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.

Botulism in Cattle: A Case Report of an Outbreak in Sardinia (Italy).

Clostridium botulinum is the main causative agent of botulism in humans and animals. The ingestion of the botulinum neurotoxin, usually types C and D, has been shown to produce disease (neurological symptoms) in most botulism cases in cattle. We report an outbreak in Southern Sardinia that involved a livestock farm with 120 animals, 39 of which died. The aim of this report is to describe the course of this outbreak and the progression of symptoms up to the death of some animals; we also describe the therapeutic approach applied in this case and the analytical techniques used to diagnose the disease. Finally, we emphasize the importance of promptly proceeding with the sampling of several matrixes when a suspicion of botulism arises.

A Chlamydia psittaci Outbreak in Psittacine Birds in Sardinia, Italy.

Chlamydia psittaci is an intracellular bacterium belonging to the Chlamydiaceae family. It is the ethiologic agent of psittacosis, an occupational zoonotic disease that mainly concerns people who work in close contact with birds that represent the main infection route for human transmission. In Italy, information about this disease is lacking. This study is the first case of avian chlamydiosis reported from a pet shop in Sardinia, Italy. Chlamydia psittaci detected in psittacine birds by molecular analysis, direct immunofluorescence test together with anatomo-pathological observed lesions, highlighted the importance of focusing the attention over this underestimated zoonosis in a "One Health" prospective.

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.

Vibrio splendidus clade associated with a disease affecting Paracentrotus lividus (Lamarck, 1816) in Sardinia (Western Mediterranean).

This work reports for the first time the so-called bald sea urchin disease of the populations of Paracentrotus lividus in Sardinia (Italy). Following the disease occurrence reported by local fishermen of about 35% on the collected specimens for commercial purpose, the phenomenon was evaluated in two areas. Animals displayed external lesions represented by loss of spines and greenish/brownish skeleton patches. Microscopy of exoskeleton and tube feet showed haemocytosis with associated bacteria; gut and intestine histopathology revealed inflammatory lesions of different type and intensity. Microbiological and molecular analysis revealed the presence of the strains belonging to the Vibrio splendidus clade. Due to the important ecological role of Paracentrotus lividus in the Mediterranean Sea, this report represents a wake-up call for the entire basin worth of further insights.

African Swine Fever in Smallholder Sardinian Farms: Last 10 Years of Network Transmission Reconstruction and Analysis.

African swine fever (ASF) is a viral disease of suids that frequently leads to death. There are neither licensed vaccines nor treatments available, and even though humans are not susceptible to the disease, the serious socio-economic consequences associated with ASF have made it one of the most serious animal diseases of the last century. In this context, prevention and early detection play a key role in controlling the disease and avoiding losses in the pig value chain. Target biosecurity measures are a strong strategy against ASF virus (ASFV) incursions in farms nowadays, but to be efficient, these measures must be well-defined and easy to implement, both in commercial holdings and in the backyard sector. Furthermore, the backyard sector is of great importance in low-income settings, mainly for social and cultural practices that are highly specific to certain areas and communities. These contexts need to be addressed when authorities decide upon the provisions that should be applied in the case of infection or decide to combine them with strict preventive measures to mitigate the risk of virus spread. The need for a deeper understanding of the smallholder context is essential to prevent ASFV incursion and spread. Precise indications for pig breeding and risk estimation for ASFV introduction, spread and maintenance, taking into account the fact that these recommendations would be inapplicable in some contexts, are the keys for efficient target control measures. The aim of this work is to describe the 305 outbreaks that occurred in domestic pigs in Sardinia during the last epidemic season (2010-2018) in depth, providing essential features associated with intensive and backyard farms where the outbreaks occurred. In addition, the study estimates the average of secondary cases by kernel transmission network. Considering the current absence of ASF outbreaks in domestic pig farms in Sardinia since 2018, this work is a valid tool to specifically estimate the risk associated with different farm types and update our knowledge in this area.

