A recent view about encephalomyocarditis virus circulating in compartmentalised animal population in Northern Italy.

Encephalomyocarditis virus (Picornaviridae, Cardiovirus A) is the causative agent of the homonymous disease, which may induce myocarditis, encephalitis and reproductive disorders in various mammals, especially in swine. Despite the disease occurred endemically in pig farms since 1997, the recent increase of death experimented in Northern Italy prompted to furtherly investigate the evolution of the virus and the actual spread of the infection. Italian EMC viruses, collected between 2013 and 2019, showed an overall antigenic stability. The in-house ELISA Monoclonal Antibodies based, able to reveal changes in seven different antigenic sites, showed only sporadic and occasional mutations in considered samples and the subsequent phylogenetic analysis confirmed antigenic panel's remarks. All the isolates could be classified within a unique lineage, which comprise other European strains and confirm that the viruses currently circulating in Italy developed from a unique common ancestor. Despite the demonstrated stability of virus, some putative newly emerged variants were detected through antigenic profile analysis and phylogenesis. Finally, the serosurvey proved that spread of EMCV is greater than the diffusion of fatal infections would suggest, due to subclinical circulation of EMCV. It demonstrated an increase in the proportion of seropositive farms, if compared with previous data with no remarkable differences between farms with and without clinical evidence of disease.

ESßL E. coli isolated in pig's chain: Genetic analysis associated to the phenotype and biofilm synthesis evaluation.

Resistance to new generation cephalosporins is an important public health problem globally, in terms of economic and social costs, morbidity and mortality. Βeta-lactamase enzymes are mainly responsible for the antibiotic resistance of Gram negative bacteria and extended-spectrum-ß-lactamases (ESßLs) are one of the major determinants of resistance against oxymino-cephalosporins in Enterobacteriaceae. Food-producing animals represent one of the sources of antibiotic resistant bacteria, including pigs. Here we analysed the presence of E. coli resistant to III generation cephalosporins isolated from different matrices collected from intensively bred pigs. A total of 498 E. coli were isolated from faeces and carcasses of pigs at slaughterhouse as well as from pork meat and sausages. Among these, 73 were phenotypically confirmed to be ESßL producers. Genetic analysis revealed that all except two harboured at least one of the three selected genes: blaCTX-M, blaTEM, and blaSHV. Furthermore, six of the E. coli ESßL isolated from faeces and carcasses swabs, were also able to produce biofilm, highlighting the virulence potential of these strains. The presence of Multi-Drug-Resistance patterns (MDR) recorded by the 73 ESßL E. coli was significant (60% of the strains were resistant to more than six antibiotics in MIC test). Results from the present study show that the transmission of resistant bacteria is possible along the food chain, including production of pork, one the most highly consumed meats around the world. Transmission is possible through the ingestion of raw meat products, and following cross-contamination between raw and cooked foods during preparation. The potential risk for human health demonstrated here, associated with the consumption of pork contaminated with bacterial strains characterized by multidrug resistance patterns, and the ability to produce ESßL and biofilm, is cause for concern. It is imperative to study future control strategies to avoid or limit as much as possible the transmission of these highly pathogenic strains through food consumption and/or contact with the environment.

Occurrence of putative virulence genes on Arcobacter butzleri isolated from three different environmental sites throughout the dairy chain.

AIMS: This comparative study investigated the occurrence of cadF, cj1349, ciaB, pldA, tlyA, hecA, hecB, mviN, irgA and IroE genes in 212 Arcobacter butzleri isolated from three different environmental sites linked to the dairy chain (farms, industrial and artisanal dairy plants) located in three Italian regions (Lombardy, Emilia-Romagna and Calabria). METHODS AND RESULTS: According to the presence of these genes, different pathotypes (P-types) were determined. The main genes detected were ciaB, mviN, tlyA, cj1349, pldA and cadF, while the least common genes were iroE, hecA, hecB and irgA. TlyA, irgA, hecA, hecB and iroE, which were significantly more frequent in isolates recovered in industrial dairy plants. Twelve P-types were detected. The occurrence of the most frequently detected P-types (P-types 1, 2, 3 and 5) differed significantly (P < 0·001) in relation to both the environmental site and geographical area of isolation. The highest diversity in P-types was observed in industrial dairy plants and in the Calabria region. CONCLUSIONS: The results of this study show a correlation between the occurrence of putative virulence genes and virulence genotype variability depending on the environmental site and geographical origin of the isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides insights into the similar distribution of putative virulence genes in a dairy chain and other sources' isolates and also into a geographical distribution of some P-types. We have shown that industrial dairy plants may represent an environmental site favouring a selection of the isolates with a higher pathogenetic pattern.

