Presence and Characterisation of Porcine Respirovirus 1 (PRV1) in Northern Italy.

Porcine Respirovirus 1 (PRV1) is an enveloped, single-stranded, negative-sense RNA virus belonging to the genus Respirovirus within the Paramyxoviridae family. Since its first detection in China in 2013, PRV1 has been identified in several American and European countries. Although its pathogenicity is uncertain, recent studies have suggested that it may play a role in the Porcine Respiratory Disease Complex (PRDC) because of its capacity to replicate in the upper and lower respiratory tracts. This study aimed to determine the spread of PRV1 in Northern Italy and the phylogeny of the isolates. Therefore, PRV1 was investigated using real-time RT-PCR in 902 samples collected from September 2022 to September 2023 from pigs with respiratory symptoms in North Italy. Fourteen (1.55%) samples tested as PRV1-positive. The full-length fusion (F) gene, which codifies for a major surface protein, was amplified and used for phylogenetic analysis to help carry out molecular epidemiological studies on this virus. In addition, swine influenza virus (SIV) and porcine reproductive and respiratory syndrome virus (PRRSV) infections were detected in most of the PRV1-positive samples. In conclusion, we report the detection of PRV1 in Italy and discuss its potential role as a co-factor in causing the Porcine Respiratory Disease Complex.

Tick-Borne Encephalitis, Lombardy, Italy.

Tick-borne encephalitis was limited to northeast portions of Italy. We report in Lombardy, a populous region in the northwest, a chamois displaying clinical signs of tickborne encephalitis virus that had multiple virus-positive ticks attached, as well as a symptomatic man. Further, we show serologic evidence of viral circulation in the area.

Understanding West Nile virus transmission: Mathematical modelling to quantify the most critical parameters to predict infection dynamics.

West Nile disease is a vector-borne disease caused by West Nile virus (WNV), involving mosquitoes as vectors and birds as maintenance hosts. Humans and other mammals can be infected via mosquito bites, developing symptoms ranging from mild fever to severe neurological infection. Due to the worldwide spread of WNV, human infection risk is high in several countries. Nevertheless, there are still several knowledge gaps regarding WNV dynamics. Several aspects of transmission taking place between birds and mosquitoes, such as the length of the infectious period in birds or mosquito biting rates, are still not fully understood, and precise quantitative estimates are still lacking for the European species involved. This lack of knowledge affects the precision of parameter values when modelling the infection, consequently resulting in a potential impairment of the reliability of model simulations and predictions and in a lack of the overall understanding of WNV spread. Further investigations are thus needed to better understand these aspects, but field studies, especially those involving several wild species, such as in the case of WNV, can be challenging. Thus, it becomes crucial to identify which transmission processes most influence the dynamics of WNV. In the present work, we propose a sensitivity analysis to investigate which of the selected epidemiological parameters of WNV have the largest impact on the spread of the infection. Based on a mathematical model simulating WNV spread into the Lombardy region (northern Italy), the basic reproduction number of the infection was estimated and used to quantify infection spread into mosquitoes and birds. Then, we quantified how variations in four epidemiological parameters representing the duration of the infectious period in birds, the mosquito biting rate on birds, and the competence and susceptibility to infection of different bird species might affect WNV transmission. Our study highlights that knowledge gaps in WNV epidemiology affect the precision in several parameters. Although all investigated parameters affected the spread of WNV and the modelling precision, the duration of the infectious period in birds and mosquito biting rate are the most impactful, pointing out the need of focusing future studies on a better estimate of these parameters at first. In addition, our study suggests that a WNV outbreak is very likely to occur in all areas with suitable temperatures, highlighting the wide area where WNV represents a serious risk for public health.

Epidemiological Features of the Highly Pathogenic Avian Influenza Virus H5N1 in a Densely Populated Area of Lombardy (Italy) during the Epidemic Season 2021-2022.

In the last two years, there have been three major epidemic seasons in the territory of the European Union and the HPAI epizootic in 2021-2022 is the most severe in recent history. In Italy, the disease was introduced to dense poultry areas with serious economic consequences for the entire sector. In Lombardy, the analysis of the risk factors was carried out, also taking into account the density of domestic birds. In the most affected areas, 66.7% of the outbreaks occurred in the areas with the highest poultry density and the likelihood of an outbreak occurring increased with an increase in the density of birds per km2. In cells 10 × 10 km with a density greater than 10,000 birds/km2, the probability of outbreak occurrence was over 66.7%. The provinces involved in the last epidemic were the same involved in previous epidemics and, given the risk factors present in the area, it is plausible that the risk remains high also for future epidemic seasons. Therefore, to avoid the repetition of similar events, certain control measures shall be strengthened and vaccination considered as a complementary tool for the control of HPAI virus in risk areas.

