Active and passive surveillance for bat lyssaviruses in Italy revealed serological evidence for their circulation in three bat species.

The wide geographical distribution and genetic diversity of bat-associated lyssaviruses (LYSVs) across Europe suggest that similar viruses may also be harboured in Italian insectivorous bats. Indeed, bats were first included within the passive national surveillance programme for rabies in wildlife in the 1980s, while active surveillance has been performed since 2008. The active surveillance strategies implemented allowed us to detect neutralizing antibodies directed towards European bat 1 lyssavirus in six out of the nine maternity colonies object of the study across the whole country. Seropositive bats were Myotis myotis, M. blythii and Tadarida teniotis. On the contrary, the virus was neither detected through passive nor active surveillance, suggesting that fatal neurological infection is rare also in seropositive colonies. Although the number of tested samples has steadily increased in recent years, submission turned out to be rather sporadic and did not include carcasses from bat species that account for the majority of LYSVs cases in Europe, such as Eptesicus serotinus, M. daubentonii, M. dasycneme and M. nattereri. A closer collaboration with bat handlers is therefore mandatory to improve passive surveillance and decrypt the significance of serological data obtained up to now.

Frequency of DEA 1 antigen in 1037 mongrel and PUREBREED dogs in ITALY.

BACKGROUND: The prevalence of dog erythrocyte antigen (DEA 1) in canine population is approximately 40-60%. Often data are limited to a small number of breeds and/or dogs. The aims of this study were to evaluate frequency of DEA 1 in a large population of purebred and mongrel dogs including Italian native breeds and to recognize a possible association between DEA 1 and breed, sex, and genetic and phenotypical/functional classifications of breeds. Frequencies of DEA 1 blood group collected from screened/enrolled blood donors and from healthy and sick dogs were retrospectively evaluated. The breed and the sex were recorded when available. DEA 1 blood typing was assessed by immunocromatographic test on K3EDTA blood samples. The prevalence of DEA 1 antigen was statistically related to breed, gender, Fédération Cynologique Internationale (FCI) and genotypic grouping. RESULTS: Sixty-two per cent dogs resulted DEA 1+ and 38% DEA 1-. DEA 1- was statistically associated with Dogo Argentino, Dobermann, German Shepherd, Boxer, Corso dogs, the molossian dogs, the FCI group 1, 2 and 3 and the genetic groups "working dogs" and "mastiff". DEA 1+ was statistically associated with Rottweiler, Briquet Griffon Vendéen, Bernese mountain dog, Golden Retriever, the hunting breeds, the FCI group 4, 6, 7 and 8 and the genetic groups "scent hounds" and "retrievers". No gender association was observed. CONCLUSIONS: Data obtained by this work may be clinically useful to drive blood donor enrollment and selection among different breeds.

Identification of undeclared sources of animal origin in canine dry foods used in dietary elimination trials.

Failure to respond to commercial limited antigen diets can occur in dogs kept on a dietary trial for the diagnosis of adverse food reaction (AFR). The aim of this study was to assess twelve canine dry limited antigen diets (eleven novel protein diets and one hydrolysed diet) for potential contamination by ingredients of animal origin not mentioned on the label. The validity of the two methods adopted for the detection of such food antigens was also evaluated. Each dietary product was analysed by microscopy analysis using the official method described in Commission Regulation EC 152/2009 with the aim of identifying bone fragments of different zoological classes (mammalian, avian and fish) and by polymerase chain reaction (PCR) for the identification of DNA of animal origin. Discrepancies between the results obtained by PCR and/or microscopy analysis and the ingredients listed on pet food packages were found. Only in two pet foods did the results of both analyses match the ingredients listed on the label. In the remaining ten samples, microscopy detected bone fragments from one or two unpredicted zoological classes, revealing avian fragments in six of ten samples followed by those of fish in five of ten and mammalian fragments in four of ten. In two samples, microscopy analysis identified a contamination that would have otherwise passed unobserved if only PCR had been used. However, PCR confirmed the presence of all the zoological classes detected by microscopy and also identified the DNA of an additional unexpected zoological class in two samples. Dogs might fail to respond to commercial limited antigen diets because such diets are contaminated with potential allergens. Both PCR and microscopy analysis are required to guarantee the absence of undeclared animal sources in pet foods. Before ruling out AFR, a novel protein home-made diet should be considered if the dog is unresponsive to a commercial regimen.