Paediatric HUS Cases Related to the Consumption of Raw Milk Sold by Vending Machines in Italy: Quantitative Risk Assessment Based on Escherichia coli O157 Official Controls over 7 years.

A quantitative risk assessment (RA) was developed to estimate haemolytic-uremic syndrome (HUS) cases in paediatric population associated with the consumption of raw milk sold in vending machines in Italy. The historical national evolution of raw milk consumption phenomenon since 2008, when consumer interest started to grow, and after 7 years of marketing adjustment, is outlined. Exposure assessment was based on the official Shiga toxin-producing Escherichia coli O157:H7 (STEC) microbiological records of raw milk samples from vending machines monitored by the regional Veterinary Authorities from 2008 to 2014, microbial growth during storage, consumption frequency of raw milk, serving size, consumption preference and age of consumers. The differential risk considered milk handled under regulation conditions (4°C throughout all phases) and the worst time-temperature field handling conditions detected. In case of boiling milk before consumption, we assumed that the risk of HUS is fixed at zero. The model estimates clearly show that the public health significance of HUS cases due to raw milk STEC contamination depends on the current variability surrounding the risk profile of the food and the consumer behaviour has more impact than milk storage scenario. The estimated HUS cases predicted by our model are roughly in line with the effective STEC O157-associated HUS cases notified in Italy only when the proportion of consumers not boiling milk before consumption is assumed to be 1%. Raw milk consumption remains a source of E. coli O157:H7 for humans, but its overall relevance is likely to have subsided and significant caution should be exerted for temporal, geographical and consumers behaviour analysis. Health education programmes and regulatory actions are required to educate people, primarily children, on other STEC sources.

Short communication: Investigation into Mycobacterium avium ssp. paratuberculosis in pasteurized milk in Italy.

This study investigated the presence of viable Mycobacterium avium ssp. paratuberculosis (MAP) in pasteurized milk produced by Italian industrial dairy plants to verify the prediction of a previously performed risk assessment. The study analyzed 160 one-liter bottles of pasteurized milk from 2 dairy plants located in 2 different regions. Traditional cultural protocols were applied to 500mL of pasteurized milk for each sample. The investigation focused also on the pasteurization parameters and data on the microbiological characteristics of raw milk (total bacterial count) and pasteurized milk (Enterobacteriaceae and Listeria monocytogenes). No sample was positive for MAP, the pasteurization parameters complied with European Union legislation, and the microbiological analysis of raw and pasteurized milk showed good microbiological quality. The results show that a 7-log (or >7) reduction could be a plausible value for commercial pasteurization. The combination of hygiene practices at farm level and commercial pasteurization yield very low or absent levels of MAP contamination in pasteurized milk, suggesting that pasteurized milk is not a significant source of human exposure to MAP in the dairies investigated.

Wide recognition of Culex pipiens and lack of detection of Culex torrentium through biomolecular differentiation of mosquitoes in the Emilia-Romagna region, Northern Italy.

