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.

Influenza at the animal-human interface: a review of the literature for virological evidence of human infection with swine or avian influenza viruses other than A(H5N1).

Factors that trigger human infection with animal influenza virus progressing into a pandemic are poorly understood. Within a project developing an evidence-based risk assessment framework for influenza viruses in animals, we conducted a review of the literature for evidence of human infection with animal influenza viruses by diagnostic methods used. The review covering Medline, Embase, SciSearch and CabAbstracts yielded 6,955 articles, of which we retained 89; for influenza A(H5N1) and A(H7N9), the official case counts of t he World Health Organization were used. An additional 30 studies were included by scanning the reference lists. Here, we present the findings for confirmed infections with virological evidence. We found reports of 1,419 naturally infected human cases, of which 648 were associated with avian influenza virus (AIV) A(H5N1), 375 with other AIV subtypes, and 396 with swine influenza virus (SIV). Human cases naturally infected with AIV spanned haemagglutinin subtypes H5, H6, H7, H9 and H10. SIV cases were associated with endemic SIV of H1 and H3 subtype descending from North American and Eurasian SIV lineages and various reassortants thereof. Direct exposure to birds or swine was the most likely source of infection for the cases with available information on exposure.

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.

An SYBR Green-based real-time RT-PCR assay for the detection of H5 hemagglutinin subtype avian influenza virus.

Increasing diversity among H5 hemagglutinin (HA) subtype avian influenza (AI) viruses has resulted in the need of novel sensitive and specific molecular assays. In this study, an SYBR Green-based real-time reverse transcription-PCR (RRT-PCR) assay was developed for the detection of H5 subtype AI virus. Sequence analysis of the Mexican lineage H5N2 isolates (subgroup B) revealed several mismatches in the primer/hydrolysis probe set reported in the commonly used RRT-PCR assay for the detection of H5 North American lineage. The present assay was designed to circumvent the challenge that these viruses represent for the specific detection of H5 subtype AI viruses. This RRT-PCR assay successfully detected a range of different H5 subtype AI strains from both Eurasian and North American lineages representing different avian H5 HA clades from diverse geographical locations. The sensitivity of the present method was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of titrated AI viruses. High sensitivity levels were obtained, with limits of detection of 10(0) 50% egg infectious dose (EID50)/mL and 4.2 gene copies/µl. The linear ranges of the assay span within 10(6)-10(0) EID50/mL and 10(6)-10(0) gene copies/µl. The results obtained from this method were directly compared with those of the H5 RRT-PCR assay recommended by the OIE. The comparison was performed with 110 tracheal and cloacal swabs from various bird species collected during field and laboratory investigations in Eurasia and Africa in 2006 and 2008 and showed 100% agreement. This assay is recommended as an alternative method, also allowing a 'double check' approach detection, to be use mainly in outbreak scenarios with higher risk of poultry infections by Central American/Caribbean H5 AI viruses.

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.

Transboundary spread of highly pathogenic avian influenza through poultry commodities and wild birds: a review.

The extensive circulation of the H5N1 highly pathogenic avian influenza (HPAI) virus in animals and the human health implications which it poses have led to extensive research in unexplored fields and thus a re-assessment of our understanding of this infection. Moreover, widespread infection of poultry has raised concerns about the food safety and trade implications of this infection, necessitating revised international trade regulations. The role of wild birds has been much debated and resources have been invested to clarify the role that they may play in the spread of infection. It is now clear that wild birds may be responsible for primary introduction in a previously free area. To date it is still unclear whether HPAI infection may be maintained in wild bird populations for extended periods of time. This paper reviews existing knowledge on the transboundary spread of HPAI through poultry and poultry commodities and summarises evidence of spread through wild birds.

Characterization of pseudorabies virus of wild boar origin from Europe.

