Susceptibility of Mediterranean Buffalo (Bubalus bubalis) following Experimental Infection with Lumpy Skin Disease Virus.

Lumpy skin disease (LSD) is a viral disease of cattle and water buffalo characterized by cutaneous nodules, biphasic fever, and lymphadenitis. LSD is endemic in Africa and the Middle East but has spread to different Asian countries in recent years. The disease is well characterized in cattle while little is known about the disease in buffaloes in which no experimental studies have been conducted. Six buffaloes and two cattle were inoculated with an Albanian LSD virus (LSDV) field strain and clinically monitored for 42 days. Only two buffaloes showed fever, skin nodules, and lymphadenitis. All samples collected (blood, swabs, biopsies, and organs) were tested in real-time PCR and were negative. Between day 39 and day 42 after inoculation, anti-LSDV antibodies were detected in three buffaloes by ELISA, but all sera were negative by virus neutralization test (VNT). Cattle showed severe clinical signs, viremia, virus shedding proven by positive real-time PCR results, and seroconversion confirmed by both ELISA and VNT. Clinical findings suggest that susceptibility in buffaloes is limited compared to in cattle once experimentally infected with LSDV. Virological results support the hypothesis of buffalo resistance to LSD and its role as an accidental non-adapted host. This study highlights that the sensitivity of ELISA and VNT may differ between animal species and further studies are needed to investigate the epidemiological role of water buffalo.

Detection of Potential Zoonotic Agents Isolated in Italian Shelters and the Assessment of Animal Welfare Correlation with Antimicrobial Resistance in Escherichia coli Strains.

Welfare conditions in shelters, where dogs might be housed for a long period of time, may have a possible correlation with the occurrence of bacterial pathogens and their antimicrobial resistance (AMR). In this study, we assessed the occurrence of AMR in 54 strains of Escherichia coli isolated from dogs housed in 15 Italian shelters and we correlated the resistance patterns to animal welfare. We also aimed to evaluate the presence of specific pathogens with zoonotic potential in sheltered dogs. Thus, nasopharyngeal, rectal, and oral swabs were collected from a group of 20 dogs in each shelter and totaled 758 swabs. We identified 9 Staphylococcus pseudointermedius, 1 Pasteurella multocida, 9 Staphylococcus aureus, 12 Campylobacter spp., 54 Escherichia coli, 2 Salmonella enterica, and 246 Capnocytophaga spp. The antimicrobial susceptibility was assessed for the E. coli isolates using a panel of 14 antibiotics. The highest level of relative AMR was recorded for ampicillin and sulfamethoxazole. The association found between AMR and the levels of animal welfare scores in shelters was evident although not statistically significant. These results support the hypothesis that the good management of shelters can increase the level of animal welfare, thus reducing the use of antibiotics and, as a consequence, the AMR occurrence found in dogs that share their domestic environment with humans.

Species of mosquitoes present in Abruzzo and Molise and their possible role as vector of Usutu and West Nile viruses.

In 2019, entomological survey on mosquitoes was carried out in Abruzzo and Molise regions in central Italy to obtain data on local mosquito fauna. Collection sites were selected based on a previous ecoregion classification of the territory.  From 2019 to 2021 virological surveillance for West Nile virus (WNV) and Usutu virus (USUV) on mosquitoes was carried out in the same regions, selecting ecoregions where virus circulation and vector presence were more likely,  all mosquitoes were collected and identified, and the female mosquitoes were sorted in 3046 pools and tested for the presence of WNV and USUV by Real-time PCR. All pools tested negative for WND, while USUV was detected in 7 pools of Aedes caspius collected in Molise region, 17 pools of Culex pipiens s.l. (2 collected in Molise, 15 in Abruzzo), and 1 pool of Culiseta longiareolata collected in Molise. These results suggests the presence of an USUV enzootic cycle, maintained by Culex pipiens s.l. and Aedes caspius in both Italian regions, as well as providing a useful picture in terms of species presence and abundance for both regions. Ecoregions proved to be a very valuable tool in determining high risk areas for vector borne diseases.

Rapid increase in neuroinvasive West Nile virus infections in humans, Italy, July 2022.