The Culex pipiens complex includes species with reported differences in vector competence for arthropod-borne viruses, many of which are of significant importance to human health such as the West Nile virus and the Sindbis virus. This group of mosquitoes is difficult to distinguish morphologically; particularly as adult females. In Europe, the two species of the complex, Culex pipiens Linnaeus 1758 and Culex torrentium Martini 1925, are often found sympatrically. With the aim to characterize the presence and spread of both species in the Emilia-Romagna region, Northern Italy, mosquitoes of the complex - collected during the West Nile virus surveillance plans - were tested by multiplex real-time PCR for the detection of the two species Cx. pipiens and Cx. torrentium. A total of 24 165 mosquitoes, collected between 2012 and 2014 from 105 sites, and sorted in 204 pools, were tested. All tested pools were found to be composed of Cx. pipiens, whereas Cx. torrentium was not detected. These results indicate a likely absence of Cx. torrentium mosquitoes within the surveyed territory, whereas Cx. pipiens is widely distributed in the area mentioned. This is in line with previous reports, which describe a pre-alpine distribution of Cx. torrentium in Italy.

Antimicrobial resistance of F4+ Escherichia coli isolated from Swine in Italy.

Four-hundred and forty-two F4+ pathogenic Escherichia coli were isolated in a period of 10 years (2002-2011), from pigs that were suffering from diarrhoea belonging to Italian swine herds. The strains were analysed for their susceptibility to 12 antimicrobials using the disc diffusion method. During the study period, a statistically significant proportion of isolates resistant to enrofloxacin (14.5-89.3%), marbofloxacin (5.4-60.7%), flumequine (49.1-92.9%), danofloxacin (21.6-80%), florfenicol (9.8-64.3%), thiamphenicol (50-92%) and cefquinome (3.8-44%) was recorded. An increase in resistance (not statistically significant) to gentamicin (63.6-85.7%), apramycin (61.8-82.1%), trimethoprim-sulphamethoxazole (75-89.3%), tetracycline (97-100%) and erythromycin (92.4-100%) was also observed. Based on antimicrobial multiresistance, the strains were collected into three groups: I. resistant to 2-5 antimicrobials; II. resistant to 6-8 antimicrobials; III. resistant to 9-12 antimicrobials. The number of isolates belonging to the first group showed a statistically significant decrease (P < 0.05; R(2)  = 0.896; r = -0.9608), while the isolates belonging to the second and third groups showed a statistically significant increase in resistance (P < 0.05; R(2)  = 0.753; r = 0.8890 and P < 0.05; R(2)  = 0.727; r = 0.8701, respectively) over the period of study. The results of this study suggest the need for continued monitoring of the development of resistance.

Sequence types and pleuromutilin susceptibility of Brachyspira hyodysenteriae isolates from Italian pigs with swine dysentery: 2003-2012.

Swine dysentery is a mucohaemorrhagic colitis of pigs caused by infection with Brachyspira hyodysenteriae. The disease can be controlled by treatment with antimicrobial agents, with the pleuromutilins tiamulin and valnemulin being widely used. In recent years, the occurrence of B. hyodysenteriae with reduced susceptibility to these drugs has been increasing. The aim of this study was to determine temporal changes in genetic groups and pleuromutilin susceptibility amongst B. hyodysenteriae isolates from Italy. Multilocus sequence typing (MLST) was performed on 108 isolates recovered from 87 farms in different regions of Italy from 2003 to 2012, and their minimum inhibitory concentrations (MICs) for tiamulin and valnemulin were determined. Logistic regression was performed to assess associations between susceptibility to the two antimicrobial agents and genetic group, year and region of isolation. The isolates were allocated to 23 sequence types (STs), with five clonal clusters (Ccs) and seven singletons. More than 50% of isolates were resistant to both pleuromutilins (MIC >2.0 µg/mL for tiamulin and >1.0 µg/mL for valnemulin). All 10 isolates in ST 83 were resistant; these were first isolated in 2011 and came from nine farms, suggesting recent widespread dissemination of a resistant strain. Significant associations were found between the proportion of pleuromutilin susceptible isolates and the genetic group and year of isolation. Although resistant isolates were found in all Ccs, isolates in Ccs 2 and 7 were over five times more likely to be susceptible than those in the other Ccs. A significant trend in the reduction of susceptibility over time also was observed.

Human and entomological surveillance of Toscana virus in the Emilia-Romagna region, Italy, 2010 to 2012.

