Nodular gill disease in Northeastern Italy: An investigation on the prevalence of the disease and the risks of introduction in rainbow trout farms.

Nodular Gill Disease (NGD) is an emerging pathogenic condition that causes gill damage and mainly affects farmed freshwater fish, rainbow trout (Oncorhynchus mykiss) in particular, and this inevitably generates noticeable economic losses for the industry. The present study aimed to assess the prevalence of NGD in the Autonomous Province of Trento, a highly productive area located in Northeastern Italy, traditionally suited to rainbow trout production, and to identify possible risk factors for the introduction of this disease in trout farms. The necessary data were obtained through a questionnaire and the collection of fish samples. According to the data analysis, 42% of the investigated farms tested positive for NGD. The two possible risk factors identified for its introduction in farms are the presence of other diseases in the same farm (OR = 17.5; 95% CI = 2.7; 111.5) and having farms located 5 km upstream (OR = 24.8; 95% CI = 2.9; 211.1). These results highlight (i) a possible impairment of the immune system caused by other diseases as a predisposing factor to the manifestation of the pathology and (ii) the role of water in spreading pathogens.

Lack of in vivo cross-protection of two different betanodavirus species RGNNV and SJNNV in European sea bass Dicentrachus labrax.

Viral encephalopathy and retinopathy (VER) is a severe infective disease characterized by neuropathological changes in several fish species associated with high mortality. The etiological agent is a virus belonging to the Nodaviridae family, genus Betanodavirus. To date, four different betanodavirus species have been officially recognized by International Committee on Taxonomy of Viruses (ICTV), namely the red-spotted grouper- (RGNNV), the striped jack- (SJNNV), the barfin flounder- (BFNNV) and the tiger puffer nervous necrosis virus (TPNNV). Moreover, two reassortants RGNNV/SJNNV and SJNNV/RGNNV have been described. Betanodaviruses can be classified into three different serotypes (A, B and C) that are antigenically different, so none (between serotype A and C) or partial (between serotype B and C) cross-immunoreactivity has been detected in vitro. In this study we investigated the in vivo cross-protection of the two main betanodavirus species (RGNNV and SJNNV), which belong to distinct serotype, by immunizing intraperitoneally (IP) juvenile sea bass with formalin inactivated RGNNV and SJNNV vaccines, followed by a challenge with RGNNV. Fish IP vaccinated with inactivated RGNNV showed a high protection value (85%). Serological analyses highlighted a great specific anti-NNV immunoglobulin M (IgM) production against the homologous virus, while a good seroconversion with low neutralization property was highlighted against the heterologous virus. In fish IP vaccinated with inactivated SJNNV the protection recorded was equal to 25%, significantly lower respect to the one provided by RGNNV IP vaccine. ELISA test detected good IgM production against the homologous virus, and a lower, but still detectable IgM production against the heterologous one. By contrast, serum neutralization test highlighted a poorly detectable antibody production unable to neutralize either the homologous or the heterologous virus. These results confirm that the two serotypes are not cross-protective in vivo. According to these findings, the production of multivalent formulation, or at least the provision of different types of vaccines based on both fish and virus species requirement, should be recommended in order to broaden the range of protection.

Lumpfish (Cyclopterus lumpus, Linnaeus) is susceptible to viral nervous necrosis: Result of an experimental infection with different genotypes of Betanodavirus.

In recent years, the use of cleaner fish for biological control of sea lice has increased considerably. Along with this, a number of infectious diseases have emerged. The aim of this study was to investigate the susceptibility of lumpfish (Cyclopterus lumpus) to Betanodavirus since it was detected in asymptomatic wild wrasses in Norway and Sweden. Three betanodaviruses were used to challenge lumpfish: one RGNNV genotype and two BFNNV genotypes. Fish were injected and monitored for 4 weeks. Brain samples from clinically affected specimens, from weekly randomly selected fish and survivors were subjected to molecular testing, viral isolation, histopathology and immunohistochemistry. Reduced survival was observed but was attributed to tail-biting behaviour, since no nervous signs were observed throughout the study. Betanodavirus RNA was detected in all samples, additionally suggesting an active replication of the virus in the brain. Viral isolation confirmed molecular biology results and revealed a high viral titre in BFNNV-infected groups associated with typical lesions in brains and eyes of survivor fish. We concluded that lumpfish are susceptible to Betanodavirus, as proven by the high viral titre and brain lesions detected, but further studies are necessary to understand if Betanodavirus can cause clinical disease in this species.