Assessment of the Costs Related to West Nile Virus Monitoring in Lombardy Region (Italy) between 2014 and 2018.

In Italy, the West Nile Virus surveillance plan considers a multidisciplinary approach to identify the presence of the virus in the environment (entomological, ornithological, and equine surveillance) and to determine the risk of infections through potentially infected donors (blood and organ donors). The costs associated with the surveillance program for the Lombardy Region between 2014 and 2018 were estimated. The costs of the program were compared with a scenario in which the program was not implemented, requiring individual blood donation nucleic acid amplification tests (NAT) to detect the presence of WNV in human samples throughout the seasonal period of vector presence. Considering the five-year period, the application of the environmental/veterinary surveillance program allowed a reduction in costs incurred in the Lombardy Region of 7.7 million EUR. An integrated surveillance system, including birds, mosquito vectors, and dead-end hosts such as horses and humans, can prevent viral transmission to the human population, as well as anticipate the detection of WNV using NAT in blood and organ donors. The surveillance program within a One Health context has given the possibility to both document the expansion of the endemic area of WNV in northern Italy and avoid most of the NAT-related costs.

Distribution of Phlebotomine Sand Flies (Diptera: Psychodidae) in the Lombardy Region, Northern Italy.

This study investigated the species composition and density of sand flies in the Lombardy region (Northern Italy). Sand flies were collected using CDC traps baited with CO2 (CO2-CDC traps) between June and August 2021. A total of 670 sand flies were collected. The specimens were identified as seven species belonging to two genera, Phlebotomus and Sergentomyia, namely, S. minuta, Ph. perniciosus, Ph. perfiliewii, Ph. neglectus, Ph. mascitti, Ph. papatasi, and Ph. ariasi. Phlebotomus perniciosus was the most abundant species (87.76%), followed by Ph. perfiliewii (7.31%), Ph. neglectus (3.13%), S. minuta (0.75%), Ph. mascitti (0.6%), Ph. papatasi (0.3%), and Ph. ariasi, for which only one specimen was identified. Among these identified species, five are considered vectors of Leishmania, which causes cutaneous and visceral leishmaniasis. As vector presence increases the risk of vector-borne leishmaniasis, these results suggest that Northern Italy could be a potential area of pathogen circulation over the next few years. These preliminary results suggest that the risk of borne leishmaniasis is high in this region of Northern Italy. Monitoring the distribution of sand fly species in areas suitable for their persistence is important for control programs aimed at reducing the risk of leishmaniasis infection.

Analysis of Bee Population Decline in Lombardy during the Period 2014-2016 and Identification of High-Risk Areas.

The first events of bee decline in Italy were reported during 1999. Since then, population decline has frequently been reported in Lombardy. In this study, the association between bee decline and the type of land surrounding the apiary was evaluated. A risk map was developed to identify areas with the highest risk of decline. Apiaries in Lombardy were selected from the national beekeeping database (BDA). The study period was from 2014 to 2016. Apiaries were deemed "declined" if they reported at least one event of decline or tested positive for plant protection products; apiaries were "not declined" if they did not report any events of bee decline during the study period. Out of 14,188 apiaries extracted from the BDA, 80 were considered declined. The probability of an apiary being declined increases by 10% in orchards and by 2% in arable land for each additional km2 of land occupied by these crops. The study showed an association between bee decline and the type of territory surrounding the apiaries, and the areas at the greatest risk of decline in Lombardy were identified. This information can be used by Veterinary Services as a predictive parameter for planning prevention and control activities.

Enhanced West Nile Virus Circulation in the Emilia-Romagna and Lombardy Regions (Northern Italy) in 2018 Detected by Entomological Surveillance.

