Integration of genetic and epidemiological data to infer H5N8 HPAI virus transmission dynamics during the 2016-2017 epidemic in Italy.

Between October 2016 and December 2017, several European Countries had been involved in a massive Highly Pathogenic Avian Influenza (HPAI) epidemic sustained by H5N8 subtype virus. Starting on December 2016, also Italy was affected by H5N8 HPAI virus, with cases occurring in two epidemic waves: the first between December 2016 and May 2017, and the second in July-December 2017. Eighty-three outbreaks were recorded in poultry, 67 of which (80.72%) occurring in the second wave. A total of 14 cases were reported in wild birds. Epidemiological information and genetic analyses were conjointly used to get insight on the spread dynamics. Analyses indicated multiple introductions from wild birds to the poultry sector in the first epidemic wave, and noteworthy lateral spread from October 2017 in a limited geographical area with high poultry densities. Turkeys, layers and backyards were the mainly affected types of poultry production. Two genetic sub-groups were detected in the second wave in non-overlapping geographical areas, leading to speculate on the involvement of different wild bird populations. The integration of epidemiological data and genetic analyses allowed to unravel the transmission dynamics of H5N8 virus in Italy, and could be exploited to timely support in implementing tailored control measures.

H7N7 Highly Pathogenic Avian Influenza in Poultry Farms in Italy in 2016.

After the H7N7 highly pathogenic (HP) avian influenza (AI) outbreak in 2013, and a single case of H5N8 HPAI in 2014, in April 2016, a H7N7 HPAI virus was detected in northeastern Italy. The case occurred in an organic free-range laying hen farm located in proximity with one of the highest densely populated poultry areas (DPPAs) in Italy. Control measures provided by the Council of the European Union in directive 2005/94/CE were promptly applied, and enhanced surveillance activities were implemented in the DPPAs. On May 16, 2016, a second case was confirmed in a fattening turkey farm within the protection zone of the previous outbreak. Following an epidemiologic inquiry, another turkey farm was considered at risk of transmission and was subjected to preemptive culling. Epidemiologic data and phylogenetic analyses indicated that the virus was likely introduced from wild birds as a low pathogenicity AI strain, through direct contact. The rapid containment of the outbreak proves the level of preparedness of the veterinary public health sector in Italy. Nevertheless, the recurrent introductions from wild birds indicate the need of improving both the biosecurity levels in the DPPA and the surveillance activities in wild birds to quickly detect the presence of AI in the territory.

Two strains of Mycoplasma synoviae from chicken flocks on the same layer farm differ in their ability to produce eggshell apex abnormality.

Mycoplasma synoviae (Ms) is considered to be an economically important poultry pathogen. Although the full economic costs of infection in layer chickens are still under debate, the prevalence of Ms is known to be high in some countries and earlier reports have shown a correlation between infection and Eggshell Apex Abnormality (EAA). This work is a continuation of an earlier study of a clinical case of EAA on a layer hen farm where the presence of two different strains of Ms, based on the sequence of the 5' end of the vlhA gene, was demonstrated. Both strains could be detected in the trachea but only one (designated strain PASC8) appeared able to colonize the oviduct, while the other (designated TRACH) was not found in the oviduct and has not been related to EAA. The PASC8 partial vlhA gene sequence differs from that of the TRACH in having a 39 nucleotide deletion in the proline rich region and three point mutations in the RIII region. Based on this information an experimental infection was performed in SPF chickens using groups infected with either the PASC8 or the TRACH strain and a non-infected control group. Both Ms strains were detected in the trachea of infected birds, but only the PASC8 strain was found in the oviduct. Furthermore, EAA developed only in the group infected with PASC8 strain. Compared to the control group, both strains produced an adverse impact on egg production: a decrease in the numbers laid and in their average weight (P<0.05) This work demonstrates a difference in oviduct tropism between two Ms strains and a possible relationship to the production of EAA in experimental conditions.

Comparative pathogenicity study of ten different betanodavirus strains in experimentally infected European sea bass, Dicentrarchus labrax (L.).