A formalin-inactivated immunogen against viral encephalopathy and retinopathy (VER) disease in European sea bass (Dicentrarchus labrax): immunological and protection effects.

The European sea bass (Dicentrarchus labrax) is an important farmed fish species in the Mediterranean area, very sensitive to the infection by encephalopathy and retinopathy virus (VERv), or Betanodavirus, which causes massive mortalities. Effective vaccines to fight the pathology are not yet available and in this work we describe a promising intraperitoneal immunization route against VERv of sea bass juveniles. We performed intraperitoneal and immersion immunization trials with a VERv (isolate 283.2009 RGNNV) inactivated by formalin, ß-propiolactone and heat treatment. Interestingly, the intraperitoneal immunization with formalin-inactivated VERv induced a significant antigen-specific IgM production, differently from other inactivation protocols. However, the same formalin-inactivated antigen resulted in very low IgM antibodies when administered by immersion. Following the intraperitoneal injection with formalin-inactivated virus, the quantitative expression of the antiviral MxA gene showed a modulation of transcripts in the gut after 48 h and on head kidney after 24 h, whereas ISG12 gene was significantly up-regulated after 48 h on both tissues. In immersion immunization with formalin-inactivated VERv, a modulation of MxA and ISG12 genes after 24 h post-treatment was detected in the gills. An effective uptake of VERv particles in the gills was confirmed by immunohistochemistry using anti-VERv antibodies. Lastly, in challenge experiments using live VERv after intraperitoneal immunization with formalin-inactivated VERv, we observed a significant increase (81.9%) in relative survival percentage with respect to non-immunized fish, whereas immersion immunization resulted in no protection. Our results suggest that intraperitoneal immunization with formalin-inactivated VERv could be a safe and effective strategy to fight Betanodavirus infection in European sea bass.

Water temperature affects pathogenicity of different betanodavirus genotypes in experimentally challenged Dicentrarchus labrax.

Betanodaviruses are the causative agents of a highly infectious disease of fish known as viral nervous necrosis (VNN). To date, 4 different nervous necrosis virus (NNV) genotypes have been described, but natural reassortant viruses have also been detected, which further increase viral variability. Water temperature plays an important role in determining the appearance and the severity of VNN disease. We assessed the effect of temperature (20°, 25° and 30°C) on mortality and virus load in the brain of European sea bass Dicentrarchus labrax experimentally infected with 4 genetically different betanodaviruses, namely red-spotted grouper NNV (RGNNV), striped jack NNV (SJNNV) and the reassortant strains RGNNV/SJNNV and SJNNV/RGNNV. The RGNNV/SJNNV virus possesses the polymerase gene of RGNNV and the coat protein gene of SJNNV, and vice versa for the SJNNV/RGNNV virus. The obtained results showed that the RGNNV strain is the most pathogenic for juvenile sea bass, but clinical disease and mortality appeared only at higher temperatures. The SJNNV strain is weakly pathogenic for D. labrax regardless of the temperature used, while virus replication was detected in the brain of survivors only at 20°C. Finally, reassortant strains caused low mortality, independent of the temperature used, but the viral load in the brain was strongly influenced by water temperature and the genetic type of the polymerase gene. Taken together, these data show that nodavirus replication in vivo is a composite process regulated by both the genetic features of the viral strain and water temperatures.

Productive and reproductive performances of dairy cattle herds in Treviso province, Italy (2009-2012): an assessment of the potential impact of Schmallenberg virus epidemic.