Toscana virus (TOSV), transmitted by phlebotomine sandflies, is recognised as one of the most important causes of viral meningitis in summer in Mediterranean countries. A surveillance plan based on both human and entomological surveys was started in 2010 in the Emilia-Romagna region, Italy. Clinical samples from patients with neurological manifestations were collected during 2010 to 2012. The surveillance protocol was improved during these years, allowing the detection of 65 human infections. Most of these infections were recorded in hilly areas, where sandflies reach the highest density. Entomological sampling around the homes of the patients resulted in a low number of captured sandflies, while later sampling in a hilly area with high number of human cases (n=21) resulted in a larger number of captured sandflies. Using this approach, 25,653 sandflies were sampled, of which there were 21,157 females, which were sorted into 287 pools. TOSV RNA was detected by real-time PCR in 33 of the pools. The results highlighted the role of Phlebotomus perfiliewi as the main vector of TOSV and a potential link between vector density and virus circulation. This integrated system shows that an interdisciplinary approach improves the sensitiveness and effectiveness of health surveillance.

The experience of West Nile virus integrated surveillance system in the Emilia-Romagna region: five years of implementation, Italy, 2009 to 2013.

Predicting West Nile virus (WNV) circulation and the risk of WNV epidemics is difficult due to complex interactions of multiple factors involved. Surveillance systems that timely detect virus activity in targeted areas, and allow evidence-based risk assessments may therefore be necessary. Since 2009, a system integrating environmental (mosquitoes and birds) and human surveillance has been implemented and progressively improved in the Emilia-Romagna region, Italy. The objective is to increase knowledge of WNV circulation and to reduce the probability of virus transmission via blood, tissue and organ donation. As of 2013, the system has shown highly satisfactory results in terms of early detection capacity (the environmental surveillance component allowed detection of WNV circulation 3­4 weeks before human cases of West Nile neuroinvasive disease (WNND) occurred), sensitivity (capacity to detect virus circulation even at the enzootic level) and area specificity (capacity to indicate the spatial distribution of the risk for WNND). Strong correlations were observed between the vector index values and the number of human WNND cases registered at the province level. Taking into consideration two scenarios of surveillance, the first with environmental surveillance and the second without, the total costs for the period from 2009 to 2013 were reduced when environmental surveillance was considered (EUR 2.093 million for the first scenario vs EUR 2.560 million for the second). Environmental surveillance helped to reduce costs by enabling a more targeted blood unit testing strategy. The inclusion of environmental surveillance also increased the efficiency of detecting infected blood units and further allowed evidence-based adoption of preventative public health measures.

A screening sampling plan to detect Mycobacterium avium subspecies paratuberculosis-positive dairy herds.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic contagious bacterial disease primarily affecting dairy cattle. Paratuberculosis represents a dual problem for the milk production chain: in addition to economic losses to affected herds, MAP may have zoonotic potential. Infected herds must be identified in order to implement programs designed to reduce the incidence of disease within and between herds and to prevent MAP from entering the food chain. The objective of this study was to evaluate the sensitivity and specificity of a screening sampling plan (SSP) to detect MAP-positive dairy herds by repetitive analysis of bulk tank milk (BTM) samples by ELISA and in-line milk filter (ILMF) samples by PCR. Samples from BTM and ILMF were collected twice from 569 dairy herds in southern Italy. Additionally, 12,016 individual milk samples were collected: 9,509 from 102 SSP-positive herds (SSP MAP-positive) and 2,507 from 21 randomly selected SSP-negative herds (SSP MAP-negative). There was a total of 126 SSP MAP-positive herds (i.e., 21.3% SSP MAP-positive herds; 95% confidence interval=18.0-24.9); the within-herd apparent prevalence (AP) ranged between 0.00 and 22.73% (mean 6.07%). A significant difference in within-herd AP was shown between SSP MAP-positive herds and SSP MAP-negative herds. A highly significant association was shown between the median AP herd status (>5%) and positivity to at least one ILMF or BTM sample. The SSP detected a minimum of 56.25% of low AP herds (AP ≤ 2.0%) up to a maximum of 100% of herds with a within-herd AP ≥ 8.0%. Overall, the SSP detected 85.57% of herds in which at least one individual milk sample was positive by ELISA. The proposed SSP was an inexpensive and useful tool to detect MAP-positive herds with a higher risk of infection diffusion and milk contamination. Although the SSP cannot be used for MAP-free certification of herds, it could be useful to prioritize appropriate control measures aimed at reducing the prevalence of infection in dairy herds and milk contamination.

