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.

The economic implications of sylvatic rabies eradication in Italy.

After more than 10 years of absence, sylvatic rabies re-appeared in Italy in 2008. To prevent disease spread, three oral rabies vaccination (ORV) campaigns targeting red foxes were performed through manual distribution of vaccine baits between January and September 2009. As these campaigns proved unsuccessful, at the end of December 2009, baits started being distributed using helicopters, allowing uniform coverage of larger areas in a shorter time period. From winter 2009 to autumn 2016, a total of 15 ORV campaigns (four emergency, four regular and seven preventive ORV) were implemented through aerial distribution of baits. In this study, we assessed the costs of the aerial ORV campaigns, which were aimed at eradicating the disease and reobtaining the rabies-free status. Cumulative costs per km2 were estimated at €59.45 during emergency campaigns and ranged between €51.94 and €65.67 in the regular vaccinations. The main portion of costs for ORV programmes were related to baits supply and distribution: €49.24 (82.83%) in emergency campaigns and from €40.33 to € 43.35 in regular ORVs (71.97% and 66.02%, respectively). At the end of each ORV campaign, the efficacy of vaccination activities was estimated by assessing the proportion of foxes testing positive for tetracycline biomarker in jawbone, indicating bait intake. Results revealed that the proportion of foxes that ingested baits varied between 70.97% and 95.51%. Statistical analysis indicated that reducing the density of dropped baits could potentially lead to a cost-saving of 22.81%, still maintaining a satisfactory level of bait intake by the fox population.

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.

Factors associated with pre-slaughter mortality in turkeys and end of lay hens.

Pre-slaughter transportation may affect poultry welfare and mortality rates. A retrospective analysis was conducted to examine the effect of environmental, management and individual factors on the percentage of dead birds during pre-slaughter transportation (dead-on-arrival, DOA). The variables accounted for in the analyses included: environmental temperature, travel duration, genetic line, gender, crate type and crate stocking density. Among the 41 452 loads of turkeys (34 696 388 birds) and 3241 of end of lay hens (21 788 124 birds) transported to three large abattoirs in northern Italy in a 3-year period, the median DOA was 0.14% in turkeys, and 0.38% in hens. In turkeys, travel duration longer than 30 min, temperature higher than 26°C and high in-crate densities were associated with increased DOA. In winter (⩽2°C), high stocking densities did not reduce the mortality risk from cold stress; on the contrary, for stocking densities either near to or just above the maximum density in EC Reg. 1/2005, the DOA risk was greater than for loads with densities of 10 kg/m2 less than the EC maximum. Male birds and specific genetic lines also showed a higher DOA. In hens, transportation lasting longer than 2 h and the brown-feathered breed were associated with higher DOA. Dead-on-arrival progressively increased with travel duration, remaining constant between 4 and 6 h and peaking at 8 h (median: 0.57%). The maximum DOA increase was detected during winter. These results show that several species-specific factors may lead to increased risk of mortality.

Retrospective space-time analysis methods to support West Nile virus surveillance activities.

The steep increase in human West Nile virus (WNV) infections in 2011-2012 in north-eastern Italy prompted a refinement of the surveillance plan. Data from the 2010-2012 surveillance activities on mosquitoes, equines, and humans were analysed through Bernoulli space-time scan statistics, to detect the presence of recurrent WNV infection hotspots. Linear models were fit to detect the possible relationships between WNV occurrence in humans and its activity in mosquitoes. Clusters were detected for all of the hosts, defining a limited area on which to focus surveillance and promptly identify WNV reactivation. Positive relationships were identified between WNV in humans and in mosquitoes; although it was not possible to define precise spatial and temporal scales at which entomological surveillance could predict the increasing risk of human infections. This stresses the necessity to improve entomological surveillance by increasing both the density of trapping sites and the frequency of captures.

Application of an integrated outbreak management plan for the control of leptospirosis in dairy cattle herds.

