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.

The Long-Jumping of African Swine Fever: First Genotype II Notified in Sardinia, Italy.

African swine fever (ASF) is a devastating infectious disease of domestic pigs and wild boar that is spreading quickly around the world and causing huge economic losses. Although the development of effective vaccines is currently being attempted by several labs, the absence of globally recognized licensed vaccines makes disease prevention and early detection even more crucial. ASF has spread across many countries in Europe and about two years ago affected the Italian susceptible population. In Italy, the first case of ASF genotype II in wild boar dates back to January 2022, while the first outbreak in a domestic pig farm was notified in August 2023. Currently, four clusters of infection are still ongoing in northern (Piedmont-Liguria and Lombardy), central (Lazio), and southern Italy (Calabria and Campania). In early September 2023, the first case of ASFV genotype II was detected in a domestic pig farm in Sardinia, historically affected by genotype I and in the final stage of eradication. Genomic characterization of p72, p54, and I73R/I329L genome regions revealed 100% similarity to those obtained from isolates that have been circulating in mainland Italy since January 2022 and also with international strains. The outbreak was detected and confirmed due to the passive surveillance plan on domestic pig farms put in place to provide evidence on genotype I's absence. Epidemiological investigations suggest 24 August as the most probable time of ASFV genotype II's arrival in Sardinia, likely due to human activities.

Correction: Socio-economic factors as indicators for various animal diseases in Sardinia.

[This corrects the article DOI: 10.1371/journal.pone.0217367.].

Socio-economic factors as indicators for various animal diseases in Sardinia.

The need to consider the role of social factors in the efficacy of farm management and, consequently, in the onset and persistence of diseases typical to animal farms is increasingly being realized increasingly worldwide. Many risk analysis studies have been conducted to assess the role of various factors in the development of animal diseases; however, very few have accounted for the role of social factors. The aim of this work was to bridge this gap, with the main hypothesis that different socio-economic factors could be valid indicators for the occurrence of different animal diseases. A socio-economic analysis was performed using demographic characteristics of the farmers and data from 44 social indicators released by the Italian Statistician National Institute of Statistics (ISTAT) database. African swine fever (ASF) in wild boars (WB) and domestic pigs and other endemic animal diseases and zoonoses in Sardinia were considered, such as cistic echinococcosis (CE), contagious agalactia (CA), trichinellosis, West Nile disease (WND), and bluetongue (BT). Seven different negative binomial regression models were fitted using the number of cases between 2011-2017. Three indicators-cultural demand, employment rate, and legality-showed a statistically significant association with risk for all the diseases considered, but with varying effects. Some indicators, such as the age and sex of the farmer, material deprivation index, number of farms and animals, micro-criminality index, and rate of reported thefts were common to ASF, CA, trichinellosis, and CE cases. Others such as the forest surface and the energy produced from renewable sources were common to BT, WND, and ASF in WB. Tourism in seasons other than summer was a valid predictor of ASF and trichinellosis, while out-of-region hospital use had a statistically significant role in CE risk identification. These results may help understand the social context in which these diseases may occur and thus guide the design and implementation of additional risk management measures that go beyond well-known veterinary measures.

Surveillance and control of African Swine Fever in free-ranging pigs in Sardinia.

African swine fever (ASF) is a notifiable infectious disease, caused by the ASF virus (ASFV), which is a DNA virus belonging to the family Asfarviridae, genus Asfivirus. This disease has gained importance in the last decade after its spread in several countries in Eastern and Central Europe, and more recently, in China. Despite the efforts made to eradicate it, ASF is still present on the Mediterranean island of Sardinia (Italy) and has been since 1978. ASF risk factors on the island have been analysed in previous studies; the role of free-ranging pigs in virus persistence has been suggested, but has not been fully elucidated. The most recent eradication plan provides more stringent measures to combat free-ranging pigs and any kind of illegality in the pig sector. From December 2017 to June 2018, a total of 29 depopulation actions were performed in 13 municipalities in central Sardinia, during which 2,281 free-ranging pigs were culled and more than 50% of them were tested for ASFV and antibody presence (1,218 and 1,416, respectively). A total of 651 pigs were seropositive, with a mean seroprevalence of 53.4% (CI 95% = 50.6-56.3), and 38 were ASFV positive (virus prevalence = 2.6%; CI 95% = 2.1-3.0). To the best of our knowledge, the present study is the first to provide a complete evaluation of this millennial system of pig farming and ASFV prevalence in free-ranging pigs. Furthermore, it has emphasised the necessity of combining the maintenance of an epidemiological surveillance program with continuous education of farmers and other people involved in pig husbandry, based on cultural and economic aspects.