Comparative assay of fluorescent antibody test results among twelve European National Reference Laboratories using various anti-rabies conjugates.

Twelve National Reference Laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies conjugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive brain tissues infected with various lyssavirus species as well as negative samples were analyzed blindly using a standardized FAT procedure. Conjugates B, C, D, and E were found to be significantly more effective than conjugate A for GS7 (French RABV) diluted samples (1/8 and 1/100) while the frequency of concordant results of conjugates C and D differ significantly from conjugates A, B and E for CVS 27. For detection of EBLV-1 strains, conjugates C and D also presented a significantly lower frequency of discordant results compared to conjugates A, B and E. Conjugates B, C and D were found to be significantly more effective than conjugates E and A for EBLV-2 and ABLV samples. In view of these results, conjugates C and D set themselves apart from the others and appeared as the most effective of this 5-panel conjugates. This study clearly demonstrates that the variability of conjugates used by National Reference Laboratories can potentially lead to discordant results and influence assay sensitivity. In case of false negative results this could have a dramatic impact if the animal under investigation is responsible for human exposure. To avoid such situations, confirmatory tests should be implemented.

Expression of the aryl hydrocarbon receptor pathway and cyclooxygenase-2 in dog tumors.

In humans, the aryl hydrocarbon receptor (AHR) gene battery constitutes a set of contaminant-responsive genes, which have been recently shown to be involved in the regulation of several patho-physiological conditions, including tumorigenesis. As the domestic dog represents a valuable animal model in comparative oncology, mRNA levels of cytochromes P450 1A1, 1A2 and 1B1 (CYP1A1, 1A2 and 1B1), AHR, AHR nuclear translocator (ARNT), AHR repressor (AHRR, whose partial sequence was here obtained) and cyclooxygenase-2 (COX2) were measured in dog control tissues (liver, skin, mammary gland and bone), in 47 mast cell tumors (MCTs), 32 mammary tumors (MTs), 5 osteosarcoma (OSA) and related surgical margins. Target genes were constitutively expressed in the dog, confirming the available human data. Furthermore, their pattern of expression in tumor biopsies was comparable to that already described in a variety of human cancers; in particular, both AHR and COX2 genes were up-regulated and positively correlated, while CYP1A1 and CYP1A2 mRNAs were generally poorly expressed. This work demonstrated for the first time that target mRNAs are expressed in neoplastic tissues of dogs, thereby increasing the knowledge about dog cancer biology and confirming this species as an useful animal model for comparative studies on human oncology.

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.

Histology as a valid and reliable tool to differentiate fresh from frozen-thawed fish.

Selling fish products as fresh when they have actually been frozen and thawed is a common fraudulent practice in seafood retailing. Unlike fish products frozen to protect them against degenerative changes during transportation and to extend the product's storage life, fish intended for raw consumption in European countries must be previously frozen at -20° C for at least 24 h to kill parasites. The aim of this study was to use histological analysis to distinguish between fresh and frozen-thawed fish and to evaluate this method for use as a routine screening technique in compliance with the requirements of European Commission Regulation No. 882/2004 on official food and feed controls. Method performance (i.e., accuracy and precision) was evaluated on tissue samples from three common Mediterranean fish species; the evaluation was subsequently extended to include samples from 35 fish species in a second experiment to test for method robustness. Method accuracy was tested by comparing histological results against a "gold standard" obtained from the analysis of frozen and unfrozen fish samples prepared for the study. Method precision was evaluated according to interrater agreement (i.e., three laboratories with expertise in histopathology in the first experiment and three expert analysts in the second experiment) by estimating Cohen's kappa (and corresponding 95 % confidence intervals) for each pair of laboratories and experts and the combined Cohen's kappa for all three experts and laboratories. The observed interrater agreement among the three laboratories and the three experts indicated high levels of method accuracy and precision (high sensitivity and specificity) and method reproducibility. Our results suggest that histology is a rapid, simple, and highly accurate method for distinguishing between fresh and frozen-thawed fish, regardless of the fish species analyzed.