SUMMARY: Two outbreaks of Leptospira borgpetersenii serovar Hardjo infection in dairy cattle herds were managed through the application of enhanced biosecurity measures, whole-herd antibiotic treatment and vaccination. Micro-agglutination test antibody titres were determined in paired serum samples at 3 weeks (T1: n = 125, 97% seropositivity, median 800, range 100-12 800) and 24 weeks (T2: n = 110, 88% seropositivity, median 200, range 100-6400) after vaccination and studied in relation to cows' age, herd of origin and sampling time. From T1 to T2, vaccine-elicited antibody titres decreased by 84·7% (95% CI 76·2-90·1). Consistent with increasing immunocompetence in calves (aged <12 months) and immunosenescence in adult cows (aged >36 months) associated with ageing, antibody titres correlated positively with calves' age and negatively with adult cows' age. No cow had cultivable, (histo)pathologically detectable and/or PCR-detectable leptospires in urine or kidney samples after treatment and vaccination. Vaccination together with proper biosecurity measures and chemoprophylaxis are an affordable insurance to control bovine leptospirosis.

Raw milk-associated foodborne infections: a scoring system for the risk-based categorisation of raw dairy farms.

A pilot study was implemented in the Veneto Region of Italy, aimed at classifying dairy farms which produce milk to be commercialised unpasteurised on the basis of their risk of faecal contamination of milk, which is directly correlated to the probability of a foodborne pathogen, if present in the herd and eliminated through faecal excretion, to contaminate the raw product. Factors considered to be relevant in the definition of the risk of pathogens potentially present in animal faeces to be transmitted to milk, were hierarchically structured, weighted through the application of experts elicitation methods (Analytic Hierarchy Process, Delphi) and used to categorise farms through the application of a herd questionnaire. The probability of faecal contamination of milk, and thus the risk of pathogens transfer appears to be modulated more by farm management than by the structure of the farm or the health status of the herd. Such a method, combined with the microbiological evaluation of the prevalence of faecal excretion of such pathogens, can be used to implement a risk-based surveillance programme and to apply targeted control measures.

Old and new diagnostic approaches for Q fever diagnosis: correlation among serological (CFT, ELISA) and molecular analyses.

The objective of this study was to evaluate the performance of the complement fixation test (CFT) with respect to ELISA for the serological diagnosis of Q fever and to assess the role of serology as a tool for the identification of the shedder status. During 2009-2010, sera from 9635 bovines and 3872 small ruminants (3057 goats and 815 sheep) were collected and analyzed with CFT and ELISA. In addition, 2256 bovine, 139 caprine and 72 ovine samples (individual and bulk tank milk samples, fetuses, vaginal swabs and placentae) were analyzed with a real-time PCR kit. The relative sensitivity (Se) and specificity (Sp) of CFT with respect to ELISA were Se 26.56% and Sp 99.71% for cattle and Se 9.96% and Sp 99.94% for small ruminants. To evaluate the correlation between serum-positive status and shedder status, the ELISA, CFT and real-time PCR results were compared. Due to the sampling method and the data storage system, the analysis of individual associations between the serological and molecular tests was possible only for some of the bovine samples. From a statistical point of view, no agreement was observed between the serological and molecular results obtained for fetus and vaginal swab samples. Slightly better agreement was observed between the serological and molecular results obtained for the individual milk samples and between the serological (at least one positive in the examined group) and molecular results for the bulk tank milk (BTM) samples. The CFT results exhibited a better correlation with the shedder status than did the ELISA results.

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.

Emergency oral rabies vaccination of foxes in Italy in 2009-2010: identification of residual rabies foci at higher altitudes in the Alps.

Following a resurgence of fox rabies in northeastern Italy in 2008-2009, two emergency oral rabies vaccination (ORV) campaigns were performed in the Alpine mountain ranges in 2009 and 2010 using aerial distribution to prevent the disease from spreading further inland. Vaccine baits were distributed only below the freezing point altitude, 1000 m above sea level (a.s.l.) in December 2009-January 2010 and 1500 m a.s.l. in April-May 2010, to avoid repeated freeze-thaw cycles. Spatial analysis unexpectedly identified fox rabies hotspots above the threshold altitudes, probably representing local residual rabies foci which may have contributed to maintaining the infectious cycle in areas not vaccinated at higher altitudes. Based on the results obtained, in May 2010, the second ORV campaign was extended to include threshold altitudes of up to 2300 m a.s.l. to eliminate residual foci. The observations made may help in the formulation of ORV strategies in countries sharing similar topographical features.

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.

Design and results of an intensive monitoring programme for avian influenza in meat-type turkey flocks during four epidemics in northern Italy.

