invasion and metastasis in human and canine neoplasia. Matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and vascular endothelial growth factor-A (VEGF-A) are key mediators of these respective processes. Mast cell tumour (MCT) is the most common malignant cutaneous tumour in dogs. MCTs are always considered potentially malignant, but their true metastatic potential is unknown. In the present study, samples from seven grade 1, 22 grade 2 and six grade 3 MCTs were subjected to quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) to evaluate MMP-2, MMP-9, membrane-type 1 MMP (MT1-MMP), TIMP-2 and VEGF-A mRNA and protein expression. Gelatin zymography (GZ) was also performed to evaluate MMP-2 and MMP-9 activity. MMP-9 and VEGF-A mRNA increased with histological grade, while TIMP-2 decreased with increasing grade. Gene expression data obtained for MMP-9, VEGF-A and TIMP-2 were confirmed by IHC for evaluation of the respective proteins. In contrast, MMP-2 and MT1-MMP had variable, but similar, expression for both mRNA and protein. Despite the high variability observed, there was correlation between MMP-2 and MT1-MMP mRNA expression (r=+0.91, P<0.0001). The MMP-2:TIMP-2 and MMP-9:TIMP-1 mRNA ratios showed an imbalance between MMPs and their specific inhibitors in MCTs, which increased with the histological grade. Finally, the activities of both latent and active forms of MMP-2 and MMP-9 were evaluated by GZ and there were significant increases in their activities with increasing histological grade and immunohistochemical expression. This study demonstrates that MMP-9, TIMP-2 and VEGF-A expression is related to histological grade and suggests that these markers are possible indicators of malignancy and targets for therapeutic strategies.

Matrix metalloproteinases and their inhibitors in canine mammary tumors

BackgroundMalignant canine mammary tumors represent 50% of all neoplasms in female dogs. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in tumor progression, and they are also associated with the reactive stroma, which provides structural and vascular support for tumor growth.ResultsMMP-2, MMP-9 and MT1-MMP were expressed at both the mRNA and protein levels in tumor samples. MMP-2 and MMP-9 immunohistochemical reactions were evident both in the epithelial tumor cells and in the stromal compartment to varying degrees; in particular, the intensity of the MMP-2 staining was stronger in the stromal fibroblasts close to epithelial tumor cells in simple carcinomas than in adenomas. These data were supported by gelatin-zymography; bands for the active form of MMP-2 were found in 94% of carcinoma samples, compared with 17% of benign tumor samples. The gene expression and immunohistochemical results for MT1-MMP were comparable to those for MMP-2. The immunoreactivity for MMP-13 and TIMP-2 was lower in carcinomas than in adenomas, confirming the mRNA data for MMP-13 and the other MMP inhibitors that were evaluated. The active form of MMP-9, but not the active form of MMP-2, was identified in the plasma of all of the tested dogs.ConclusionsOur findings suggest that MMP-9, MMP-2 and MT1-MMP, which are synthesized by epithelial cancer cells and cancer-associated fibroblasts, play an important role in malignant canine mammary tumors. The reduction of MMP-13 and TIMP-2 could also be a significant step in malignant transformation. MMP-2 and MT1-MMP could be further evaluated as future biomarkers for predicting the progression and prognosis of canine mammary tumors.

Honey bee colony losses in Italy

Franco Mutinelli, Cecilia Costa, Marco Lodesani, Alessandra Baggio, Piotr Medrzycki, Giovanni Formato and Claudio Porrini Istituto Zooprofilattico Sperimentale delle Venezie, National Reference Laboratory for Beekeeping, Viale dell’Universita’ 10, 35020 Legnaro (PD), Italy. Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Unita di Ricerca di Apicoltura e Bachicoltura (CRA-API), Via di Saliceto 80, 40128 Bologna, Italy. Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Via Appia Nuova 1411, 00178 Roma, Italy. Dipartimento di Scienze e Tecnologie Agroambientali (DiSTA), Universita di Bologna, Viale G. Fanin 42, 40127 Bologna, Italy.