As in 2018, when a large West Nile virus (WNV) epidemic occurred, the 2022 vector season in Italy was marked by an early onset of WNV circulation in mosquitoes and birds. Human infections were limited until early July, when we observed a rapid increase in the number of cases. We describe the epidemiology of human infections and animal and vector surveillance for WNV and compare the more consolidated data of June and July 2022 with the same period in 2018.

West Nile Virus Lineage 2 Overwintering in Italy.

In January 2022, West Nile virus (WNV) lineage 2 (L2) was detected in an adult female goshawk rescued near Perugia in the region of Umbria (Italy). The animal showed neurological symptoms and died 15 days after its recovery in a wildlife rescue center. This was the second case of WNV infection recorded in birds in the Umbria region during the cold season, when mosquitoes, the main WNV vectors, are usually not active. According to the National Surveillance Plan, the Umbria region is included amongst the WNV low-risk areas. The necropsy evidenced generalized pallor of the mucous membranes, mild splenomegaly, and cerebral edema. WNV L2 was detected in the brain, heart, kidney, and spleen homogenate using specific RT-PCR. Subsequently, the extracted viral RNA was sequenced. A Bayesian phylogenetic analysis performed through a maximum-likelihood tree showed that the genome sequence clustered with the Italian strains within the European WNV strains among the central-southern European WNV L2 clade. These results, on the one hand, confirmed that the WNV L2 strains circulating in Italy are genetically stable and, on the other hand, evidenced a continuous WNV circulation in Italy throughout the year. In this report case, a bird-to-bird WNV transmission was suggested to support the virus overwintering. The potential transmission through the oral route in a predatory bird may explain the relatively rapid spread of WNV, as well as other flaviviruses characterized by similar transmission patterns. However, rodent-to-bird transmission or mosquito-to-bird transmission cannot be excluded, and further research is needed to better understand WNV transmission routes during the winter season in Italy.

West Nile and Usutu Virus Introduction via Migratory Birds: A Retrospective Analysis in Italy.

The actual contribution of migratory birds in spreading West Nile (WNV) and Usutu virus (USUV) across Europe and from Africa to old countries is still controversial. In this study, we reported the results of molecular and serological surveys on migrating birds sampled during peaks of spring and autumn migration at 11 Italian sites located along important flyways, from 2012 to 2014. A total of 1335 specimens made of individual or pooled sera, and organs from 275 dead birds were tested for WNV and USUV RNA by real time PCR (RT-PCR). Furthermore, sera were tested by serum neutralization assay for detecting WNV and USUV neutralizing antibodies. Molecular tests detected WNV lineage 2 RNA in a pool made of three Song Thrush (Turdus philomelos) sera sampled in autumn, and lineage 1 in kidneys of six trans-Saharan birds sampled in spring. Neutralizing antibodies against WNV and USUV were found in 5.80% (n = 72; 17 bird species) and 0.32% (n = 4; 4 bird species) of the tested sera, respectively. Our results do not exclude the role of migratory birds as potential spreaders of WNV and USUV from Africa and Central Europe to Mediterranean areas and highlight the importance of a more extensive active surveillance of zoonotic viruses.

Epidemiological and Evolutionary Analysis of West Nile Virus Lineage 2 in Italy.

West Nile virus (WNV) is a mosquito-borne virus potentially causing serious illness in humans and other animals. Since 2004, several studies have highlighted the progressive spread of WNV Lineage 2 (L2) in Europe, with Italy being one of the countries with the highest number of cases of West Nile disease reported. In this paper, we give an overview of the epidemiological and genetic features characterising the spread and evolution of WNV L2 in Italy, leveraging data obtained from national surveillance activities between 2011 and 2021, including 46 newly assembled genomes that were analysed under both phylogeographic and phylodynamic frameworks. In addition, to better understand the seasonal patterns of the virus, we used a machine learning model predicting areas at high-risk of WNV spread. Our results show a progressive increase in WNV L2 in Italy, clarifying the dynamics of interregional circulation, with no significant introductions from other countries in recent years. Moreover, the predicting model identified the presence of suitable conditions for the 2022 earlier and wider spread of WNV in Italy, underlining the importance of using quantitative models for early warning detection of WNV outbreaks. Taken together, these findings can be used as a reference to develop new strategies to mitigate the impact of the pathogen on human and other animal health in endemic areas and new regions.