Fatal case of human rabies imported to Italy from India highlights the importance of adequate post-exposure prophylaxis, October 2011.

In October 2011, an Indian man resident in Italy was admitted to a hospital in Mantua, Italy with symptoms of acute encephalitis. Due to a recent history of bite by a suspected rabid dog in India, where he had received incomplete post-exposure treatment, rabies was suspected. The patient died after 22 days of intensive care treatment and rabies was confirmed post mortem. This report stresses the need of appropriate post-exposure prophylaxis in rabies-endemic countries.

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.

Viral encephalopathy and retinopathy outbreak in freshwater fish farmed in Italy.

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a neuropathological condition affecting > 40 species of fish. Although VER affects mainly marine fish, the disease has also been detected in certain species reared in freshwater environments. There are relatively few reports concerning the disease in freshwater species, and there is not much information on clinical signs. Nevertheless, the most common clinical findings reported from affected freshwater species are consistent with the typical signs observed in marine species. In this paper we describe the main clinical signs and the laboratory results associated with the detection of a betanodavirus in hybrid striped bass x white bass (Morone saxatilis x Morone chrysops) and largemouth bass Micropterus salmoides, reared in a freshwater environment. We also detected the virus by real-time PCR and isolated it in cell culture from a batch of pike-perch Sander lucioperca farmed in the same system.

The spread of pathogens through trade in honey bees and their products (including queen bees and semen): overview and recent developments.

International trade in bees and bee products is a complex issue, affected bytheir different origins and uses. The trade in bees, which poses the main risk for disease dissemination, is very active and not all transactions may be officially registered by the competent authorities. Globally, bee health continues to deteriorate as pathogens, pests, parasites and diseases are spread internationally through legitimate trade, smuggling and well-intentioned but ill-advised bee introductions by professionals. International trade rules strengthen the ability of many countries to protect bee health while trading but also carry obligations. Countries that are Members of the World Trade Organization (WTO) should only restrict imports to protect against identifiable health risks. If imports are safe, trade should be permitted. The trading rules of the WTO have given greater importance to the international standards applicable to bee health, developed by the World Organisation for Animal Health, which aims to prevent the spread of animal diseases while facilitating international trade in animals and animal products.

Sudden deaths and colony population decline in Greek honey bee colonies.

During June and July of 2009, sudden deaths, tremulous movements and population declines of adult honey bees were reported by the beekeepers in the region of Peloponnesus (Mt. Mainalo), Greece. A preliminary study was carried out to investigate these unexplained phenomena in this region. In total, 37 bee samples, two brood frames containing honey bee brood of various ages, eight sugar samples and four sugar patties were collected from the affected colonies. The samples were tested for a range of pests, pathogens and pesticides. Symptomatic adult honey bees tested positive for Varroa destructor, Nosema ceranae, Chronic bee paralysis virus (CBPV), Acute paralysis virus (ABPV), Deformed wing virus (DWV), Sacbrood virus (SBV) and Black queen cell virus (BQCV), but negative for Acarapis woodi. American Foulbrood was absent from the brood samples. Chemical analysis revealed that amitraz, thiametoxan, clothianidin and acetamiprid were all absent from symptomatic adult bees, sugar and sugar patty samples. However, some bee samples, were contaminated with imidacloprid in concentrations between 14 ng/g and 39 ng/g tissue. We present: the infection of Greek honey bees by multiple viruses; the presence of N. ceranae in Greek honey bees and the first record of imidacloprid (neonicotonoid) residues in Greek honey bee tissues. The presence of multiple pathogens and pesticides made it difficult to associate a single specific cause to the depopulation phenomena observed in Greece, although we believe that viruses and N. ceranae synergistically played the most important role. A follow-up in-depth survey across all Greek regions is required to provide context to these preliminary findings.

Impact of emergency oral rabies vaccination of foxes in northeastern Italy, 28 December 2009-20 January 2010: preliminary evaluation.

Fox rabies re-emerged in northeastern Italy in 2008, in an area bordering Slovenia. In 2009, the infection spread westward to Veneto region and in 2010 to the provinces of Trento and Bolzano. Aerial emergency oral fox vaccination was implemented in the winter 2009-10. Since this vaccination was performed at altitudes below the freezing level, a statistical analysis was conducted to evaluate its impact. Of the foxes sampled following the vaccination campaign, 77% showed a rabies antibody titre of >or=0.5 IU/ml.

