Whole-Genome Sequencing of Two Canine Herpesvirus 1 (CaHV-1) Isolates and Clinicopathological Outcomes of Infection in French Bulldog Puppies.

Canine herpesvirus 1 (CaHV-1) infects dogs, causing neonatal death and ocular, neurological, respiratory, and reproductive problems in adults. Although CaHV-1 is widespread in canine populations, only four studies have focused on the CaHV-1 whole genome. In such context, two CaHV-1 strains from both the kidney and spleen of 20-day-old deceased French Bulldog puppies were recently isolated in Sardinia, Italy. The extracted viral DNA underwent whole-genome sequencing using the Illumina MiSeq platform. The Italian CaHV-1 genomes were nearly identical (>99%), shared the same tree branch, and clustered near the ELAL-1 (MW353125) and BTU-1 (KX828242) strains, enlarging the completely separated clade discussed by Lewin et al., in 2020. This study aims to provide new insights on the evolution of the CaHV-1, based on high-resolution whole-genome phylogenetic analysis, and on its clinicopathological characterization during a fatal outbreak in puppies.

Passive Surveillance as a Key Tool for African Swine Fever Eradication in Wild Boar: A Protocol to Find Carcasses Tested and Validated in the Mediterranean Island of Sardinia.

African swine fever (ASF) is one of the most important and serious contagious hemorrhagic viral diseases affecting domestic pigs and wild boar and is associated with high mortality rates while also having an extensive sanitary and socioeconomic impact on the international trade of animal and swine products. The early detection of the disease is often hampered by inadequate surveillance. Among the surveillance strategies used, passive surveillance of wild boars is considered the most effective method for controlling the African swine fever virus (ASFV). Otherwise, the design of a sufficiently sensitive ASF surveillance system requires a solid understanding of the epidemiology related to the local eco-social context, especially in the absence of virus detection. Even if the number of carcasses needed to demonstrate ASF eradication has been established, the scientific context lacks detail compared to protocols applied in the active search for wild boar carcasses. The aim of this study was to describe the protocol applied in the active search for carcasses, providing detailed information on the number of people and dogs as well as the amount of time and space used within the Mediterranean area. Using a specific tool developed to record, trace, and share field data (the GAIA observer app), a total of 33 active searches for wild boar carcasses were organized during 2021-2023. Most of these searches were planned to find carcasses that had previously been reported by hunters. A total of 24 carcasses were found, with only 2 carcasses not previously reported. The final protocol applied involved four people, with an average speed of 1.5 km/h. When a carcass had been previously reported, about 2 km of distance had to be covered in about 1.5 h to find the carcass, and even less time was spent when a dog (untrained) was present. In conclusion, it can be stated that, when searching for carcasses, solid collaboration with local hunters or other forest visitors is necessary to ensure carcasses are reported. The process involves small groups of experts actively searching for carcasses, possibly with the use of hunting dogs without special training. The data presented could be of valid support for those countries characterized by Mediterranean vegetation that are faced with the need to plan active carcass searches.

The illegal rearing and slaughtering of pigs in the wild on the Mediterranean island of Sardinia favor an increase in the biomass of Trichinella britovi in wild boars (Sus scrofa) but do not affect the serological prevalence of infection.

BACKGROUND: Worms of the nematode genus Trichinella are zoonotic pathogens with a worldwide distribution. The first report of Trichinella on the Mediterranean island of Sardinia was for Trichinella britovi, one of the four species of this genus circulating in Europe, which was identified in 2005 following an outbreak of trichinellosis in humans due to the consumption of pork from pigs reared in the wild. Since then, T. britovi larvae have been repeatedly isolated from free-ranging pigs, foxes (Vulpes vulpes) and wild boars (Sus scrofa) sampled in the central-eastern region of the island (Orgosolo municipality), but have never been isolated from samples from other areas of the island. The aim of this study was to investigate the parasitological and serological prevalence of T. britovi infection in wild boars in Sardinia over space [eight wild boar hunting management units (HMUs)] and time (seven wild boar hunting seasons). METHODS: Muscle and serum samples of boars killed in the 2014-2015 to 2020-2121 hunting seasons were collected from eight HMUs of central and south-western Sardinia. Trichinella sp. larvae were detected by artificial digestion of predilection muscles. A total of 4111 serum samples of wild boar were collected from the investigated HMUs and tested by enzyme-linked immunosorbent assay as a screening test and by western blot as a confirmatory test using excretory/secretory antigens. RESULTS: Trichinella britovi muscle larvae were detected in six (0.03%) of the 17,786 wild boars tested. All of the Trichinella sp.-positive wild boars had been hunted in Orgosolo municipality (central-eastern area of the island), except for one, hunted in a neighboring municipality. An overall serological prevalence of 3.8% (95% confidence interval, 3.3-4.5) was detected by western blot. No statistical differences were detected between the HMUs where T. britovi larvae were detected in wild boars, foxes, and free-ranging pigs and those where wild boars, foxes and free-ranging pigs tested negative. CONCLUSIONS: The serological prevalence did not vary between the wild boar populations in which the larval load was detectable by artificial digestion (Orgosolo municipality) and those in which the larval load was below the detection limit. Furthermore, the serological prevalence of anti-Trichinella immunoglobulin G in the wild boar populations remained constant during the study period, which covered seven wild boar hunting seasons. As the transmission events (i.e., the serological prevalence) are stable, the high biomass of the parasite in Orgosolo municipality can only have arisen as a consequence of factors independent of its natural cycle, i.e., the presence of a high number of free-ranging pigs, and the concomitant presence of African swine fever, due to illegal pig slaughtering in the field. This epidemiological situation suggests that the natural cycle of T. britovi may be influenced by inappropriate pig husbandry and slaughtering practices.