BACKGROUND: Schmallenberg virus (SBV) has spread across Europe since mid-2011, causing unspecific and transitory symptoms in ruminants and congenital malformations in their offspring. Evidence for the impact of SBV on cattle (re)productive performance is limited. Using a comprehensive data set from a SBV-affected province in North-East Italy, this study aimed at assessing the potential impact of SBV emergence on 11 productive and reproductive performance indicators of dairy cattle herds, accounting for weather conditions and other herd-level factors that could also influence these indicators. RESULTS: A total of 127 farms with an average of 71 cows per farm (range 29-496) were monitored monthly from January 2009 to June 2012. Mixed-effects linear models for longitudinal data were used to assess the average variation in herds' performance indicators over semesters (Jan-Jun 2009, Jul-Dec 2009, Jan-Jun 2010, Jul-Dec 2010, Jan-Jun 2011, Jul-Dec 2011, Jan-Jun 2012) and trimesters therein. Taking the second semester of 2011 as reference, significant decreases in the average lactation length (-6 days, on average) and calving-to-conception interval (-4 days, on average) were observed relative to the same semesters of the years 2010 and 2009, respectively. Similarly, during the last trimester of 2011, which is most likely to cover the SBV infection period in the study area, there was an average decrease of -4 days (lactation length) and -7 days (calving-to-conception interval) compared to the same trimesters of the years 2010 and 2009, respectively. However, the observed decreases actually represent a positive outcome that is not as such imputable to SBV emergence, but rather reflects other beneficial changes in farm management. None of the other indicators showed significant variations, confirming the relatively mild expression of SBV infection in cattle. CONCLUSIONS: Although the emergence of SBV might have significantly affected the (re)productive performance of some individual farms, we concluded that overall at the province level there were no significant variations attributable to SBV, at least not in a way that would lead to negative effects on farm profitability.

Farmers' Biosecurity Awareness in Small-Scale Alpine Dairy Farms and the Crucial Role of Veterinarians.

BACKGROUND: Biosecurity measures are essential to improve animal health and welfare, tackle antimicrobial resistance (AMR), minimize the burden of infectious diseases, and enhance the safety, security, and quality of sustainable agricultural products. However, the implementation of these measures in small mountain farms can be influenced by several factors, which probably differ from the main variables that affect intensive farming systems. METHODS: This study investigated the awareness among farmers regarding the implementation of biosecurity measures at the local level in small dairy farms located in the Autonomous Province of Bolzano/Bozen (Italy). In order to determine to what extent these measures are in line with the recommendations, a questionnaire was conceptualized and sent by post to a representative group of local farmers. The initiative was voluntary and anonymous. RESULTS: A total of 50 farmers responded to the questionnaire, resulting in a response rate of 27.5%. This study confirms that most of the farmers are familiar with biosecurity, and 38% of them know its comprehensive definition. The results indicate that 77% of respondents are willing to implement preventive measures to reduce the use of antimicrobials, and 76% of them acknowledge that they would follow the recommendations provided by veterinarians. In agreement with other studies, the role of the veterinarian as a person of trust among farmers is confirmed. CONCLUSIONS: Main strengths and areas of improvement have been identified. Additional data and effective tools are needed to better investigate drivers toward biosecurity and to implement impactful and practical measures for mountain farming.

Experimental infection of poults and guinea fowl with genetically distinct avian astroviruses.

Avian astroviruses, of the genus Avastrovirus, are recognized as being the cause of enteritis in different bird species worldwide. In particular, turkeys are very susceptible and can be severely affected by this viral agent. More recently, astroviruses were detected in diseased guinea fowl in Italy but whether or not they were the causative agents of the clinical disease was not established. Despite the distribution and relevance of Avastrovirus infection, very little information on pathogenesis or factors influencing the pathogenicity of astroviruses is available. To increase available data on the pathogenesis of these viruses and to test the hypothesis of possible interspecies transmission, experimental infections were carried out in turkeys and guinea fowl with two genetically distinct avian astroviruses, namely TK-6363 and GF-5497, originating respectively from diseased turkey poults and guinea fowl. Data obtained in our study show that both of the viruses selected were able to infect young birds of the species in which they were originally detected. Additionally, these viruses were able to infect young birds of different species causing clinical signs, thus providing experimental evidence for the infection of distinct avian astroviruses in different avian species.