Pseudorabies virus (PrV) infections appear to be more widely distributed in the European wild boar (Sus scrofa) population than assumed. In Europe, attempts to isolate and characterize the causative agents have been limited so far. We therefore collected and examined a total of 35 PrV isolates obtained from wild boar or hunting dogs in Germany, France, Spain, Italy, Slovakia and Hungary between 1993 and 2008. Restriction enzyme analysis of genomic DNA using BamHI showed that all isolates, except one, belonged to genogroup I but different subtypes were evident. For further investigations of the phylogenetic relationships, a 732-bp fragment of the glycoprotein C (gC) gene was amplified by PCR. Sequence analysis revealed about 40 variant positions within this fragment. Comparison of the nucleotide sequences supported the separation into a clade containing isolates from North-Rhine Westphalia, Rhineland-Palatinate (Germany), France and Spain (clade B) and an apparently more variable clade comprising isolates from Brandenburg, Baden-Wurttemberg, Saxony, Saxony-Anhalt (Germany), Slovakia, Hungary, Italy and France (clade A).

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.

Essential veterinary education in avian medicine: a global perspective.

Avian medicine is a relatively recent discipline in the veterinary curriculum, and is definitely not considered a topical issue. However, in the face of a growing demand for poultry meat worldwide, and in view of the health issues surrounding wild, exotic and pet birds, the relevance of avian medicine should be acknowledged and taken into account when revising curricula.

Vaccination as a tool to combat introductions of notifiable avian influenza viruses in Europe, 2000 to 2006.

In late 2000, Italy was the first country of the European Union (EU) to implement an emergency vaccination programme against notifiable avian influenza. Vaccination with a conventional vaccine containing a seed strain with a different neuraminidase subtype from that of the field virus was used to complement biosecurity and restriction measures as part of an overall eradication strategy. This vaccination technique, in line with the Differentiating Infected from Vaccinated Animals system (DIVA), was applied several times until March 2008. This strategy enabled the identification of field exposed flocks and ultimately the eradication of low pathogenic H7N1, H7N3 and H5N2 infections. Italy was also the first country to implement a bivalent H5/H7 prophylactic vaccination programme of defined poultry populations, which was discontinued in December 2006. Following the incursion of highly pathogenic H5N1 into Europe, in 2005 and 2006, two other EU Member States, namely France and the Netherlands, implemented preventive vaccination programmes in 2006 but they targeted selected poultry populations different from those targeted in Italy and were implemented for short periods of time. Data generated during six years of experience with vaccination against avian influenza in Italy indicate that it is a useful tool to limit secondary spread and possibly prevent the introduction of low pathogenic avian influenza viruses in a susceptible population. The experience of France and the Netherlands provides data on vaccination of ducks and hobby poultry respectively and monitoring programmes associated with vaccination and difficulties related to their application. The advantages and disadvantages of vaccination need to be considered in the decision-making process, including the financial aspects of vaccination.

Avian influenza infection in birds: a challenge and opportunity for the poultry veterinarian.

Influenza A viruses infecting poultry can be divided into 2 groups. The extremely virulent viruses cause highly pathogenic avian influenza (HPAI), with flock mortality as great as 100%. These viruses have been restricted to subtypes H5 and H7, although not all H5 and H7 viruses cause HPAI. All other viruses cause a milder, primarily respiratory, disease (LPAI) unless exacerbated. Until recently, HPAI viruses were rarely isolated from wild birds, but for LPAI viruses, extremely great isolation rates have been recorded in surveillance studies. Influenza viruses may infect all types of domestic or captive birds in all areas of the world, with the frequency with which primary infections occur in any type of bird usually depending on the degree of contact of the bird with feral birds. Secondary spread is usually associated with human involvement, either by bird or bird product movement, or by transferring infective feces from infected to susceptible birds, but potentially wild birds could be involved. In recent years, the frequency of HPAI outbreaks appears to have increased, and there have been particularly costly outbreaks of HPAI in densely populated poultry areas in Italy, the Netherlands, and Canada. In each, millions of birds were slaughtered to bring the outbreaks under control. Since the 1990s, avian influenza infections attributable to 2 subtypes have been widespread in poultry across a large area of the world. The LPAI H9N2 virus appears to have spread across the whole of Asia in that time and has become endemic in poultry in many of the affected countries. However, these outbreaks have tended to be overshadowed by the H5N1 HPAI virus, which, although initially isolated in China, has now spread in poultry, wild birds, or both throughout Asia and into Europe and Africa, resulting in the death or culling of hundreds of millions of poultry and posing a significant zoonotic threat. To date, control methods seem to have been unsuccessful on the larger scale, and HPAI H5N1 outbreaks continue to be reported.