Haemophilus parasuis serovars isolated from pathological samples in Northern Italy.

From January 2007 to December 2011, a total of 106 Haemophilus parasuis strains isolated from pigs were serotyped by agar gel diffusion test (DG). Serovar 4 was the most prevalent (24.5%), followed by serovar 13 (19.8%) and serovar 5 (11.3%). Twenty-nine strains were non-typeable (27.3%). The strains were divided into two groups, depending on whether they were isolated from specific pathological lesions of systemic disease such as polyserositis, arthritis or meningitis (73 cases of 106) or from the lower respiratory tract of pigs suffering from bronchopneumonia (33 cases of 106). Serovars 4 and 13 had a higher prevalence in systemic infection (polyserositis) than in respiratory disease only. Pasteurella multocida (14/106), Streptococcus suis (7/106), Actinobacillus pleuropneumoniae (4/106), Bordetella bronchiseptica (3/106) and Arcanobacterium pyogenes (3/106) were isolated in association with H. parasuis.

Environmental methicillin-resistant Staphylococcus aureus contamination in pig herds in relation to the productive phase and application of cleaning and disinfection.

The aim of the study is to better understand MRSA environmental contamination in pig holdings in relation to the productive phase and the application of environmental cleaning and disinfection practices (C&D). This study was carried out on six herds. Dust samples from farrowing crates, weaning boxes, growing and finishing pens were collected from each herd environment prior to and after C&D and cultured for MRSA. Ten samples were collected at the end of each productive phase with the animals present and 10 additional samples were collected after C&D, prior to introduction of a new stock. The proportion of positive samples prior to C&D was lower in fattening than in other phases. The proportion of positive samples prior to and after C&D was 121/240 (50%) and 46/240 (19%) respectively. In the mixed effects logistic regression analysis it was shown not only that both productive phase and C&D were associated with the probability of having a positive sample but also that the C&D effect was different in the different productive phases. In particular the effect of C&D was stronger in farrowing crates than in the other productive phases. The results of this study show that, although current practices of cleaning and disinfection reduce MRSA environmental contamination, they are likely to be inadequate to the elimination of the microorganism. However, a strict application of hygienic protocols can lead to a marked reduction of MRSA environmental contamination.

Quantitative risk assessment of verocytotoxin-producing Escherichia coli O157 and Campylobacter jejuni related to consumption of raw milk in a province in Northern Italy.

A quantitative risk assessment was developed to describe the risk of campylobacteriosis and hemolytic uremic syndrome (HUS) linked to consumption of raw milk sold in vending machines in Northern Italy. Exposure assessment considered the microbiological status of dairy farms, expected milk contamination, storage conditions from bulk tank to home storage, microbial growth during storage, destruction experiments, consumption frequency of raw milk, age of consumers, serving size, and consumption preference. The differential risk between milk handled under regulation conditions (4°C throughout all phases) and the worst field handling conditions was considered. The probability of Campylobacter jejuni infection was modeled with a single-hit dose-response beta-Poisson model, whereas for HUS an exponential dose-response model was chosen and two probabilities were used to model the higher susceptibility of children younger than 5 years old. For every 10,000 to 20,000 consumers each year, the models predicted for the best and worst storage conditions, respectively, 2.12 and 1.14 campylobacteriosis cases and 0.02 and 0.09 HUS cases in the 0- to 5-year age group and 0.1 and 0.5 HUS cases in the >5-year age group. The expected pediatric HUS cases do not differ considerably from those reported in Italy by the Minister of Health. The model developed may be a useful tool for extending the assessment of the risk of campylobacteriosis and HUS due to raw milk consumption at the national level in Italy. Considering the epidemiological implications of this study, the risk of illness linked to raw milk consumption should not be ignored and could be reduced by the use of simple measures. Boiling milk before consumption and strict control of temperatures by farmers during raw milk distribution have significant effects on campylobacteriosis and HUS and are essential measures for risk management.