Some Considerations about Winter Colony Losses in Italy According to the Coloss Questionnaire.

The Italian beekeeping industry has grown steadily during the last decade, according to data from the national beekeeping registry, which came into existence in February 2015. Winter colony losses remain a matter of concern for beekeepers in Italy, and administration of the questionnaire defined by the Coloss Association could contribute to a better understanding of this phenomenon. To evaluate the percentage trends over time in honeybee colony losses arising from various causes, we used the quasi-binomial generalized linear modelling (GzLM) approach, taking the year as an independent variable. We set our level of significance at 5% and performed the data analysis only for the seven regions that sent data continuously from 2014 to 2020. We considered the percentage of losses due to queen-related problems, natural disasters, and dead or empty colonies, given that these questions remained unchanged over the years. The survey also revealed that the percentage trend for respondents using drone brood removal showed a significant increase. In general, the percentage of colony losses due to queen-related problems remained lower than 8%, and the percentage of colony losses associated with natural disasters was very low (<2%). The mean percentages of losses due to dead or empty colonies ranged from 6 to 17% in the considered period. In addition, we took account of the responses relating to treatments against Varroa mite infestation, given the importance attributed to this honeybee parasite. Unlike the other variables, we calculated the percentages related to the types of beekeeper treatments against Varroa destructor based on the respondents, not on the colonies. What emerged was that almost every beekeeper used at least one type of treatment against V. destructor. In general, the trend of respondents appeared stable at 0.3% during the last four years.

Donkey assisted interventions: A pilot survey on service providers in North-Eastern Italy.

CONTEXT: Animal Assisted Interventions (AAI), which are generally defined as 'pet therapy', focus on the improvement of human health and wellbeing through a multidisciplinary approach and the involvement of domestic animals. An Italian survey conducted in 2017 revealed that donkeys were the third most involved species in AAI. Nevertheless, data on the main features of the providers of Donkey Assisted Interventions (DAI), especially in Italy, and their organisational models are scarce. OBJECTIVE: This questionnaire-based pilot study aimed to collect information about the features of DAI providers in the Veneto Region to have a general framework about the field. STUDY METHOD: In this descriptive mixed-method pilot study, we interviewed DAI providers through a computer assisted telephone interview. Respondents were selected by integrating data from the Veneto Region Livestock Database and from the Italian official register of AAI professionals. The questionnaire was designed following the advice from Equator Network. MAIN OUTCOME MEASURES: Our outcome measures included the prevalent organisational system adopted by our respondents, main type of AAI programs, and users. Furthermore, we collected qualitative data about economic and income and respondents' personal opinions about DAI (strengths and weaknesses). MAIN RESULTS: Data analysis revealed a heterogeneous picture of the DAI field with a prevalence of non-profit organisations providing mainly Donkey Assisted Activities to a large range of user categories. Despite the poor economic framework and low income from DAI, DAI providers were strongly motivated and spent more time and efforts in delivering this service.

Pathogenicity of Different Betanodavirus RGNNV/SJNNV Reassortant Strains in European Sea Bass.

European sea bass (Dicentrarchus labrax) is an important farmed marine species for Mediterranean aquaculture. Outbreaks of betanodavirus represent one of the main infectious threats for this species. The red-spotted grouper nervous necrosis virus genotype (RGNNV) is the most widely spread in Southern Europe, while the striped jack nervous necrosis virus genotype (SJNNV) has been rarely detected. The existence of natural reassortants between these genotypes has been demonstrated, the RGNNV/SJNNV strain being the most common. This study aimed to evaluate the pathogenicity of different RGNNV/SJNNV strains in European sea bass. A selection of nine European reassortants together with parental RGNNV and SJNNV strains were used to perform in vivo experimental challenges via intramuscular injection. Additional in vivo experimental challenges were performed by bath immersion in order to mimic the natural infection route of the virus. Overall, results on survival rates confirmed the susceptibility of European sea bass to reassortants and showed different levels of induced mortalities. Results obtained by RT-qPCR also highlighted high viral loads in asymptomatic survivors, suggesting a possible reservoir role of this species. Our findings on the comparison of complete genomic segments of all reassortants have shed light on different amino acid residues likely involved in the variable pathogenicity of RGNNV/SJNNV strains in European sea bass.