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.

Conventional H5N9 vaccine suppresses shedding in specific-pathogen-free birds challenged with HPAI H5N1 A/chicken/Yamaguchi/7/2004.

Avian influenza represents one of the greatest concerns for public health that has emerged in recent times. Highly pathogenic avian influenza viruses belonging to the H5N1 subtype are endemic in Asia and are spreading in Europe and Africa. Vaccination is now considered a tool to support eradication efforts, provided it is appropriately managed. This study was carried out to establish the degree of clinical protection and reduction of viral shedding induced by a high-specification, commercially available avian influenza vaccine of a different lineage and containing a strain with a heterologous neuraminidase (H5N9 subtype) to the challenge virus isolate A/chicken/Yamaguchi/7/2004 (H5N1 subtype).

Identification of sensitive and specific avian influenza polymerase chain reaction methods through blind ring trials organized in the European Union.

Many different polymerase chain reaction (PCR) protocols have been used for detection and characterization of avian influenza (AI) virus isolates, mainly in research settings. Blind ring trials were conducted to determine the most sensitive and specific AI PCR protocols from a group of six European Union (EU) laboratories. In part 1 of the ring trial the laboratories used their own methods to test a panel of 10 reconstituted anonymized clinical specimens, and the best methods were selected as recommended protocols for part 2, in which 16 RNA specimens were tested. Both panels contained H5, H7, other AI subtypes, and non-AI avian pathogens. Outcomes included verification of 1) generic AI identification by highly sensitive and specific M-gene real-time PCR, and 2) conventional PCRs that were effective for detection and identification of H5 and H7 viruses. The latter included virus pathotyping by amplicon sequencing. The use of recommended protocols resulted in improved results among all six laboratories in part 2, reflecting increased sensitivity and specificity. This included improved H5/H7 identification and pathotyping observed among all laboratories in part 2. Details of these PCR methods are provided. In summary, this study has contributed to the harmonization of AI PCR protocols in EU laboratories and influenced AI laboratory contingency planning following the first European reports of H5N1 highly pathogenic AI during autumn 2005.

Diagnosing avian influenza infection in vaccinated populations by systems for differentiating infected from vaccinated animals (DIVA).

Vaccination against avian influenza is recommended as a tool to support control measures in countries affected by avian influenza. Vaccination is known to increase the resistance of susceptible birds to infection and also to reduce shedding; however, it does not always prevent infection. Vaccinated infected flocks can therefore be a source of infection and thus be responsible for the perpetuation of infection. To avoid the spread of infection in a vaccinated population, immunization strategies must allow differentiation of infected from vaccinated animals (DIVA), combined with an appropriate monitoring system. Vaccinated exposed flocks must be identified and managed by restriction policies that include controlled marketing and stamping-out. Several vaccines and diagnostic tests to detect infection in vaccinated populations are available, the tests having various properties and characteristics. In order to achieve eradication, the most appropriate DIVA vaccination strategy must be identified and an appropriate monitoring programme be designed, taking into account risk factors, the epidemiological situation and the socioeconomic implications of the policy.

Vaccination for notifiable avian influenza in poultry.

Notifiable avian influenza (NAI) is a listed disease of the World Organisation for Animal Health (OIE) that has become a disease of great importance both for animal and human health. Prior to 2000, vaccination against NAI was discouraged and used to aid control of only a limited number of outbreaks, without reaching the goal of eradication. Pivotal work on the application of a vaccination programme aimed at, and resulting in, eradication was carried out in Italy, and was followed by other research, e.g. in Hong Kong and the United States of America. Given the spread of Asian lineage highly pathogenic avian influenza (HPAI) H5N1 to three continents, vaccination is now being used on a wide scale under different conditions, which in most cases are not ideal. Although in some countries, a lack of infrastructure and resources can greatly limit the overall success of control programmes that encompass vaccination, it is imperative that international organisations set guidelines to 'accredit' control strategies. These guidelines should include recommendations on seed strains to be used in vaccine preparations, the characteristics of the vaccine, the most appropriate field strategy to apply in the different phases of a control/eradication programme, and models of exit strategies. The availability of harmonised protocols would greatly facilitate the achievement of tangible results and would save time and avoid unnecessary wastage of resources.