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a severe pathological condition caused by RNA viruses belonging to the Nodaviridae family, genus Betanodavirus. The disease, described in more than 50 fish species worldwide, is considered as the most serious viral threat affecting marine farmed species in the Mediterranean region, thus representing one of the bottlenecks for further development of the aquaculture industry. To date, four different genotypes have been identified, namely red-spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus and barfin flounder nervous necrosis virus, with the RGNNV genotype appearing as the most widespread in the Mediterranean region, although SJNNV-type strains and reassortant viruses have also been reported. The existence of these genetically different strains could be the reason for the differences in mortality observed in the field. However, very little experimental data are available on the pathogenicity of these viruses in farmed fish. Therefore, in this study, the pathogenicity of 10 isolates has been assessed with an in vivo trial. The investigation was conducted using the European sea bass, the first target fish species for the disease in the Mediterranean basin. Naive fish were challenged by immersion and clinical signs and mortality were recorded for 68 days; furthermore, samples collected at selected time points were analysed to evaluate the development of the infection. Finally, survivors were weighed to estimate the growth reduction. The statistically supported results obtained in this study demonstrated different pathogenicity patterns, underlined the potential risk represented by different strains in the transmission of the infection to highly susceptible species and highlighted the indirect damage caused by a clinical outbreak of VER/VNN.

The effectiveness of domestic cook on inactivation of murine norovirus in experimentally infected Manila clams (Ruditapes philippinarum).

AIM: The aim of this work was to evaluate the efficacy of domestic cooking in inactivating Manila clams experimentally infected with murine norovirus (MNV). METHODS AND RESULTS: A cooking pan was modified to enable electronic temperature probes to be positioned to record both flesh and environment temperature. Manila clams were infected with 10(4) TCID 50% ml(-1) of MNV. The infected whole-in-shell clams, divided into three replicates, were cooked on an electric stove, and groups of nine clams were removed from the pan at fixed intervals. Pools of three digestive glands were examined by virus isolation to ascertain residual viral load. CONCLUSION: Results showed that 10 min of cooking by a traditional domestic method at a temperature close to 100°C, for at least 2 min, can completely devitalize the MNV in infected clams. This is generally the time needed for the majority of valves to open up. SIGNIFICANCE AND IMPACT OF THE STUDY: At present, it is highly recommended to label all lagoon products as 'requiring cooking before consumption', but no specifications are given on how long and at what temperature they should be cooked. Our results can provide the consumer with useful indications on how to cook clams to prevent any risk of foodborne illness.

Cross-clade protection against H5N1 HPAI strains recently isolated from commercial poultry in Egypt with a single dose of a baculovirus based vaccine.

The current avian influenza epidemic in Egypt caused by circulation of genetically and antigenically diverse H5N1 HPAI viruses in poultry is controlled by applying vaccination among other measures. In this context, the use of a DIVA (differentiating infected from vaccinated animals) vaccination strategy utilizing a vaccine capable of inducing protection against multiple antigenic variants may result as an additional control tool to the existing ones. In this study the efficacy of a single-shot recombinant baculovirus-based vaccine in specific-pathogen-free chickens was tested by experimental challenge with genetically and antigenically diverse H5N1 HPAI viruses belonging to clades 2.2.1 and 2.2.1.1, which have been circulating in Egypt since 2010. A single dose of vaccine, administration at 10 days of age, was shown to confer 100% clinical protection, with a decrease or suppression of virus shedding.

Antigenic and genetic analyses of isolate APMV/wigeon/Italy/3920-1/2005 indicate that it represents a new avian paramyxovirus (APMV-12).

Isolate wigeon/Italy/3920-1/2005 (3920-1) was obtained during surveillance of wild birds in November 2005 in the Rovigo province of Northern Italy and shown to be a paramyxovirus. Analysis of cross-haemagglutination-inhibition tests between 3920-1 and representative avian paramyxoviruses showed only a low-level relationship to APMV-1. Phylogenetic analysis of the whole genome and each of the six genes indicated that while 3920-1 grouped with APMV-1 and APMV-9 viruses, it was quite distinct from these two. In the whole-genome analysis, 3920-1 had 52.1 % nucleotide sequence identity to the closest APMV-1 virus, 50.1 % identity to the APMV-9 genome, and less than 42 % identity to representatives of the other avian paramyxovirus groups. We propose isolate wigeon/Italy/3920-1/2005 as the prototype strain of a further APMV group, APMV-12.

Evidence of West Nile virus lineage 2 circulation in Northern Italy.