Improving heat stability of haemagglutinating antigens for avian influenza.

The increasing demand for avian influenza diagnostic reagents worldwide, has included requests for significant supplies of product to developing countries. Difficulties in dispatching to remote areas and tropical countries are a major concern to suppliers, international organisations and donors as delays in forwarding parcels often result in storage at non-optimal or inadequate temperatures results in loss in titre and thus wastage of resources. In this study we demonstrate that the heat stability of avian influenza haemagglutination inhibition antigens of the H5, H7 and H9 subtype following 14 days of exposure to 37°C and 45°C is significantly increased by adding D-(+)-Trehalose to the freshly prepared antigen. Increased stability was detected both for freeze-dried antigens over an extended period of 6 months and also in heat exposed antigens that were then stored at +4C for up to 35 days post reconstitution.

Fast and simultaneous analysis of carbamate pesticides and anticoagulant rodenticides used in suspected cases of animal poisoning.

Carbamate pesticides (CBs) are reported as one of the main causes of intentional or accidental poisoning of animals. Anticoagulant rodenticides (ARs) form the main class of poisons implicated in analyzed poisoned baits. These two groups of pesticide compounds include multiple substances, and thus, the development of a simple and rapid multiclass/multiresidue analytical method for simultaneous identification of both toxicant classes should be a useful strategy for analytical laboratories to reduce analysis time and cost. The present study aimed to elaborate and validate a rapid method to simultaneously determine 11 CBs and 8 ARs in samples of real matrices (bait, stomach content, and liver) from suspected animal poisoning cases. QuEChERS sample treatment and liquid chromatography coupled to hybrid high resolution mass spectrometry were used. The method resulted in good linearity (R2 ≥ 0.98) for all compounds, recovery was between 70% and 120% for CBs and 40-90% for ARs, and precision was ≤ 20% for all compounds. The method was successfully applied to the analysis of 871 real samples originating from suspected cases of animal poisoning, collected from April 2019 to October 2020. Furthermore, full scan dependent data acquisition allowed qualitative retrospective data analysis of an additional 15 compounds outside the scope of the method to be performed; these compounds could potentially be involved in unresolved poisoning cases.

Equine-Assisted Interventions (EAIs) for Children with Autism Spectrum Disorders (ASD): Behavioural and Physiological Indices of Stress in Domestic Horses (Equus caballus) during Riding Sessions.

Equine-assisted interventions (EAIs) are well-known complementary practices combining physical activity with emotional/cognitive stimulation. They are especially suited for children with autism spectrum disorders (ASD) who need a high degree of physical and psychological enrichment. Even though EAIs have become a common practice, stress responses in horses interacting with individuals that can manifest inappropriate behaviours, such as ASD children, have not been thoroughly investigated. Our multicentre study aimed to investigate behavioural and physiological indices of stress in horses involved in EAI standardised sessions with children with ASD compared to typically developing (TD) children. A controlled within-subject design with repeated measurements involving 19 horses and 38 children was adopted. Stress-related behaviours, heart rate, heart rate variability, and eye temperature were recorded during the riding sessions. Moreover, blood samples were collected from horses before and after each session to monitor changes in blood adrenocorticotropic hormone (ACTH), cortisol, and catecholamines. Results indicate that, in general, stress responses in horses involved in EAIs did not differ as a function of the horse being ridden by children with ASD or TD. A lower sympathetic tone in horses involved in ASD sessions was found, while in the mounting and dismounting phases, horses displayed behavioural signs of stress, independently from children's behaviour. We conclude that professionals working in EAI should increase their awareness of animal welfare and refine riding practices, taking into account horse's needs.

Canine leishmaniosis in the Italian northeastern Alps: A survey to assess serological prevalence in dogs and distribution of phlebotomine sand flies in the Autonomous Province of Bolzano - South Tyrol, Italy.