The Status of Honey Bee Health in Italy: Results from the Nationwide Bee Monitoring Network

In Italy a nation-wide monitoring network was established in 2009 in response to significant honey bee colony mortality reported during 2008. The network comprised of approximately 100 apiaries located across Italy. Colonies were sampled four times per year, in order to assess the health status and to collect samples for pathogen, chemical and pollen analyses. The prevalence of Nosema ceranae ranged, on average, from 47–69% in 2009 and from 30–60% in 2010, with strong seasonal variation. Virus prevalence was higher in 2010 than in 2009. The most widespread viruses were BQCV, DWV and SBV. The most frequent pesticides in all hive contents were organophosphates and pyrethroids such as coumaphos and tau-fluvalinate. Beeswax was the most frequently contaminated hive product, with 40% of samples positive and 13% having multiple residues, while 27% of bee-bread and 12% of honey bee samples were contaminated. Colony losses in 2009/10 were on average 19%, with no major differences between regions of Italy. In 2009, the presence of DWV in autumn was positively correlated with colony losses. Similarly, hive mortality was higher in BQCV infected colonies in the first and second visits of the year. In 2010, colony losses were significantly related to the presence of pesticides in honey bees during the second sampling period. Honey bee exposure to poisons in spring could have a negative impact at the colony level, contributing to increase colony mortality during the beekeeping season. In both 2009 and 2010, colony mortality rates were positively related to the percentage of agricultural land surrounding apiaries, supporting the importance of land use for honey bee health.

Detection of Nosema ceranae in Apis mellifera from Bosnia and Herzegovina.

Istituto Zooprofilattico Sperimentale delle Venezie, National Reference Laboratory for Beekeeping, Legnaro (Padova), Italy. Received 28 September 2009, accepted subject to revision 14 October 2009, accepted for publication 4 November 2009. *Corresponding author: Email: santracv@veterinarskiinstitutrs.com

Global gene expression analysis of canine cutaneous mast cell tumor: could molecular profiling be useful for subtype classification and prognostication?

Prognosis and therapeutic management of dogs with cutaneous mast cell tumors (MCTs) depend on clinical stage and histological grade. However, the prognostic value of this latter is still questionable. In the present study, MCT transcriptome was analyzed to identify a set of candidate genes potentially useful for predicting the biological behavior of MCTs. Fifty-one canine MCT biopsies were analyzed. Isolated and purified total RNAs were individually hybridized to the Agilent Canine V2 4x44k DNA microarray. The comparison of reference differentiated and undifferentiated MCT transcriptome revealed a total of 597 differentially expressed genes (147 down-regulated and 450 up-regulated). The functional analysis of this set of genes provided evidence that they were mainly involved in cell cycle, DNA replication, p53 signaling pathway, nucleotide excision repair and pyrimidine metabolism. Class prediction analysis identified 13 transcripts providing the greatest accuracy of class prediction and divided samples into two categories (differentiated and undifferentiated), harboring a different prognosis. The Principal Component Analysis of all samples, made by using the selected 13 markers, confirmed MCT classification. The first three components accounted for 99.924% of the total variance. This molecular classification significantly correlated with survival time (pu200a=u200a0.0026). Furthermore, among all marker genes, a significant association was found between mRNA expression and MCT-related mortality for FOXM1, GSN, FEN1 and KPNA2 (p<0.05). Finally, marker genes mRNA expression was evaluated in a cohort of 22 independent samples. Data obtained enabled to identify MCT cases with different prognosis. Overall, the molecular characterization of canine MCT transcriptome allowed the identification of a set of 13 transcripts that clearly separated differentiated from undifferentiated MCTs, thus predicting outcome regardless of the histological grade. These results may have clinical relevance and warrant future validation in a prospective study.