A West Nile virus (WNV) strain belonging to lineage 2 was for the first time detected in two pools of Culex pipiens collected in the province of Udine and in tissues of a wild collared dove (Streptopelia decaocto) found dead in the province of Treviso, in North East of Italy. It was molecularly identified by group and WNV lineage specific RT-PCRs and characterized by partial sequencing of the NS3 and NS5 genes. When compared with the sequences of same fragments of NS3 and NS5 of the WNV lineage 2 strain isolated from birds of prey in Hungary (2004), the phylogenetic analysis of these sequences revealed 100% and 99% similarity, respectively. As the Hungarian strain, the NS3 selected sequence differed from the 2010 Greek isolate by one amino-acid located at 249 site which is the site involved in genetic modulation of WNV pathogenicity. The Italian and Hungarian strains have histidine rather than proline at this site. The presence of a lineage 2 strain in regions where the lineage 1 strain is still circulating, creates a new scenario with unpredictable consequences. In this situation comprehensive investigations on the occurrence, ecology, and epidemiology of these different WNV strains circulating in Italy become the highest priority.

Effect of high hydrostatic pressure on murine norovirus in Manila clams.

AIMS: Eating raw or insufficiently cooked bivalve molluscs contaminated with human noroviruses (NVs) can result in acute cases of gastroenteritis in humans. Manila clams (Ruditapes philippinarum) are particularly prone to exposure to NVs due to the brackish environment in which they are farmed which is known to be susceptible to human faecal contamination. High hydrostatic pressure processing (HHP) is a food treatment technique that has been shown to inactivate NV. METHODS AND RESULTS: In this study we investigated the ability of HHP to inactivate murine norovirus (MNV-1), a recognised surrogate for NV, in experimentally contaminated manila clams. Pools of contaminated live clams were subjected to hydrostatic pressure ranging from 300 to 500 MPa for different time intervals of between one and 10 min. The trial was repeated three times, at monthly intervals. CONCLUSIONS: Virus vitality post-treatment was assessed and the data obtained indicates that the use of high hydrostatic pressures of at least 500 MPa for 1 min was effective in inactivating MNV-1. SIGNIFICANCE AND IMPACT OF THE STUDY: HHP results to be an effective technique that could be applied to industrial process to obtain safe Manila clams ready to eat.

Epidemiology and control of low pathogenicity avian influenza infections in rural poultry in Italy.

We analyzed the involvement of the rural poultry sector in outbreaks of low pathogenicity avian influenza (AI) in Italy in 2007-2009 and discuss possible measures for improving monitoring and control. A description of how the rural poultry sector is organized also is provided. Data were obtained by the AI surveillance system established in the areas affected by the outbreaks. The surveillance activities identified two H7N3 epidemics, in 2007 and 2009, both of which mainly involved the rural sector, yet these activities did not allow for the prompt eradication of the disease. Additional strategies could be adopted to avoid the persistence of AI within the rural sector, based on the regulation and control of poultry holdings at the top of the production chain.

West Nile virus circulation in Veneto region in 2008-2009.

West Nile virus (WNV) was detected in Italy, in late summer 2008 in horses and birds in the Po valley. As a consequence, an intense WNV surveillance was implemented in that area involving Emilia-Romagna, Veneto and Lombardy. This paper presents the results of the September 2008-November 2009 surveillance on equines, mosquitoes, wild birds, dogs and cattle in Veneto. WNV was detected in equines and dogs, and, to a lesser extent in cattle and wild birds. Simultaneous circulation of Usutu virus was detected by testing wild birds found dead. Usutu virus but not WNV was also found in mosquitoes monitored during 2009. Equine practices monitoring allowed the definition of an area of WNV circulation and the 2008-2009 westward and northward spread of the infection. Although a relatively low number of human cases and a low virus circulation in vectors and birds detected in Veneto region could be considered favourable conditions for a limited risk of human exposure, it remains difficult to predict the possible evolution of the epidemiological situation.

Field evidence of the efficacy of vaccination to control low pathogenicity avian influenza in meat turkeys.

This paper analyzes the efficacy of vaccination to control low pathogenicity avian influenza outbreaks using information collected during four epidemics occurring in Italy between 2000 and 2005. Different vaccination strategies and protocols for meat-turkey immunization are also considered.

Low pathogenicity avian influenza in Italy during 2007 and 2008: epidemiology and control.

