Ciccone, Elena (2026) Integrated control of cystic echinococcosis: innovative epidemiological, diagnostic and treatment strategies, including microRNAs. [Tesi di dottorato]

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Abstract

Cystic echinococcosis (CE), caused by the larval stages of Echinococcus granulosus sensu lato (s.l.)., is a global public health priority disease due to its impact on human and animal health. Echinococcosis belongs to the group of 21 neglected tropical diseases (NTDs), recognized by the World Health Organization (WHO) as being of major public health importance and requiring effective control efforts. The taxonomy of E . granulosus s.l. currently numbers five species: E. granulosus sensu stricto (s.s.), E. equinus, E. ortleppi,E. canadensis, and E. felidis. Among these species, E. granulosus s.s. is the most common , with a worldwide distribution. The lifecycle of E. granulosus s.s. is essentially based on the consumption of sheep viscera by dogs, particularly affecting pastoral and poor rural communities where people raise sheep or goats in close contact with dogs. The economic impact of this infection on the livestock industry is multifaceted, affecting the quality of meat, milk and wool, reduced birth rate, delayed performance and growth, as well as post-mortem rejections of infected organs at slaughtering. CE has a broad geographic range, but it exhibits the highest prevalence in communities where pastoral activities predominate, as in the Mediterranean and Balkan areas. Key challenges in CE control include gaps in diagnosis, prevention, and management both in intermediate and definitive hosts. These stem from inadequate mapping of baseline infection prevalence in endemic areas, difficulties in delivering effective deworming programs to rural dog populations, limited diagnostic and therapeutic tools, insufficient infrastructure and resources in remote regions, and poor community compliance with control measures. Effective disease management in animal and human populations demands a coordinated, multidisciplinary approach involving veterinary, research, and public health stakeholders. In order to support the development of sustainable and effective strategies to control CE, the present thesis entitled “Integrated control of cystic echinococcosis: innovative epidemiological, diagnostic and treatment strategies,including microRNAs” investigated innovative epidemiological methods,advancing diagnostic techniques, and exploring novel treatment strategies,with a particular focus on the role of microRNAs in disease management and therapy optimization. The PhD thesis consists of two parts, according to the European standard requirements. The first part, entitled “Literature Review”, summarizes scientific updates from the literature on the infection caused by E. granulosus in ani mals and humans. The second part, entitled “Own research”, presents the general and specific objectives of the thesis, followed by seven original studies dealing with the update of the epidemiology of CE and the innovative strategies to improve the control of the infection in southern Italy, such as new diagnostic and therapeutic tools. The literature review in Chapter 1 provides an overview of Cystic Echinococcosis. Data on morphology, lifecycle, transmission, clinical signs, symptoms, diagnosis, treatment, control and prevention of the infection are discussed in detail. This chapter highlighted the urgent need for high spatial resolution studies to assess CE epidemiology and emphasized the importance of exploring innovative strategies and technologies to improve the effectiveness of CE control programs in areas highly endemic for E. granulosus infections. Chapter 2 presents an updated analysis of the epidemiological scenario of CE in various ruminant intermediate hosts (sheep, goats, cattle, and buffaloes), along with an evaluation of shepherds’ knowledge about the disease,in four highly endemic Mediterranean countries (i.e., Algeria, Greece, Italy,and Tunisia). This study aimed to identify high-risk endemic areas and practices that could favor the persistence of E. granulosus infection in these areas.A systematic examination of peer-reviewed literature on CE prevalence data published in the period 2017–2023 was carried out and the results were used to map an updated spatial distribution of CE prevalence in the study area, using a Geographical Information Systems (GIS). A total of 219 farmers were interviewed in depth to collect information about their management systems, the awareness of CE and sources of parasite’s transmission. The questionnaire survey revealed that some risky practices (e.g. irregular deworming of dogs,use of ineffective or incorrect drugs and dosages, administration of raw animal offal to dogs) are still observed and that farmers’ awareness of CE needs to be raised. The collection of published data on CE prevalence in livestock,a GIS-based identification of the areas with the highest CE incidence and the assessment of the level of knowledge of the local population are among the first steps to thoroughly understand the epidemiology of the disease and limitits transmission. Chapter 3 presents a panel of microsatellite targets for population genetic analyses to describe the genetic diversity of E. granulosus sensu stricto (s.s.)at the global to farm scale. A total of 145 isolates from 11 countries were used to evaluate the panel at the international level. Additionally, 131 samplesfrom sheep and cattle across six regions in southern Italy were analyzed to assess the panel’s usefulness at both the regional and farm scale. Population genetics showed high genotypic diversity. The microsatellite panel provided data relevant for E. granulosus s.s. population genetics from global to farm scale, making it a low-cost, simple alternative to full mitochondrial genome sequencing for analyzing numerous samples and obtaining