Impact of climate change on risk of incursion of Crimean-Congo haemorrhagic fever virus in livestock in Europe through migratory birds.

AIMS: To predict the risk of incursion of Crimean-Congo haemorrhagic fever virus (CCHFV) in livestock in Europe introduced through immature Hyalomma marginatum ticks on migratory birds under current conditions and in the decade 2075-2084 under a climate-change scenario. METHODS AND RESULTS: A spatial risk map of Europe comprising 14 282 grid cells (25 × 25 km) was constructed using three data sources: (i) ranges and abundances of four species of bird which migrate from sub-Saharan Africa to Europe each spring, namely Willow warbler (Phylloscopus trochilus), Northern wheatear (Oenanthe oenanthe), Tree pipit (Anthus trivialis) and Common quail (Coturnix coturnix); (ii) UK Met Office HadRM3 spring temperatures for prediction of moulting success of immature H. marginatum ticks and (iii) livestock densities. On average, the number of grid cells in Europe predicted to have at least one CCHFV incursion in livestock in spring was 1·04 per year for the decade 2005-2014 and 1·03 per year for the decade 2075-2084. In general with the assumed climate-change scenario, the risk increased in northern Europe but decreased in central and southern Europe, although there is considerable local variation in the trends. CONCLUSIONS: The absolute risk of incursion of CCHFV in livestock through ticks introduced by four abundant species of migratory bird (totalling 120 million individual birds) is very low. Climate change has opposing effects, increasing the success of the moult of the nymphal ticks into adults but decreasing the projected abundance of birds by 34% in this model. SIGNIFICANCE AND IMPACT OF THE STUDY: For Europe, climate change is not predicted to increase the overall risk of incursion of CCHFV in livestock through infected ticks introduced by these four migratory bird species.

Survey of pleuritis and pulmonary lesions in pigs at abattoir with a focus on the extent of the condition and herd risk factors.

Cranioventral pulmonary consolidation (enzootic pneumonia-like lesions) and chronic pleuritis (CP) are common findings in slaughtered pigs. Pleural lesions involving dorsocaudal lobes are suggestive of pleuropneumonia due to Actinobacillus pleuropneumoniae. In this report the results of an abattoir survey of pleuritis and pulmonary lesions in pigs is presented with a focus on herd risk factors. A total of 4889 animals, ranging in age from 9 to 10 months, from 48 batches of pigs belonging to an equal number of herds, were included in the study. Bronchopneumonic lesions suggestive of enzootic pneumonia (EP-like lesions) were detected in 46.4% of the examined lungs. The EP-like lesion average value for all lungs was 1.03 (95% CI 0.98-1.08), ranging from 0.17 to 2.56 among the 48 batches; 47.5% of lungs showed chronic pleuritis. Dorsocaudal pleuritis suggestive of recovered pleuropneumonia (SPES score ≥2) was found in 25.1% of the lungs. The mean SPES (slaughterhouse pleuritis evaluation system) value of the overall 4889 lungs was 0.83 (95% CI 0.78-0.86). The mean SPES value of the batches ranged from 0.04 to 1.87. The mean Actinobacillus pleuropneumoniae index of all studied batches was 0.61 (95% CI 0.51-0.71), ranging from 0 to 1.84. Blood samples were collected from each herd to evaluate antibody titres to Mycoplasma hyopneumoniae, A. pleuropneumoniae, Aujeszky's disease virus, porcine reproductive and respiratory syndrome virus (PRRSV), and swine influenza virus. Herd characteristics were recorded using a questionnaire given to the farmers. A multivariable analysis was conducted to identify risk factors for pleuritis and EP-like lesions. High dorsocaudal pleuritis was associated with A. pleuropneumoniae seroprevalence and history of A. pleuropneumoniae isolation from pneumonic lungs of dead animals. Vaccination of weaners at 3-5 weeks of age against PRRS using a modified live vaccine was associated with a reduction in the percentage of cranioventral pulmonary consolidation (EP-like lesions).