Marker vaccines and the impact of their use on diagnosis and prophylactic measures.

Molecular biology and technical advances in DNA recombination have ushered in a new era in vaccinology. This article examines the recent development of specific marker vaccines and examines the impact of their use on the diagnosis and prevention of major infectious diseases. Gene-deleted vaccines, DIVA strategies (differentiating infected from vaccinated animals) and similar methods have been successfully applied in the control and eradication of Aujeszky's disease, infectious bovine rhinotracheitis, classical swine fever, foot and mouth disease and, recently, avian influenza. The efficacy and performance of existing marker vaccines and their companion diagnostic tools (which should be assesed by an independent body) are discussed, as are the ways in which these tools are deployed by competent authorities. The limits and the advantages of the use of marker vaccines are carefully analysed in the light of practical experiences. Although these vaccines can limit the speed and the extent of virus dissemination and thus reduce the number of animals slaughtered, marker vaccines are no substitute for sanitary measures. Early detection and warning systems and the quick implementation of sanitary measures, including stamping out, remain key issues in the control of highly contagious diseases.

Avian influenza--past, present and future challenges.

Avian influenza (AI) is an OIE-listed disease that has become of great importance both for animal and for human health. The increased relevance of AI in the fields of animal and human health, has highlighted the lack of scientific information on several aspects of the disease; this has hampered the adequate management of some of the recent crises, thus resulting in millions of dead animals and concern over loss of human lives and over management of the pandemic potential. The present paper aims to identify areas of knowledge of veterinary competence that need to be explored to generate information to support the global AI crisis. Given the current situation, it is imperative that close collaboration is sought and achieved by public health officials involved in the veterinary and medical aspects of the disease. In fact, only through the exchange of data, experiences, views and information will it be possible to combat this zoonosis that represents a major threat to public health and animal well-being.

Molecular diagnosis of avian influenza during an outbreak.

Virus isolation (VI) in specific-pathogen-free (SPF) embryonated eggs or cell cultures is traditionally considered the method of choice for the detection and identification of avian influenza (AI) viruses. However, its value is limited because it is time-consuming and not cost-effective. AI is a highly contagious disease, able to spread in a susceptible population in a short period of time. Therefore, the prompt identification of an infected flock is crucial for control and eradication purposes. During an AI outbreak, the sample processing times using the above mentioned methods are often not compatible with the demands of the poultry industry. In addition, the delay in moving birds from a premises, whilst awaiting the VI result, often results in animal welfare issues. For this reason, when dealing with an epidemic of AI, rapid and reliable laboratory tests, such as RT-PCR and real-time PCR, should be available to reveal direct evidence of infection in the flocks. Scientific reports have been published in the recent past, evaluating their use during AI monitoring and surveillance programmes and epidemics. Based on the available information, the advantages and limits concerning the application of molecular methods during AI outbreaks are discussed in this paper.

Control of avian influenza in Italy: from stamping out to emergency and prophylactic vaccination.

Since 1997, north-eastern Italy has been repeatedly challenged by avian influenza (AI) infections caused by viruses of the H5 and H7 subtypes. The penetration of such infections in the industrial circuit in densely populated poultry areas (DPPAs) resulted in massive spread, and early attempts to control AI only by stamping-out and restriction policies resulted in death or culling of millions of birds. The re-emergence or the introduction of AI viruses in the same DPPA resulted in the development of an emergency vaccination programme based on the use of heterologous vaccination and a companion discriminatory test. By enabling the detection of field exposure in vaccinated animals, the application of this system, in conjunction with a monitoring programme and a well-defined territorial strategy has resulted in the eradication of H7N1 and H7N3 epidemics that occurred between 2000 and 2004. Retrospective analysis of the AI outbreaks in north-eastern Italy coupled with surveillance programmes in wild birds and in hobby flocks indicating that certain areas are at continuous high risk of infection, was the rationale for developing and implementing a bivalent H5/H7 pilot vaccination programme in a restricted area of the DPPA. Laboratory and field evidence indicate that vaccinated animals are more resistant to challenge and shed lower amounts of virus, thus acting as a tool for both prevention and control, thus limiting the impact of AI infections.