a detailed over view of genetic diversity, an input that is useful for cystic echinococcosis control programs. The microsatellite panel developed in this study has proved to efficiently reveal the high genetic diversity found in CE livestock samples, which was already unveiled previously using the two EgSca6 and EgSca11 targets. Chapter 4 presents the evaluation of the effectiveness of ultrasound (US) in sheep compared with the necropsy (gold standard) as an in-vivo monitoring tool used in a long-term control program of CE. The study involved 10 randomly-selected farms, enrolled over 6 years in an endemic area of southern Italy. All sheep of ten farms destined for slaughter underwent liver and lung US followed by necropsy, during the study period. Overall, the US showed a Sensitivity (SE) of 87.9 %, a Specificity (SP) of 72.3 % as well as a positive- (PPV) and negative-predictive-value (NPV), and Accuracy of 74.4 %, 86.7 %, and 79.7 %, respectively. Although technical and logistical challenges have to be addressed for its use, a US conscious integration into continuous surveillance program might promote the early in vivo identification of infected farms, limiting necropsy dependence for CE monitoring. In Chapter 5, the distribution of anthelmintic PZQ baits against wild and not owned definitive hosts is explored as new action to control CE in the Mediterranean area. Despite their crucial role in sustaining CE transmission,free-roaming canids have not yet been included in control programs against CE in southern Italy. Deworming dogs with praziquantel (PZQ) is currently considered the most effective method for limiting CE transmission. The study presented aimed to identify the most suitable bait laced with PZQ for deworming unowned dogs. In particular, three types of PZQ baits were tested by evaluating different parameters (integrity over time, attractiveness and palatability for dogs, and mechanical resistance after release to different altitudes). In addition, the bait acceptance in the field by the target animals, i.e. not owned dogs, was tested by using camera traps. The double PZQ-laced baits proved highly durable, maintained attractiveness for up to 10 days, and withstood drone drops from 25 meters, making them ideal for aerial delivery. Field trials showed 82.2% consumption by unowned dogs, confirming their effectiveness for targeting free-ranging dog populations and supporting their use in controlling E. granulosus transmission in endemic rural areas. Chapter 6 presents an innovative strategy for deworming the owned and not owned dogs in a pilot area of southern Italy. The collection of detailed data on the movements and spatial behavior of definitive (e.g. dogs) and intermediate hosts (e.g. sheep) is crucial for planning effective control strategies against CE. Geospatial technologies and GPS dataloggers offer an ideal solution for tracking animal movements with high spatial and temporal accuracy. Five sheep farms with confirmed CE cases were selected, and shepherd dogs were treated with praziquantel every two months. Additionally, 15 GPS dataloggers were used to track the movements of selected sheep and dogs over a month; their home ranges were mapped using minimum convex polygon (MCP) analysis. GPS data collected from sheep and dogs over a month showed similar daily walking distances, with most movements within 1500 meters of the farms and activity areas under 250 hectares. The study estimated high-risk areas for canid access around each farm’s centroid and proposed a scheme for delivering praziquantel-laced baits to treat unowned dogs freely roaming near grazing areas. Chapter 7 presents a review that explores the potential of microRNAs (miRNAs) as diagnostic and therapeutic targets to enhance control programs for cystic echinococcosis (CE), a zoonotic parasitic disease posing significant threats to global human and animal health. Scientific evidence has demonstrated that Echinococcus-derived miRNAs are present in the biofluids of infected hosts, underscoring their potential for CE diagnosis. Additionally,specific miRNAs from Echinococcus granulosus sensu lato have been shown to influence host–parasite interactions, making them promising therapeutic targets for CE. Chapter 8 presents an integrated molecular approach employed to investigate hydatid cysts samples collected in southern Italy from different ruminant species (sheep, cattle, and water buffaloes), naturally infected with CE, with the ultimate goal of establishing a more comprehensive picture of miRNA expression patterns in these intermediate hosts. The bioinformatic analysis of hydatid cyst samples revealed 168 mature miRNAs identified. Among these, egr-miR-10-5p, egr-let-7-5p, and egr-miR-71-5p were the most abundant, with egr-miR-10-5p showing particularly high expression levels in both PSCs and germinal membranes. The bioinformatic analysis revealed no significant differences in miRNA’s abundance between host species, in contrast, when focusing on the comparison between PSCs and sterile membranes, 24 miRNAs were found to be differentially expressed. Targeted qRT-PCR of four selected miRNAs (egr-miR-71-5p, egr-let-7-5p, egr-miR-125-5p, and egr-miR-10-5p) showed clear overexpression in PSCs and in fertile germinal membranes compared with sterile ones. The differential miRNA expression patterns provide insight into the molecular mechanisms controlling the parasite’s lifecycle and may guide the development of novel intervention methods to enhance CE control in endemic areas. Chapter 9 is an overall discussion regarding future perspectives for integrated control of cystic echinococcosis based on miRNAs and other innovative technologies. It is emphasised that a multidisciplinary and multi- institutional One Health effort is fundamental for the development of CE control programmes at different geographic levels.

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