Histopathological and Molecular Study of Pacific Oyster Tissues Provides Insights into V. aestuarianus Infection Related to Oyster Mortality.

Consumer preference for healthy and sustainable food products has been steadily increasing in recent years. Bivalve mollusks satisfy these characteristics and have captured ever-increasing market shares. However, the expansion of molluscan culture in worldwide and global trade have favored the spread of pathogens around the world. Combined with environmental changes and intensive production systems this has contributed to the occurrence of mass mortality episodes, thus posing a threat to the production of different species, including the Pacific oyster Crassotrea gigas. In the San Teodoro lagoon, one of the most devoted lagoons to extensive Pacific oyster aquaculture in Sardinia, a mortality outbreak was observed with an estimated 80% final loss of animal production. A study combining cultural, biomolecular and histopathological methods was conducted: (1) to investigate the presence of different Vibrio species and OsHV-1 in selected oyster tissues (digestive gland, gills, and mantle); (2) to quantify Vibrio aestuarianus and to evaluate the severity of hemocyte infiltration in infected tissues; (3) to produce post-amplification data and evaluating ToxR gene as a target for phylogenetic analyses. Results provide new insights into V. aestuarianus infection related to oyster mortality outbreaks and pave the way to the development of tools for oyster management.

From the traditional Chinese medicine plant Schisandra chinensis new scaffolds effective on HIV-1 reverse transcriptase resistant to non-nucleoside inhibitors.

HIV-1 reverse transcriptase (RT) is still an extremely attractive pharmaceutical target for the identification of new inhibitors possibly active on drug resistant strains. Medicinal plants are a rich source of chemical diversity and can be used to identify novel scaffolds to be further developed by chemical modifications. We investigated the ability of the main lignans from Schisandra chinensis (Turcz.) Baill. fruits, commonly used in Traditional Chinese Medicine, to affect HIV-1 RT functions. We purified 6 lignans from Schisandra chinensis fruits and assayed their effects on HIV-1 RT and viral replication. Among the S. chinensis fruit lignans, Schisandrin B and Deoxyschizandrin selectively inhibited the HIV-1 RT-associated DNA polymerase activity. Structure activity relationship revealed the importance of cyclooctadiene ring substituents for efficacy. In addition, Schisandrin B was also able to impair HIV-1 RT drug resistant mutants and the early phases of viral replication. We identified Schisandrin B and Deoxyschizandrin as new scaffold for the further development of novel HIV-1 RT inhibitors.

Development and Field Testing of a Real-Time PCR Assay for Caprine Arthritis-Encephalitis-Virus (CAEV).

Caprine arthritis/encephalitis (CAE) of goats and occasionally sheep are persistent virus infections caused by a lentivirus (CAEV). This viral infection results in arthritis in adult animals and encephalitis in kids. Prognosis for the encephalitic form is normally poor, with substantial economic loss for the farm. In this context an early/fast laboratory diagnosis for CAEV infection could be useful for effective prophylactic action. In this work we performed a quantitative real time PCR designed on the CAEV env gene to detect/quantify in goat/sheep samples, viral RNA or proviral DNA forms of CAEV. This procedure was validated in 15 sheep, experimentally infected with CAEV or with a highly correlated lentivirus (visna maedi, MVV); in addition, a total of 37 clinical goat specimens recruited in CAEV positive herds were analyzed and compared using serological analysis (Elisa and AGID). All samples infected with MVV resulted negative. In sheep experimentally infected with CAEV, proviral DNA was detectable 15 days post infection, whereas the serological methods revealed an indicative positivity after 40-60 days.This method showed a sensitivity of 10(2) env fragments/PCR) with a linear dynamic range of quantitation from 10(3) to 10(7)env fragments/PCR; the R2 correlation coefficient was 0.98. All subjects with a clinical diagnosis for Caprine Arthritis-Encephalitis (CAE) resulted CAEV DNA positive.