Brucella ceti Infection in Striped Dolphins from Italian Seas: Associated Lesions and Epidemiological Data.

Brucella ceti infections have been increasingly reported in cetaceans. In this study, we analyzed all cases of B. ceti infection detected in striped dolphins stranded along the Italian coastline between 2012 and 2021 (N = 24). We focused on the pathogenic role of B. ceti through detailed pathological studies, and ad hoc microbiological, biomolecular, and serological investigations, coupled with a comparative genomic analysis of the strains. Neurobrucellosis was observed in 20 animals. The primary histopathologic features included non-suppurative meningoencephalitis (N = 9), meningitis (N = 6), and meningoencephalomyelitis (N = 5), which was also associated with typical lesions in other tissues (N = 8). Co-infections were detected in more than half of the cases, mostly involving Cetacean Morbillivirus (CeMV). The 24 B. ceti isolates were assigned primarily to sequence type 26 (ST26) (N = 21) and, in a few cases, ST49 (N = 3). The multilocus sequence typing (cgMLST) based on whole genome sequencing (WGS) data showed that strains from Italy clustered into four genetically distinct clades. Plotting these clades onto a geographic map suggests a link between their phylogeny and the topographical distribution. These results support the role of B. ceti as a primary neurotropic pathogen for striped dolphins and highlight the utility of WGS data in understanding the evolution of this emerging pathogen.

Potential SARS-CoV-2 Susceptibility of Cetaceans Stranded along the Italian Coastline.

Due to marine mammals' demonstrated susceptibility to SARS-CoV-2, based upon the homology level of their angiotensin-converting enzyme 2 (ACE2) viral receptor with the human one, alongside the global SARS-CoV-2 occurrence and fecal contamination of the river and marine ecosystems, SARS-CoV-2 infection may be plausibly expected to occur also in cetaceans, with special emphasis on inshore species like bottlenose dolphins (Tursiops truncatus). Moreover, based on immune and inflammatory responses to SARS-CoV-2 infection in humans, macrophages could also play an important role in antiviral defense mechanisms. In order to provide a more in-depth insight into SARS-CoV-2 susceptibility in marine mammals, we evaluated the presence of SARS-CoV-2 and the expression of ACE2 and the pan-macrophage marker CD68. Aliquots of tissue samples, belonging to cetaceans stranded along the Italian coastline during 2020-2021, were collected for SARS-CoV-2 analysis by real-time PCR (RT-PCRT) (N = 43) and Immunohistochemistry (IHC) (N = 59); thirty-two aliquots of pulmonary tissue sample (N = 17 Tursiops truncatus, N = 15 Stenella coeruleoalba) available at the Mediterranean Marine Mammal Tissue Bank (MMMTB) of the University of Padua (Legnaro, Padua, Italy) were analyzed to investigate ACE2 expression by IHC. In addition, ACE2 and CD68 were also investigated by Double-Labeling Immunofluorescence (IF) Confocal Laser Microscopy. No SARS-CoV-2 positivity was found in samples analyzed for the survey while ACE2 protein was detected in the lower respiratory tract albeit heterogeneously for age, gender/sex, and species, suggesting that ACE2 expression can vary between different lung regions and among individuals. Finally, double IF analysis showed elevated colocalization of ACE2 and CD68 in macrophages only when an evident inflammatory reaction was present, such as in human SARS-CoV-2 infection.