Multilocus genotype analysis outlines distinct histories for Trichinella britovi in the neighboring Mediterranean islands of Corsica and Sardinia.

BACKGROUND: The zoonotic nematode Trichinella britovi was discovered in two neighboring Mediterranean islands of Corsica and Sardinia, almost simultaneously at the beginning of the 21st century. An epidemiological link between the two parasite populations was generally assumed. In 2015, an outbreak of trichinellosis in Nice, the South of France, was reportedly caused by the consumption of raw pork delicatessen imported from Corsica. The aims of the present study were to investigate, by multilocus genotype (MLG) analyses, the hypothesis of the common origin of the Corsican and Sardinian T. britovi foci and to trace "from fork to farm" the origin of the pork product, which caused a trichinellosis outbreak in mainland France in 2015. METHODS: Sixty-three T. britovi isolates were collected from animals and pork products of Sardinia and Corsica islands and from mainland of Italy, France and Spain. We analyzed genetic variability at four polymorphic microsatellite loci by two independent algorithms, the Bayesian and multivariate analyses, to evaluate the genetic relationships of 1367 single larvae. RESULTS: Trichinella britovi isolates of the two islands showed different genetic structures and the Bayesian analysis revealed a different membership of the two insular populations. Furthermore, two geographically separate genetic groups were identified among Corsican isolates. Lastly, the origin of the pork delicatessen marketed in Nice was linked to a breeder-butcher in Corsica. CONCLUSIONS: The low level of genetic admixture of the insular T. britovi isolates suggests that this pathogen colonized the two islands by separate events. On the other hand in Corsica, although the isolates share the same genetic structure, geographically separate isolates showed different membership. We suggest the MLG analysis as a suitable method in supporting epidemiological investigations to trace "from fork to farm" insular populations of T. britovi.

Indirect versus direct detection methods of Trichinella spp. infection in wild boar (Sus scrofa).

BACKGROUND: Trichinella spp. infections in wild boar (Sus scrofa), one of the main sources of human trichinellosis, continue to represent a public health problem. The detection of Trichinella spp. larvae in muscles of wild boar by digestion can prevent the occurrence of clinical trichinellosis in humans. However, the analytical sensitivity of digestion in the detection process is dependent on the quantity of tested muscle. Consequently, large quantities of muscle have to be digested to warrant surveillance programs, or more sensitive tests need to be employed. The use of indirect detection methods, such as the ELISA to detect Trichinella spp. infections in wild boar has limitations due to its low specificity. The aim of the study was to implement serological detection of anti-Trichinella spp. antibodies in meat juices from hunted wild boar for the surveillance of Trichinella spp. infections. METHODS: Two tests were used, ELISA for the initial screening test, and a specific and sensitive Western blot (Wb) as a confirmatory test. The circulation of anti-Trichinella IgG was determined in hunted wild boar muscle juice samples in 9 provinces of 5 Italian regions. RESULTS: From 1,462 muscle fluid samples, 315 (21.5%, 95% C.I. 19.51-23.73) were tested positive by ELISA. The 315 ELISA-positive muscle fluid samples were further tested by Wb and 32 (10.1%, 95% C.I. 7.29-13.99) of these were positive with a final seroprevalence of 2.2% (95% C.I 1.55-3.07; 32/1,462). Trichinella britovi larvae were detected by artificial digestion in muscle tissues of one (0.07%, 95%C.I. 0.01-0.39) out of the 1,462 hunted wild boars. No Trichinella spp. larvae were detected in Wb-negative wild boar. From 2006 to 2012, a prevalence of 0.017% was detected by muscle digestion in wild boar hunted in the whole Italian territory. CONCLUSIONS: The combined use of both serological methods had a sensitivity 31.4 times higher than that of the digestion (32/1,462 versus 1/1,462), suggesting their potential use for the surveillance of the Trichinella spp. infection in wild boar populations.