The Autonomous Province of Bolzano-South Tyrol (APB), located in the northernmost territory of the Italian eastern Alps, is still considered non-endemic for canine leishmaniosis (CanL) despite clinical cases being observed and a competent Leishmania infantum vector (Phlebotomus perniciosus) having been recorded since 2008. A serological survey of leishmaniosis among a randomly-selected subpopulation of registered owned dogs was carried in 2018, followed by entomological investigations performed in 2019 and driven by canine survey results. A total of 457 resident dogs from all over the APB territory were examined through IFAT for antibodies against L.infantum, of which 63 (13.8%) tested positive. Thirty-five seropositive cases (7.7%) were considered autochthonous to APB, i.e. dogs born and lived in the province, or imported dogs with no travel history in the past 5 years. Most of these animals showed an antibody titre at the threshold level of 1:40, suggesting a low degree of parasite transmission/contacts. In 2 autochthonous cases with moderately high IFAT titre, the infection was confirmed by nested-PCR in peripheral blood. Thirty-one georeferenced sites were monitored for sand flies by means of interception (sticky papers) and attraction (CDC miniature light traps) collection devices. Traps were set during summer approximately on monthly basis, and extended up to October for positive sites. Only 2 sites were found positive for a total of 317 phlebotomine specimens collected by sticky traps, which included a previously known P. perniciosus-endemic site near Bolzano town. Sergentomyia minuta was by far the most prevalent (98.1%) and the only recorded sand fly species in the most northerly Italian site ever investigated (Coldrano municipality in Venosta valley). For the first time, Leishmania serology and n-PCR positive dogs autochthonous to APB were identified, however the spread of sand flies competent for L. infantum transmission could not be demonstrated in several places where endemic seropositive cases were recorded. APB can be considered a territory of low CanL endemicity, however awareness and continuous monitoring are needed to detect changes in the epidemiological status of the zoonosis.

Risk factors for highly pathogenic H7N1 avian influenza virus infection in poultry during the 1999-2000 epidemic in Italy.

In 1999-2000, Italian poultry production was disrupted by an H7N1 virus subtype epidemic of highly pathogenic avian influenza (HPAI). The objectives of the present study were to identify risk factors for infection on poultry farms located in regions that had the highest number of outbreaks (Veneto and Lombardia) and the impact of pre-emptive culling as a complementary measure for eradicating infection. A Cox regression model that included spatial factors, such as the G index, was used. The results confirmed the relationship between risk of infection and poultry species, production type and size of farms. The effectiveness of pre-emptive culling was confirmed. An increased risk of infection was observed for poultry farms located near an infected farm and those at altitudes less than 150m above sea level. The measures for the control and eradication of AI virus infection need to consider species differences in susceptibility, the types of production and the density of poultry farms in the affected areas.

Animal assisted interventions in practice: mapping Italian providers.

Animal assisted interventions (AAI) have become increasingly popular in Western countries during the last fifty years, attracting a lot of attention both from the general public and the scientific community. In Italy, similarly to other countries, this evolution has been accompanied by the diffusion of associations delivering AAI. What is the current state of AAI in Italy? How many realities are dealing with AAI? What professionals and animals are involved? The results of the questionnaire here reported have the objective to answer these questions, outlining a snapshot of the distribution and the main features of AAI within Italian territory. According to the 208 respondents, the sector has seen a remarkable expansion over the last twenty years. AAI providers are located mainly in the North and Centre of the country, the majority of them are arranged in associations or AAI specialized centers and work mainly with dogs and equids in animal assisted activity (AAA) programs addressed to disabled people and children. The picture of the sector still appears fragmented in particular regarding team of professionals involved and their training; nonetheless, the recent set up of national guidelines by the Italian authorities has the objective of standardizing the field and defining best practices for each type of intervention.

Evaluation of the efficacy of intervention measures and vaccination for the control of LPAI epidemics in Verona province (Veneto, Italy).