Changes in Estimating the Wild Boar Carcasses Sampling Effort: Applying the EFSA ASF Exit Strategy by Means of the WBC-Counter Tool.

African swine fever (ASF) is a devastating disease, resulting in the high mortality of domestic and wild pigs, spreading quickly around the world. Ensuring the prevention and early detection of the disease is even more crucial given the absence of licensed vaccines. As suggested by the European Commission, those countries which intend to provide evidence of freedom need to speed up passive surveillance of their wild boar populations. If this kind of surveillance is well-regulated in domestic pig farms, the country-specific activities to be put in place for wild populations need to be set based on wild boar density, hunting bags, the environment, and financial resources. Following the indications of the official EFSA opinion 2021, a practical interpretation of the strategy was implemented based on the failure probabilities of wrongly declaring the freedom of an area even if the disease is still present but undetected. This work aimed at providing a valid, applicative example of an exit strategy based on two different approaches: the first uses the wild boar density to estimate the number of carcasses need to complete the exit strategy, while the second estimates it from the number of wild boar hunted and tested. A practical free access tool, named WBC-Counter, was developed to automatically calculate the number of needed carcasses. The practical example was developed using the ASF data from Sardinia (Italian island). Sardinia is ASF endemic from 43 years, but the last ASFV detection dates back to 2019. The island is under consideration for ASF eradication declaration. The subsequent results provide a practical example for other countries in approaching the EFSA exit strategy in the best choices for its on-field application.

Retrospective spatial analysis for African swine fever in endemic areas to assess interactions between susceptible host populations.

African Swine Fever (ASF) is one of the most complex and significant diseases from a sanitary-economic perspective currently affecting the world's swine-farming industry. ASF has been endemic in Sardinia (Italy) since 1978, and several control and eradication programmes have met with limited success. In this traditional ASF endemic area, there are three susceptible host populations for this virus sharing the same habitat: wild boar, farmed domestic pigs and non-registered free-ranging pigs (known as "brado" animals). The main goal of this study was to determine and predict fine-scale spatial interactions of this multi-host system in relation to the epidemiology of ASF in the main endemic area of Sardinia, Montes-Orgosolo. To this end, simultaneous monitoring of GPS-GSM collared wild boar and free-ranging pigs sightings were performed to predict interaction indexes through latent selection difference functions with environmental, human and farming factors. Regarding epidemiological assessment, the spatial inter-specific interaction indexes obtained here were used to correlate ASF notifications in wild boar and domestic pig farms. Daily movement patterns, home ranges (between 120.7 and 2,622.8 ha) and resource selection of wild boar were obtained for the first time on the island. Overall, our prediction model showed the highest spatial interactions between wild boar and free-ranging pigs in areas close to pig farms. A spatially explicit model was obtained to map inter-specific interaction over the complete ASF-endemic area of the island. Our approach to monitoring interaction indexes may help explain the occurrence of ASF notifications in wild boar and domestic pigs on a fine-spatial scale. These results support the recent and effective eradication measures taken in Sardinia. In addition, this methodology could be extrapolated to apply in the current epidemiological scenarios of ASF in Eurasia, where exist multi-host systems involving free-ranging pigs and wild boar.

Free-Ranging Pig and Wild Boar Interactions in an Endemic Area of African Swine Fever.

African swine fever virus (ASFV) is spreading throughout Eurasia and there is no vaccine nor treatment available, so the control is based on the implementation of strict sanitary measures. These measures include depopulation of infected and in-contact animals and export restrictions, which can lead to important economic losses, making currently African swine fever (ASF) the greatest threat to the global swine industry. ASF has been endemic on the island of Sardinia since 1978, the longest persistence of anywhere in Eurasia. In Sardinia, eradication programs have failed, in large part due to the lack of farm professionalism, the high density of wild boar and the presence of non-registered domestic pigs (free-ranging pigs). In order to clarify how the virus is transmitted from domestic to wild swine, we examined the interaction between free-ranging pigs and wild boar in an ASF-endemic area of Sardinia. To this end, a field study was carried out on direct and indirect interactions, using monitoring by camera trapping in different areas and risk points. Critical time windows (CTWs) for the virus to survive in the environment (long window) and remain infectious (short window) were estimated, and based on these, the number of indirect interactions were determined. Free-ranging pigs indirectly interacted often with wild boar (long window = 6.47 interactions/day, short window = 1.31 interactions/day) and these interactions (long window) were mainly at water sources. They also directly interacted 0.37 times per day, especially between 14:00 and 21:00 h, which is much higher than for other interspecific interactions observed in Mediterranean scenarios. The highly frequent interactions at this interspecific interface may help explain the more than four-decade-long endemicity of ASF on the island. Supporting that free-ranging pigs can act as a bridge to transmit ASFV between wild boar and registered domestic pigs. This study contributes broadly to improving the knowledge on the estimation of frequencies of direct and indirect interactions between wild and free-ranging domestic swine. As well as supporting the importance of the analysis of interspecific interactions in shared infectious diseases, especially for guiding disease management. Finally, this work illustrates the power of the camera-trapping method for analyzing interspecific interfaces.