With several human cases reported annually since 2008 and the unapparent risk of infection of blood donors, the West Nile virus (WNV) is emerging as an important health issue in Europe. Italy, as well as other European countries, experienced a recrudescence of the virus circulation in 2018, which led to an increased number of human cases. An integrated surveillance plan was activated in the Emilia-Romagna and Lombardy regions (Northern Italy) since 2008 in order to monitor the intensity and timing of WNV circulation. A fundamental part of this plan consists in entomological surveillance. In 2018, the surveillance plan made it possible to collect 385,293 mosquitoes in 163 stations in the two Regions. In total 269,147 Culex mosquitoes were grouped into 2,337 pools and tested for WNV, which was detected in 232 pools. Circulation started in the central part of the Emilia-Romagna region in the middle of June, about one month before the previous seasons. Circulation suddenly expanded to the rest of the region and reached the Lombardy region in the middle of July. WNV circulated more intensively in the eastern part of the surveyed area, as confirmed by the highest number of human cases. A relationship between the number of mosquitoes collected and the virus incidence emerged, but the data obtained highlighted that the probability of detecting the virus in a given site was less than expected with a higher number of collected mosquitoes. A significant relationship was observed between the temperature recorded one week before the sampling and the number of collected mosquitoes, as well as between the estimated number of WNV-positive mosquitoes and the temperature recorded two weeks before the sampling. The two weeks delay in the influence of temperature on the positive mosquitoes is in line with the time of the virus extrinsic incubation in the mosquito. This finding confirms that temperature is one of the principal drivers in WNV mosquito infection. The surveillance system demonstrated the ability to detect the virus circulation early, particularly in areas where circulation was more intense. This allowed evaluating the effect of mosquito abundance and weather factors on virus circulation.

Porcine Epidemic Diarrhoea Virus in Italy: Disease spread and the role of transportation.

Porcine Epidemic Diarrhoea Virus (PEDV) causes watery diarrhoea, dehydration, and a high mortality rate among suckling pigs. Recently, PEDV had a large negative economic impact on the swine industries in Asia and North America. In 2014, PEDV re-emerged in many European countries, but most countries only reported a few sporadic cases. Here, we report the epidemic wave that occurred in Italy from 2015 to 2017. During this time, PEDV was detected by real-time PCR in 438 farms located mainly in the high-density pig production area in Northern Italy. Most of the outbreaks were in farrow-to-finish, farrow-to-wean and finisher farms. Clinical signs were observed mainly in suckling and fattening animals, while mortality rates were higher in piglets, reaching 50%. A sequence analysis showed that a PEDV strain, similar to the OH851 S-INDEL strain isolated in the USA in January 2014, was responsible for the outbreaks in Italy in 2015 and 2016. However, from January 2017, a recombinant variant strain, containing a portion of the Swine Enteric Coronavirus in the S1 gene, spread and almost completely outcompeted the previous nonrecombinant strain. In total, 14.1% of the environmental swabs collected from trucks at slaughterhouses after animals were unloaded tested positive for PEDV before the trucks were cleaned and disinfected, and 46% remained positive after cleaning and disinfection processes were performed. Moreover, environmental swabs indicated that 17.3% of the empty trucks arriving at the farms to load animals were PEDV-positive. This study indicates that trucks can have an important role in the spread of PEDV in Italy.

Relevant Measures to Prevent the Spread of African Swine Fever in the European Union Domestic Pig Sector.

During the past decade, African swine fever (ASF) has spread from the Caucasus region to eastern European Union countries affecting domestic pig and wild boar populations. In order to avert ASF spread, mitigation measures targeting both populations have been established. However, despite these efforts, ASF has been reported in thirteen different countries (Georgia, Azerbaijan, Armenia, the Russian Federation, Ukraine, Belarus, Estonia, Latvia, Lithuania, Poland, Moldova, Czech Republic, and Romania). In the absence of an effective vaccine or treatment to ASF, introduction and spread of ASF onto domestic pig farms can only be prevented by strict compliance to control measures. This study systematically reviewed available measures to prevent the spread of ASF in the EU domestic pig sector distinguishing between commercial, non-commercial, and outdoor farms. The search was performed in PubMed and using a common browser. A total of 52 documents were selected for the final review process, which included scientific articles, reports, EU documents and official recommendations, among others. From this literature review, 37 measures were identified as preventive measures for the introduction and spread of ASF. Subsequently, these measures were assessed by ASF experts for their relevance in the mitigation of ASF spread on the three mentioned types of farms. All experts agreed that some of the important preventive measures for all three types of farms were: the identification of animals and farm records; strict enforcement of the ban on swill feeding; and containment of pigs, so as to not allow direct or indirect pig-pig and/or pig-wild boar contacts. Other important preventive measures for all farms were education of farmers, workers, and operators; no contact between farmers and farm staff and external pigs; appropriate removal of carcasses, slaughter residues, and food waste; proper disposal of manure and dead animals, and abstaining from hunting activities during the previous 48 h (allowing a 48 h interval between hunting and being in contact with domestic pigs). Finally, all experts identified that the important preventive measures for non-commercial and outdoor farms is to improve access of those farms to veterinarians and health services.

Surveillance of Mosquitoes and Selected Arthropod-Borne Viruses in the Context of Milan EXPO 2015.

