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.

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.

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.

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.

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.

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.

Viral encephalopathy and retinopathy of farmed marine fish species in Italy.

Viral encephalopathy and retinopathy, otherwise known as fish encephalitis or viral nervous necrosis (VNN), is an emerging problem in several farmed marine fish species in various geographic areas all over the world. Since summer 1995, heavy losses affecting mainly juvenile and adult sea bass (Dicentrarchus labrax) have been observed in several on-growing facilities in Italy. Dying fish show abnormal swimming behaviour and, at temperatures higher than 20-22 degrees C, mortality rates range between 15 and 50%. Neither significant external nor internal gross pathological signs, except frequent abnormal swim bladder hyperinflation, were detected. Histological investigations reveal vacuolations in the grey matter of the brain and spinal cord and in the granular layers of the retina. Serial tissue sections examined by an immunohistochemical method carried out with antisera against fish nodaviruses showed a positive reaction. Additionally, spherical virus-like particles 22-25 nm in diameter were detected by electron microscopy in negative stained preparations of brain tissues, and the same samples gave a positive reverse transcription-polymerase chain reaction (RT-PCR) for the T4 region of the fish nodavirus gene. These results indicate that both juvenile and adult sea bass subject to mass mortality in Italy since summer 1995 are infected with a fish nodavirus and strongly suggest that the identified virus is the cause of the observed mortality.

Mortality in Muscovy ducks (Cairina moschata) and domestic geese (Anser anser var. domestica) associated with natural infection with a highly pathogenic avian influenza virus of H7N1 subtype.

Among the 413 outbreaks of highly pathogenic avian influenza (HPAI) caused by a virus of the H7N1 subtype, which occurred in Italy during 1999 and 2000, an outbreak diagnosed in a backyard flock was characterized by mortality and nervous signs in ducks and geese. Dead geese (Anser anser var. domestica) and Muscovy ducks (Cairina moschata) were submitted to the laboratory for bacteriological, virological, histological and immunohistochemical investigations. Routine bacteriological tests resulted negative, while a HPAI virus of the H7N1 subtype was isolated from the geese. Pancreatic damage was observed in both the geese and the ducks, and the pancreas was also positive by immunohistochemistry for avian influenza in the geese. Histopathological lesions were observed in the central nervous system of both species, and this result was supported by positive immunohistochemical findings for the presence of the virus.

H7N1 avian influenza in Italy (1999 to 2000) in intensively reared chickens and turkeys.

From the end of March to the beginning of December 1999, an epidemic of low pathogenicity avian influenza (LPAI) affected the industrial poultry population of northern Italy. The virus responsible for the epidemic was subtyped as H7N1 with an intravenous pathogenicity index (IVPI) of 0.0, and a deduced amino acid sequence of the region coding for the cleavage site of the haemagglutinin molecule typical of low pathogenicity viruses. The circulation of the virus in a susceptible population for several months caused the emergence of a highly pathogenic virus with an IVPI of 3.0 and the presence of multiple basic amino acids in the deduced amino acid sequence for the cleavage site of the haemagglutinin molecule. Over 13 million birds were affected by the epidemic and, in the present paper, we report the results of the clinical, virological and histopathological investigations performed on affected chickens and turkeys. Clinical, gross and microscopic lesions caused by LPAI were more severe in turkeys than in chickens, while highly pathogenicity avian influenza (HPAI) caused similar mortality rates in both species. Current European legislation considers LPAI and HPAI as two completely distinct diseases, not requiring any compulsory eradication policy for LPAI but enforcing eradication for HPAI. In the Italian 1999 to 2000 epidemic, LPAI mutated to HPAI in a densely populated area, causing great economic losses. A reconsideration of the current European Union legislation on avian influenza, including LPAI of the H5 and H7 subtypes, could possibly be an aid to avoiding devastating epidemics for the poultry industry.

Highly pathogenic avian influenza (H7N1) in ostriches (Struthio camelus).

The clinical, virological and pathological findings observed in a natural outbreak of highly pathogenic avian influenza in intensively farmed ostriches (Struthio camelus) are reported. Clinical signs characterized by anorexia, depression, nervous and enteric signs were observed in young birds, which resulted in death of 30% of the affected birds. Virus isolation performed in accordance with the guidelines listed in European Union Directive 92/40/EEC yielded an influenza A virus of the H7N1 subtype with a deduced cleavage site motif containing multiple basic amino acids, typical of highly pathogenic viruses. Gross lesions, mainly haemorrhagic enteritis and liver degeneration and necrosis, were confirmed by histopathology and immunohistochemistry, resulting in the detection of necrotic lesions and influenza A nucleoprotein in selected organs. The findings reported indicate that ostriches are susceptible to highly pathogenic avian influenza.

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.

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.

Newcastle disease outbreaks in Italy during 2000.

Among the consequences of the epidemic of highly pathogenic avian influenza which affected Italy between 1999 and 2000 was an epidemic of Newcastle disease in northern and central Italy. It affected industrially reared poultry, dealer flocks and backyard flocks, with a total of 254 outbreaks notified up to December 31, 2000. Virological investigations yielded virulent isolates of Newcastle disease virus, which produced intracerebral pathogenicity indices ranging from 1.6 to 2.0 and which, on the basis of their monoclonal antibody binding patterns, could be classified as belonging to group C1. The clinical, gross and microscopical findings were typical of Newcastle disease, and different avian species were susceptible to different degrees. Chickens and guinea fowl appeared to be the most susceptible, followed by pheasants, turkeys and ostriches. The epidemiological inquiry highlighted the crucial role of a broiler hatchery in initiating the epidemic, and of dealers in perpetuating it. The control measures imposed by Directive 92/66/EEC are discussed with reference to the outbreaks in backyard flocks.