Novel dolphin morbillivirus (DMV) outbreak among Mediterranean striped dolphins Stenella coeruleoalba in Italian waters.

An unusual mortality event (UME) of striped dolphins Stenella coeruleoalba occurred in the period July to December 2016 along the Italian Ionian coastline. We conducted a complete postmortem examination on 28 specimens and detected dolphin morbillivirus (DMV), by means of biomolecular analyses, in the target tissues of 17 animals. Unlike previous outbreaks occurring in the Mediterranean Sea in 2011 and 2013, we observed typical pathological changes suggestive of morbilliviral infection in an acute/subacute phase and immunohistochemical reactivity. The same findings were observed in 13 other specimens beached along the Italian coastline during 2016 with no temporal and geographical relationship with the ongoing epidemic outbreak. Molecular characterization and phylogenetic analysis showed that DMV sequences detected in Italy in 2016 clustered with those identified in Portugal and Galicia (Spain), representing a novel DMV strain of Atlantic origin which entered the Mediterranean Sea and affected a naïve striped dolphin population. DMV sequences detected in the previous Mediterranean outbreaks exhibited a marked genetic relatedness and diverged from those detected in cetaceans stranded along the Galician and Portuguese coasts since 2007.

Mediterranean Fin Whales (Balaenoptera physalus) Threatened by Dolphin MorbilliVirus.

During 2011-2013, dolphin morbillivirus was molecularly identified in 4 stranded fin whales from the Mediterranean Sea. Nucleoprotein, phosphoprotein, and hemagglutinin gene sequences of the identified strain were highly homologous with those of a morbillivirus that caused a 2006-2007 epidemic in the Mediterranean. Dolphin morbillivirus represents a serious threat for fin whales.

Cetacean strandings in Italy: an unusual mortality event along the Tyrrhenian Sea coast in 2013.

An unusual mortality event involving cetaceans, mainly striped dolphins Stenella coeruleoalba (Meyen, 1833), occurred along the Tyrrhenian Sea coast of Italy during the first 3 mo of 2013. Based on post-mortem analyses carried out according to body condition on 66 dolphins (54% of stranded animals), several hypotheses to explain the causes of this mortality event were proposed. Although no definitive conclusions can be drawn, dolphin morbillivirus was deemed the most likely cause, although other infectious agents (including Photobacterium damselae damselae and herpesvirus) or environmental factors may also have contributed to this recent mortality event.

Avian influenza and animal health risk: conservation of endemic threatened wild birds in Sardinia Island.

Sardinia is a Mediterranean island with a long geological history, leading to a separation process from continental Europe during the Miocene. As a consequence, in this insular habitat some wild bird species developed endemic forms, some of which are currently threatened. The aim of this study is to evaluate the possible animal health risk associated with a potential avian influenza virus (AIV) circulation in Sardinian wild bird populations. Overall, 147 cloacal swabs were sampled in the Sardinia region from June 2009 to September 2011. Samples were obtained from 12 taxonomic orders, including 16 families and 40 species of birds. Based on the endangered host status or on the ecology of the host-virus interaction, samples were categorized into three groups of species: 1) endemic, endangered, or both (17 species); 2) potential reservoir (21 species); and 3) potential spillover (two species). Cloacal swabs were tested by reverse transcription (RT)-PCR for influenza A virus matrix gene amplification. Forty-one serum samples were tested by nucleoprotein-enzyme-linked immunosorbent assay (NP-ELISA) for antibodies against influenza A virus nucleoprotein and by hemagglutination inhibition assay for detection of seropositivity against H5 and H7 AIV subtypes. No cloacal swabs tested RT-PCR positive for AIV, whereas two weak seropositive results were detected by NP-ELISA in a mallard (Anas platyrhynchos) and in a yellow-legged gull (Larus michahellis). The low or absent AIV circulation detected in Sardinia's wild birds during the study suggests a naïve status in these avian populations. These data provide new information on AIV circulation in Sardinia's wild birds that could be applied to implement conservation strategies for threatened species.