West Nile Virus Lineage 1 in Italy: Newly Introduced or a Re-Occurrence of a Previously Circulating Strain?

In Italy, West Nile virus (WNV) appeared for the first time in the Tuscany region in 1998. After 10 years of absence, it re-appeared in the areas surrounding the Po River delta, affecting eight provinces in three regions. Thereafter, WNV epidemics caused by genetically divergent isolates have been documented every year in the country. Since 2018, only WNV Lineage 2 has been reported in the Italian territory. In October 2020, WNV Lineage 1 (WNV-L1) re-emerged in Italy, in the Campania region. This is the first occurrence of WNV-L1 detection in the Italian territory since 2017. WNV was detected in the internal organs of a goshawk (Accipiter gentilis) and a kestrel (Falco tinnunculus). The RNA extracted in the goshawk tissue samples was sequenced, and a Bayesian phylogenetic analysis was performed by a maximum-likelihood tree. Genome analysis, conducted on the goshawk WNV complete genome sequence, indicates that the strain belongs to the WNV-L1 Western-Mediterranean (WMed) cluster. Moreover, a close phylogenetic similarity is observed between the goshawk strain, the 2008-2011 group of Italian sequences, and European strains belonging to the Wmed cluster. Our results evidence the possibility of both a new re-introduction or unnoticed silent circulation in Italy, and the strong importance of keeping the WNV surveillance system in the Italian territory active.

Rift Valley fever virus: a serological survey in Libyan ruminants.

A serological survey was carried out in Libya to investigate the circulation of Rift Valley fever virus (RVFV) among domestic ruminants. A total of 857 serum samples were collected from year 2015 to 2016 in eleven provinces of Libya belonging to five branches of the country. Samples were tested for RVFV antibodies using a competitive Enzyme-Linked Immunosorbent Assay (c-ELISA). Antibodies specific for RVFV were not detected in any of the 857 samples. However, a statistical analysis was carried out to assess the maximum expected number of infected animals and the maximum expected prevalence of RVFV among Libyan ruminants' populations according to the sampled population. The overall maximum expected prevalence was estimated to be 1.8% for cattle and 0.4% for small ruminants. Results seem to exclude the circulation of RVFV, however, a surveillance plan should be implemented in areas at risk of RVFV introduction.

Preliminary results on innocuity and immunogenicity of an inactivated vaccine against Peste des petits ruminants.

Peste des petits ruminants (PPR) virus belongs to the family Paramyxoviridae and represents a major threat to small livestock industry. In recent years, outbreaks of PPR have occurred in Turkey and North Africa. In endemic areas, disease prevention is accomplished using live­attenuated vaccines. However, the use of live vaccines in non­endemic regions, such as Europe, is not approved by Veterinary Authorities. In these regions inactivated vaccines are then the only viable alternative. In this study an inactivated vaccine (iPPRVac) was formulated with either a water­in­oil emulsion (ISA 71 VG) or with delta inulin adjuvant, alone (AFSA1) or combined with a TLR9 agonist oligonucleotide (AFSA2). These formulations were then tested for immunogenicity on rats. The iPPRV formulation with AFSA2 adjuvant induced 100% seroconversion in rats after 2 injections and was subsequently evaluated in goats. Five goats were immunised twice subcutaneously, 36 days apart with iPPRVac + AFSA2. The immunised goats all seroconverted to PPR by day 9 and remained seropositive until the end of the experimental period (133 days). These data indicate that the rat model is useful in predicting vaccine responses in goats and that inactivated vaccine, when formulated with a delta inulin adjuvant, represents a promising alternative to live attenuated vaccines for PPR vaccination campaigns in non­endemic areas.

Genetic characterization of Italian field strains of Schmallenberg virus based on N and NSs genes.

Following its first identification in Germany in 2011, the Schmallenberg virus (SBV) has rapidly spread to many other European countries. Despite the wide dissemination, the molecular characterization of the circulating strains is limited to German, Belgian, Dutch, and Swiss viruses. To fill this gap, partial genetic characterization of 15 Italian field strains was performed, based on S segment genes. Samples were collected in 2012 in two different regions where outbreaks occurred during distinct epidemic seasons. The comparative sequence analysis demonstrated a high molecular stability of the circulating viruses; nevertheless, we identified several variants of the N and NSs proteins not described in other SBV isolates circulating in Europe.