Meat-type turkey farms in Verona province (Veneto, Italy) have been affected by three low-pathogenicity avian influenza (LPAI) epidemics between 2000 and 2004. Control measures implemented ranged from stamping out to controlled marketing in conjunction with restocking bans and movement restriction on live poultry, vehicles, and personnel. These measures were complemented with two emergency vaccination programs (2000-01, 2002-03) started after the beginning of the epidemics, while 2004 outbreaks occurred in vaccinated farms. The three epidemics differed in the number of outbreaks, duration, and economic impact. The aim of the investigation was to estimate the risk of infection and the effect of vaccination on the LPAI epidemics affecting turkey farms in Verona province. Farm probability to avoid infection (Ps) was calculated by Kaplan-Meier for each epidemic. The vaccination effect was evaluated for the 2000-01 and 2002-03 epidemics considering different risk before or after the emergency vaccination. The epidemics and vaccination entered as predictors in a Cox regression model and hazard ratios (HR) were calculated. Ps values at the end of the epidemics were as follows: 2000-01 = 0.66, 2002-03 = 0.51, and 2004 = 0.91. Vaccination reduced dramatically the risk of infection. The measures implemented had different effects on the three epidemics. The lower probability of being infected during the 2004 epidemic was most likely related to the protection level of the vaccinated farms acquired before the beginning of the epidemic, which was also responsible for the reduced spread of infection.

Effect of different temperatures on the stability of avian influenza reference reagents.

The production and supply of reference reagents for the diagnosis of avian influenza (AI) is one of the duties of the World Organization for Animal Health reference laboratories. The lyophilized reagents are routinely shipped on dry ice to both national and international clients. In order to determine the effect of different short-term storage temperatures on the activity of AI reference reagents, vials containing lyophilized avian influenza A antigens and antisera preparations were maintained at 4 C, 22 C, and 37 C over a 21-day period. At days 0, 3, 7, 14, and 21 the reagents were titrated using the hemagglutination test, the hemagglutination inhibition test, or the agar gel immunodiffusion test (AGID). All of the AI antigens that were kept at 4 C and 22 C retained hemagglutinating activity for at least 21 days, but when they were stored at 37 C several lost their hemagglutinating activity. All of the reference antisera tested were still able to inhibit hemagglutination after 21 days, and the antigen used in AGID also gave clear results after 21 days even after incubation at 37 C. Our results therefore indicate that lyophilized avian influenza antigens maintain their hemagglutinating activity at temperatures between 4 C and 22 C for at least 21 days, and both antisera and antigens prepared for AGID remain stable for 21 days between 4 C and 37 C. This information will allow for alternative shipping temperatures than those presently recommended, in addition to the short-term storage of these reagents at nonrefrigerated temperatures.

Intervention strategies for low-pathogenic avian influenza control in Italy.

Italian poultry production was affected by several outbreaks of low-pathogenicity avian influenza (LPAI) between 2000 and 2005. Intervention measures (IM), such as stamping out of infected and suspected farms, controlled marketing, restocking bans, movement restriction, and emergency vaccination, were put into force in the most affected areas of Lombardia and Veneto regions. These two regions also showed differences in terms of measures applied and timeliness of application. In this study we describe the epidemics and discuss the effectiveness of the IM put into effect. The regional surveillance systems provided the data on the epidemics and the IM description. The IM effectiveness was compared between the different epidemics and the Lombardia and Veneto regions, considering the number of farms involved, the duration of the epidemics, and the extension of the area affected. With regard to the IM applied, reductions in the number of outbreaks (from 388 in 2002-03 to 15 in 2005), the duration of the outbreaks (from more than 1 yr to approximately 1 mo), and the spatial extension of the outbreaks (from 89 to 8 municipalities involved) were observed. The emergency vaccination, depopulation, and pre-emptive slaughtering reduced significantly the spread of the epidemic. Comparing the dynamics of the epidemics, more effective results were observed in the Veneto region, where the IM were applied to a greater extent. Emergency vaccination and depopulation were effective in the eradication of the disease during an epidemic, but vaccination and farm density reduction showed the most effective results in controlling the spread of LPAI.