West Nile virus circulation in Emilia-Romagna, Italy: the integrated surveillance system 2009.

Following a large West Nile virus (WNV) epidemic in northeastern Italy in 2008, human and animal surveillance activities were implemented in Emilia Romagna. Human surveillance was performed by serology or genome detection on blood and cerebrospinal fluid for all suspected cases suffering from acute meningoencephalitis in the regional territory. Animal surveillance consisted of passive and active surveillance of horses and active surveillance of wild birds and mosquitoes. Between 15 June and 31 October 2009, nine of 78 possible cases of West Nile neuroinvasive disease were confirmed (three fatal). From May to October, 26 cases of neurological West Nile disease were confirmed among 46 horses. The overall incidence of seroconversion among horses in 2009 was 13%. In 2009, 44 of 1,218 wild birds yielded positive PCR results for WNV infection. The planned veterinary and entomological surveillance actions detected WNV activity from the end of July 2009, about 2-3 weeks before the onset of the first human neurological case. Passive surveillance of horses seems to be an early and suitable tool for the detection of WNV activity, but it will be less sensitive in the future, because an intensive programme of horse vaccination started in June 2009.

Usutu virus infection in a patient who underwent orthotropic liver transplantation, Italy, August-September 2009.

We report a case of Usutu virus (USUV)-related illness in a patient that underwent an orthotropic liver transplant (OLT). Post transplant, the patient developed clinical signs of a possible neuroinvasive disease with a significant loss of cerebral functions. USUV was isolated in Vero E6 cells from a plasma sample obtained immediately before the surgery, and USUV RNA was demonstrated by RT-PCR and sequencing. This report enlarges the panel of emerging mosquito-borne flavivirus-related disease in humans.

Prevalence, disease associations and risk factors for colonization with intestinal spirochaetes (Brachyspira spp.) in flocks of laying hens in north-eastern Italy.

The present study investigated the occurrence of anaerobic intestinal spirochaetes of the genus Brachyspira in laying hen flocks in Treviso province, north-eastern Italy, with respect to prevalence, spirochaete species present, disease associations and risk factors for colonization. A total of 450 faecal samples from 45 sheds on 29 laying hen farms were cultured for intestinal spirochaetes. Nineteen sheds on 12 farms contained chickens with symptoms consistent with avian intestinal spirochaetosis, including reduced egg production, wet litter and/or pasty vents. Spirochaetes were isolated from 157 (34.8%) samples from 21 (72.4%) farms, and from 32 (71.1%) sheds. From these positive samples, 189 spirochaetal isolates were speciated using three polymerase chain reaction assays and a restriction fragment polymorphism analysis of 16S rDNA polymerase chain reaction products. Overall, 52 (27.5%) isolates were identified as pathogenic Brachyspira intermedia, 26 (13.8%) as pathogenic Brachyspira pilosicoli, 93 (49.7%) as non-pathogenic (Brachyspira innocens/Brachyspira murdochii), and 18 (9.6%) were unidentified. Faeces from 14 sheds (31%) on 10 farms (34.5%) contained B. intermedia and/or B. pilosicoli, and disease consistent with avian intestinal spirochaetosis was observed in nine of these sheds on seven farms. There was a significant association (P=0.042) between the presence of spirochaetes and using deep pits rather than conveyor belts for manure disposal. Sheds housing chickens >40 weeks of age were significantly more likely to contain spirochaetes (P=0.048) and pathogenic species (P=007) than sheds housing younger chickens. A significant association (P=0.02) was found between infection with pathogenic spirochaetes and reduced egg production.