Surveillance programmes for low pathogenicity (LPAI) and high pathogenicity avian influenza (HPAI) infections in poultry are compulsory for EU Member States; yet, these programmes have rarely been evaluated. In Italy, following a 1999 HPAI epidemic, control measures, including vaccination and monitoring, were implemented in the densely populated poultry area (DPPA) where all epidemics in Italy have been concentrated. We evaluated the monitoring system for its capacity to detect outbreaks rapidly in meat-type turkey flocks. The evaluation was performed in vaccination areas and high-risk areas in the DPPA, in 2000-2005, during which four epidemics occurred. Serum samples and cloacal swabs were taken from vaccinated birds and unvaccinated (sentinel) birds. We compared the detection rate of active, passive and targeted surveillance, by vaccination status, using multinomial logistic regression. A total of 13 275 samplings for serological testing and 4889 samplings for virological testing were performed; 6315 production cycles of different bird species were tested. The outbreaks detection rate in meat-type turkeys was 61% for active surveillance (n = 222/363 outbreaks), 32% for passive surveillance and 7% for targeted surveillance. The maximum likelihood predicted values for the detection rates differed by vaccination status: in unvaccinated flocks, it was 50% for active surveillance, 40% for passive surveillance and 10% for targeted surveillance, compared to respectively 79%, 17% and 4% for vaccinated flocks. Active surveillance seems to be most effective in detecting infection, especially when a vaccination programme is in place. This is the first evaluation of the effectiveness of different types of surveillance in monitoring LPAI infections in vaccinated poultry using field data.

Impact of emergency oral rabies vaccination of foxes in northeastern Italy, 28 December 2009-20 January 2010: preliminary evaluation.

Fox rabies re-emerged in northeastern Italy in 2008, in an area bordering Slovenia. In 2009, the infection spread westward to Veneto region and in 2010 to the provinces of Trento and Bolzano. Aerial emergency oral fox vaccination was implemented in the winter 2009-10. Since this vaccination was performed at altitudes below the freezing level, a statistical analysis was conducted to evaluate its impact. Of the foxes sampled following the vaccination campaign, 77% showed a rabies antibody titre of >or=0.5 IU/ml.

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.

The effects of control measures on the economic burden associated with epidemics of avian influenza in Italy.

In 1999, Italy experienced a devastating epidemic of high-pathogenicity avian influenza (HPAI) caused by an H7N1 virus subtype. After this epidemic, a ministerial decree was passed to implement control measures for low-pathogenicity avian influenza (LPAI) due to H5 and H7 subtypes. We investigated whether these control measures have decreased the public expenditure associated with epidemics of LPAI and HPAI by comparing the direct and consequential losses of the 1999 epidemic to the losses associated with successive epidemics. The estimated total economic burden of the epidemics was about euro650 million (euro217 million in direct losses and euro433 million in consequential losses). The 1999 epidemic accounted for most of these losses (euro507 million: euro112 million in direct losses and euro395 million in consequential losses), whereas the total economic burden for the 5 successive LPAI was euro143 million (euro105 million in direct losses and euro38 million in consequential losses). These results demonstrate that the implementation of a coordinated set of disease-control measures, which included both emergency and prophylactic vaccination, was able to reduce the overall costs associated with avian influenza epidemics. The results also show that the application of adequate LPAI control measures may limit the risk of emergence of an HPAI virus in an area with a high poultry density, allowing the complete disruption of the poultry market and its huge associated costs to be avoided.

Evaluation of interventions and vaccination strategies for low pathogenicity avian influenza: spatial and space-time analyses and quantification of the spread of infection.

In recent years the control of low pathogenicity avian influenza (LPAI) viruses of the H5 and H7 subtypes has increasingly become a concern. We evaluated the measures (stamping out, controlled marketing, emergency and preventive vaccination, farm density reduction and restocking in homogenous areas) implemented to control the LPAI epidemics that occurred in Italy between 2000 and 2005, using a combination of spatial and space-time analyses and estimates of the basic reproduction ratio (R0). Clustering of infected farms decreased over the years, indicating the effectiveness of the control strategies implemented. Controlled marketing [relative risk (RR) 0.46, 95% confidence interval (CI) 0.27-0.80], emergency (RR 0.47, 95% CI 0.39-0.57) and preventive vaccination (RR 0.19, 95% CI 0.09-0.41) were the most effective measures, yet R0<1 was only for preventive vaccination. Our results are useful for identifying the most effective measures for reducing the risk of the spread of LPAI and optimizing the allocation of resources.

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.