Expression profiling of Mycobacterium tuberculosis H37Rv and Mycobacterium smegmatis in acid-nitrosative multi-stress displays defined regulatory networks.

Several studies regarding the transcriptome of Mycobacterium tuberculosis following the exposure to various in vitro simulated phagosomal stressors, have already tried to elucidate the bacterium behavior during the intracellular infection. An in vitro acid-nitrosative multi-stress was carried out for M. tuberculosis H37Rv and Mycobacterium smegmatis MC(2)155 in order to analyze by DNA-microarray the gene expression changes associated respectively to pathogenic and non-pathogenic mycobacterial species. During acid-nitrosative multi-stress both mycobacteria shift their transcriptome to allow the anaerobic respiratory state and energy pathways characteristic of starvation. M. tuberculosis counteracts the combined acid-nitrosative stress more efficiently than M. smegmatis as also shown by the up-regulation of glbN and hmp genes, that are specifically directed to NO detoxification. Moreover, the down-regulation of some virulence factors involved in phthiocerol dimycocerosates synthesis strengthens the hypothesis that these major virulence determinants may be attenuated by M. tuberculosis in the presence of reactive nitrogen species. In fact, it down-regulates other genes implicated in the synthesis of membrane structural lipids but in contrast to M. smegmatis, M. tuberculosis up-regulates many genes annotated for the synthesis of peptidoglycan. Results suggest a gene regulation of M. tuberculosis which reveals a distinctive expression pattern under stressful environment.

Unraveling the transcriptional regulatory networks associated to mycobacterial cell wall defective form induction by glycine and lysozyme treatment.

It is known that a combined glycine/lysozyme treatment is able to induce in vitro the mycobacterial conversion from the bacillary to the cell wall defective forms. These forms also naturally occur in vivo as a response to various antimicrobial factors such as lysozyme released by phagocytic cells. Although they have been successfully isolated from patients with several chronic diseases, their role in pathogenesis is still unknown, mainly due to the difficulties in handling the in vivo isolated variants. Moreover, nothing is known about the transcriptional peculiarities that may exist in comparison to the vegetative phase. Hence, in this study, we simulated in vitro the induction of the mycobacterial cell wall defective state by using a glycine and lysozyme-based treatment in order to identify the gene expression profiles of both pathogenic and non-pathogenic mycobacteria. DNA-microarray results showed that in contrast to the non-pathogenic Mycobacterium smegmatis species, glycine and lysozyme treated forms of Mycobacterium tuberculosis and Mycobacterium avium subspecies paratuberculosis regulated a repertoire of genes usually expressed in vivo during adaptation and persistence within host environments. Results suggest that the cell wall defective state may represent an important stage in the life-cycle of pathogenic mycobacteria that potentially coordinates persistence.

Characterisation of Mycoplasma capricolum P60 surface lipoprotein and its evaluation in a recombinant ELISA.

This paper reports the identification and characterisation of a 60kDa surface protein antigen (P60) of Mycoplasma capricolum subspecies capricolum (Mcc), and describes its diagnostic application. Genomic localization and presence in P60 of conserved functional domains suggested a structural and functional relationship with the immunodominant antigen P48 of Mycoplasma agalactiae, a basic membrane protein. A rP60-ELISA was developed, and it resulted in a high specificity for Mcc infections after evaluation with 125 goat sera. The comparison with an existent ELISA based on whole Mcc cell lysates showed that the two assays have comparable sensitivities, but the rP60-ELISA has the significant advantage of a greater specificity. Results indicate that P60 is a potential marker of Mcc infection, especially useful in areas where the presence of M. capricolum subspecies capripenumoniae is also reported.