Since 1999, the Italian poultry production system has experienced several outbreaks of avian influenza (AI), mainly located in northeastern Italy. This paper describes the low pathogenicity (LP) AI outbreaks detected during the surveillance activities implemented in 2007-08. From May to October 2007, ten rural and hobby poultry farms were infected by an LPAI virus of the H7N3 subtype. In August-October 2007, the H7N3 LPAI virus was introduced into the industrial poultry sector with the involvement of six meat turkey farms. Phylogenetic analysis of the hemagglutinin gene indicated that all but one of the H7N3 virus strains had a high level of homology (98.7%-99.8%). Furthermore, in August 2007, an LPAI H5N2 virus was identified in a free-range geese and duck breeder flock. The hemagglutinin and neuraminidase genes showed a high level of homology (99.8% and 99.9%, respectively) with H5N2 LPAI viruses isolated from mallards in July 2007 in the same area, suggesting a possible introduction from the wild reservoir. All the birds (in total 129,386) on the infected poultry farms were culled. The prompt implementation of AI control measures, including the enforcement of a targeted emergency vaccination plan, allowed the rapid eradication of infection. In 2008, three LPAI viruses (two H7N1 and one H5N1) were identified in dealer/rural farms. The surveillance activity implemented in this area allowed the prompt detection of LPAI viruses of the H5 and H7 subtypes in the rural sector, which, as observed in the 2007 epidemic, might be the source of infection for industrial poultry.

West Nile virus transmission in 2008 in north-eastern Italy.

After 10 years, West Nile virus (WNV) re-emerged in Italy in August 2008. As on 31 December 2008, the infection affected eight Provinces in three Regions (Emilia Romagna, Veneto, Lombardy), where a total of 794 cases of WNV infection in 251 equine stables were detected on the basis of the clinical signs and as a result of a serological screening in horses living in the area. Only 4.0% (32/794) of the serologically positive animals showed clinical signs, and the 32 clinical cases were reported in 18 different farms. The observed case-fatality rate was 15.6% (5/32). The confirmed clinical cases were detected from end August to mid October. Significant levels of positivity by RT-PCR were also observed in magpies (Pica pica) (9.1%, 95% confidence levels: 6.1-13.4%), carrion crows (Corvus corone) (7.4%, 95% confidence levels: 3.6-14.4%) and rock pigeons (Columba livia) (12.9%, 95% confidence levels: 7.6-21.2%).

Emergence of a new genetic lineage of Newcastle disease virus in West and Central Africa--implications for diagnosis and control.

Newcastle disease (ND) is an OIE listed disease caused by virulent avian paramyxovirus type 1 (APMV-1) strains, which affect many species of birds and may cause severe economic losses in the poultry sector. The disease has been officially and unofficially reported in many African countries and still remains the main poultry disease in commercial and rural chickens of Africa. Unfortunately, virological and epidemiological information concerning ND strains circulating in the Western and Central regions of Africa is extremely scarce. In the present study, sequence analysis, pathotyping and detailed genetic characterization of virulent ND strains detected in rural poultry in West and Central Africa revealed the circulation of a new genetic lineage, distinguishable from the lineages described in the Eastern and Southern parts of the continent. Several mismatches were observed in the segment of the matrix gene targeted by the primers and probe designed for the molecular detection of APMV-1, which were responsible for the false negative results in the diagnostic test conducted. Furthermore, deduced amino acid sequences of the two major antigens eliciting a protective immune response (F and HN glycoprotein) revealed protein similarities <90% if compared to some common vaccine strains. Distinct mutations located in the neutralizing epitopes were revealed, indicating the need for detailed assessment of the efficacy of the current vaccines and vaccination practices in Africa. The present investigation provides important information on the epidemiology, diagnosis and control of NDV in Africa and highlights the importance of supporting surveillance in developing countries for transboundary animal diseases.

Resistance of turkeys to experimental infection with an early 2009 Italian human influenza A(H1N1)v virus isolate.

We performed an experimental infection of 21- and 70-day-old meat turkeys with an early human isolate of the 2009 pandemic H1N1 influenza virus exhibiting an alpha-2,3 receptor binding profile. Virus was not recovered by molecular or conventional methods from blood, tracheal and cloacal swabs, lungs, intestine or muscle tissue. Seroconversion was detected in a limited number of birds with the homologous antigen only. Our findings suggest that in its present form, the pandemic H1N1 influenza virus is not likely to be transmitted to meat turkeys and does therefore not represent an animal health or food safety issue for this species.