From 1 May 2015 to 31 October 2015 over 20 million visitors from all over the world visited the Universal Exhibition (EXPO) hosted by Milan (Lombardy region, Italy), raising concerns about the possible introduction of mosquito-borne diseases from endemic countries. The entomological surveillance protocol performed in Lombardy over the last three years was implemented in the EXPO area and in the two major regional airports using both Center for Disease Control CO2 and Biogents Sentinel traps. This surveillance aimed to estimate the presence and densities of putative vectors, and also to support investigations, including the vector species involved and area of diffusion, on the local spread of Chikungunya, Dengue and West Nile viruses (WNV) by competent vectors. From 3544 mosquitoes belonging to five different species, 28 pools of Culex spp. and 45 pools of Aedes spp. were screened for the presence of WNV, and for both Chikungunya and flaviviruses, respectively. The entomological surveillance highlighted a low density of potential vectors in the surveyed areas and did not reveal the presence of Chikungunya or Dengue viruses in the local competent vectors inside the EXPO area or in the two airports. In addition, the surveillance reported a low density of Culex spp. mosquitoes, which all tested negative for WNV.

Long-Term Surveillance of Aujeszky's Disease in the Alpine Wild Boar (Sus scrofa).

Although wild boar can act as a persistent Aujeszky's disease (AD) reservoir, limited data are available on long-term epidemiology in free-ranging wild boar living in areas where industrial swine herds are limited. Hence, this study provides crucial information, which fills this knowledge gap, on the natural dynamics of AD infection. From 3260 sera sampled during eight hunting seasons, 162 (4.97%) were tested positive. Factors, including the animal's age class, and the sampling year, had significant effects on the probability of the wild boar being seropositive, while wild boar mean abundance per area, yearly abundance and the total number of pig farms, as well as interactions among age, year and sex, were not significant. In particular, a positive trend of seroprevalence was observed over the years, with values ranging from 2.1 to 10.8%. This long-term surveillance showed an increase in seroprevalence with a higher probability of being seropositive in older individuals and the independence of wild boar seropositivity from the likelihood of contact with pigs in the area.

The use of convalescent sera in immune-electron microscopy to detect non-suspected/new viral agents.

Negative staining electron microscopy methods can be employed for the diagnosis of viral particles in animal samples. In fact, negative staining electron microscopy methods are used to identify viruses, especially in minor species and wild animals, when no other methods are available and in cases of rare, emerging or re-emerging infections. In particular, immune-electron-microscopy with convalescent sera is employed to detect etiological agents when there are undiagnosed clinical outbreaks, when alternative diagnostic methods fail due to the lack of immunological reagents and primers, and when there is no indicative clinical suspect. An overview of immune-electron-microscopy with convalescent sera's use in the diagnosis of new and unsuspected viruses in animals of domestic and wild species is provided through the descriptions of the following four diagnostic veterinary cases: (I) enteric viruses of pigs: Porcine Rotavirus, Porcine Epidemic Diarrhea Virus, Porcine Circovirus and Porcine Torovirus; (II) Rotavirus and astrovirus in young turkeys with enteritis; (III) Parvovirus-like particles in pheasants; and (IV) Lagoviruses: Rabbit Hemorrhagic Disease Virus and European Brown Hare Syndrome Virus.

Evaluation of three rapid diagnostic tests used in bovine spongiform encephalopathy monitoring in Italy.

In 2001, a compulsory active surveillance system was started in the European Union to assess the prevalence of bovine spongiform encephalopathy (BSE) in the cattle population. The aim of the current study was to report on the field performances of 3 rapid tests: a Western blot (WB), a chemiluminescence enzyme-linked immunosorbent assay (ELISA), and an immunochromatographic assay, routinely used at 3 laboratories of the Istituto Zooprofilattico Sperimentale of Lombardia and Emilia Romagna, over 8 years of BSE monitoring activity. A total of 2,802,866 samples from slaughtered animals and 202,453 samples from fallen stock were tested by 1 of 3 tests. Positive results of the rapid tests were confirmed by histopathological examination, immunohistochemistry, and confirmatory WB. The field performances (i.e., initial reactive and false-positive rates) and practical aspects regarding resources and applicability of the tests to high-throughput routine testing laboratories were evaluated. The 3 tests proved to be reliable tools when applied to slaughtered samples, showing no or very low false-positive rates (<1 per 100,000 negative samples tested) and low retesting frequencies (0.02-0.26%). When samples from fallen stock were analyzed, performances of the immunochromatographic assay, and especially the chemiluminescence ELISA, were negatively affected, resulting in higher false-positive and retesting rates. On the other hand, both tests are less expensive, much easier to use, provide more rapid results, and adapt well to application in routine laboratories as compared with WB. In the authors' experience, the immunochromatographic assay was a good compromise between performance and convenience.