Pylorogastric intussusception in a Chihuahua puppy. A case report.

A three-month-old Chihuahua dog was presented with acute abdominal pain, vomiting and cardiovascular shock. Abdominal ultrasound (US) and iodated contrast gastrogram revealed suspected pylorogastric intussusception. Because of the poor prognosis the dog was euthanatized. Diagnosis of pylorogastric intussusception was confirmed at necropsy. Parasitological, virological, serological and histological examinations were also performed. This report documents the sixth case of pylorogastric (i.e. duodenogastric, gastrogastric) intussusception in the veterinary medical literature and it is the first report on a puppy dog in which US and radiographic diagnosis were confirmed post mortem.

Residues of antibacterial drugs in honey from the Italian market.

Antibacterial drugs are used worldwide for the control of American and, less often, European foulbrood. Their administration is mostly uncontrolled and applied without approved protocols and instructions for use as well as precautionary recommendations. Consequently, this practice is responsible for the contamination of beehive products and contributes to the problem of food safety. According to this situation, 4672 analyses were carried out on 5303 honeys collected from 2001 to 2007. These samples were investigated for antibacterial residues of tetracyclines, sulphonamides, streptomycin, chloramphenicol and tylosin. Honeys were classified according to their origin: imported honey and honey from the Italian market. In the last group (only for samples collected from 2001 to 2004), another type of honey was distinguished: that of local honey. A total of 6.3% of all samples were positive for the antibacterial drugs analysed; in particular, 6.8% of imported honeys and 6.1% of honeys on the Italian market. Only 1.7% of local honey had antibacterial residues. These results are indicative of a rather frequent presence of antibacterial drug residues in both Italian and imported honeys. Furthermore, the data showed that among the active substances analysed, sulphonamides are the most used antibacterial substance followed by tetracyclines, streptomycin, tylosin, and chloramphenicol. Finally, a continuous monitoring programme is needed, accompanied by an education programme to beekeepers on proper hive management.

Emergence of fox rabies in north-eastern Italy.

Italy has been classified as rabies-free since 1997. In October 2008, two foxes have been diagnosed with rabies in the Province of Udine, north-east Italy. One case of human exposure caused by a bite from one of the foxes has occurred and was properly treated.

Evaluation of indirect biomarkers for detecting corticosteroids used as illegal growth promoters in beef cattle.

The histological status of the thymus, blood cortisol concentration and circulating neutrophil:lymphocyte ratio were evaluated in 349 slaughtered beef cattle, to assess the potential of these parameters as indirect biomarkers of the illegal use of corticosteroids in meat production. The livers of 20 of the animals were analysed chemically for residues of corticosteroids. The morphology of the thymus was examined for adipose tissue infiltration, cortical atrophy and 'starry sky' appearance, and on the basis of these characteristics, the animals were considered to be negative, suspected or positive for illegal corticosteroid treatment. The animals considered to be negative had a mean cortisol concentration that was significantly higher (29 ng/ml) than that of the animals suspected for corticosteroid treatment (22 ng/ml). Using the chemical analysis as the gold standard for identifying illegally treated animals, the histological examination of the thymus had a sensitivity of 100 per cent and a specificity of 85 per cent. The samples that were positive by chemical analysis had cortisol concentrations of less than 2.0 ng/ml, whereas the mean cortisol concentration of the negative samples was 10.3 ng/ml.

Intra-pelvic chondrolipoma in a dog.

An 11-year-old male dog with dysuria-stranguria, progressing to anuria, had a multilobulated, firm mass in the perineal area, embedded in the fibroconnective adipose tissue of the pelvic cavity. The mass, which was resected surgically, was unencapsulated and composed of multiple islands of hyaline cartilage, interspersed with mature adipose tissue, and myxoid and fibrous tissues. Foci of chondroid metaplasia were present. Histological and immunohistochemical examination confirmed the mesenchymal origin of the tumour. A diagnosis of intra-pelvic chondrolipoma was made.