PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Francesco Chiesa

Curriculum vitae

Curriculum Vitae

Phd thesis

Title: Tracking Bovine Cysticercosis Outbreaks: New approaches to Molecular Surveillance of Taenia saginata

Scientific background/state of the art

Bovine cysticercosis (BC), a parasitosis caused by the larval stage of the tapeworm Taenia saginata, is a zoonotic disease of economic and public health significance. It affects cattle globally, leading to condemnation, freezing and downgrading of infected carcasses, financial losses for farmers, and potential health risks for humans consuming undercooked beef. Cattle, as intermediate hosts, ingest tapeworm eggs through contaminated feed, water or human contact and develop cysticerci (T. saginata metacestodes - Cysticercus bovis) predominantly in the heart, tongue, and skeletal muscles [1]. Control measures include meat inspection, optimized sanitation, and public health education. Current inspection procedures based on Regulation (EU) No. 2019/627 exhibit limitations in detecting low-level infections. While reported prevalence rates range from 0–7.8% in Europe and 0.02–2.4% in Italy, serological studies have indicated rates as high as 38.4% [2][3]. Recent studies show that systematic analysis of slaughterhouse data can improve the accuracy of bovine cysticercosis prevalence estimates and enhance risk assessment efforts[4][5]. However, the  integration between epidemiological data and molecular diagnostics remains still limited. Given the economic burden associated with carcass downgrading [6], along with the Piedmontese tradition of consuming raw meat and the recurrent incidence of cysticercosis in the region [7], it is crucial to improve disease monitoring and control. Furthermore, while T.saginata isolates globally exhibit relatively low genetic variability [9], subtle genetic differences may still impact transmission dynamics or detection sensitivity, highlighting the need for integrating high-resolution molecular tools into surveillance systems. By combining traditional inspection methods with epidemiological, genomic and cost-effective tools,early detection of outbreaks could be enhanced, supporting new control strategies.

Aims

This project aims to investigate BC outbreaks applying both i) traditional molecular tools and ii) innovative sequencing methods on viable and degenerated cysticerci detected at slaughter in order to explore the genetic variability of T. saginata metacestodes and differentiate bovine cysticercosis cases, thereby improving epidemiological surveillance and food safety. To reach our goal, the project will compare the use of:

1. Traditional molecular tools: PCR amplification of mitochondrial gene regions, followed by Sanger sequencing. Resulting sequences are analyzed through haplotype Network Analysis, a method used to visualize genealogical relationships among DNA haplotypes.

and

1. Innovative and reduced-representation sequencing methods, such as
   1. double digest Restriction-Site Associated DNA Sequencing (ddRADseq)
   2. low-coverage whole-genome sequencing (skim sequencing) 

to assess their suitability for rapid, reliable and cost-effective tracking of BC outbreaks.

Although preliminary applications of these Next-Generation Sequencing-based methods have already been reported to investigate the genetic variability of selected foodborne parasites [11-12], their use remains limited and, to date, largely unexplored in cestodes. This study therefore represents a novel and potentially informative approach to investigate the molecular epidemiology and population structure of T. saginata.

Materials and methods

1st Year: Sample Collection and Initial Processing

Cysticerci samples (∼120) will be collected from at least two outbreaks, to compare three molecular approachesThis sample size accounts for the low genetic variability observed in T. saginata populations and ensures sufficient statistical power (≥ 0.8; α = 0.05) for downstream population structure analyses. Sample metadata will be collected to contextualize genetic data.

2nd Year: DNA Extraction, Quantification, and Preliminary Molecular Analyses

DNA from each sample will be extracted using DNeasy Blood and Tissue Kit (Qiagen) and quantified. 

Preliminary genetic characterization will include: 

i) PCR and Sanger sequencing, followed by haplotype Network Analysis based on selected mitochondrial markers (cox1, nad1, nad5, 12S) using the software PopART (Population Analysis with Reticulate Trees), in line with previously published studies on saginata [8–10] 

3rd Year: High-Resolution Genomics and Bioinformatics Analyses

For high-resolution analysis of genetic diversity and outbreak reconstruction, two reduced-representation NGS approaches will be applied:  

iia) ddRADseq study design, which will be optimized in silico using the software ddRADseqTools [13], which simulates enzyme combinations and fragment size distributions based on reference data.  Candidate enzyme pairs will be selected along with the target fragment size, balancing genomic coverage and resolution for downstream analyses of genetic diversity and population structure. Data processing will follow Bilska-Zając et al. [11]. 

iib) Skim sequencing, which will be performed following the protocol described by Papaiakovou et al. [12] with depth chosen for genome-wide coverage, considering cost-effectiveness and resolution.

Expected results

This study is set to realize a reliable and cost-effective molecular approach to detect, track and characterize T. saginata outbreaks in areas where BC remains a recurrent issue, such as the Piedmont region. Through the application of different molecular tools, the present project seeks to address common limitations, including fragmented parasite DNA, host DNA contamination or low sample variability. Approximately 90 samples have already been collected, including lesions from multiple outbreaks.

The integration of Sanger sequencing with ddRADseq and skim sequencing will enable the precise identification of outbreak sources. Results will directly inform local risk mapping and intervention strategies, providing scientific support for veterinarians, public health institutions and food safety authorities working in endemic regions.

Bibliography

[1] Dorny P, Praet N, Deckers N, Gabriël S. Emerging food-borne parasites. Vet Parasitol. 2009;163:196–206. doi: 10.1016/j.vetpar.2009.05.026.

[2] Laranjo-González, M.; Devleesschauwer, B.; Trevisan, C.; Allepuz, A.; Sotiraki, S.; Abraham, A.; Afonso, M.B.; Blocher, J.; Cardoso,  L.; Correia da Costa, J.M.; et al. Epidemiology of Taeniosis/Cysticercosis in Europe, a Systematic Review: Western Europe.  Parasites Vectors 2017, 10, 349. doi: 10.1186/s13071-017-2280-8.

[3] Cassini, R.; Mulatti, P.; Zanardello, C.; Simonato, G.; Signorini, M.; Cazzin, S.; Tambalo, P.G.; Cobianchi, M.; Pietrobelli, M.;  Capelli, G. Retrospective and Spatial Analysis Tools for Integrated Surveillance of Cystic Echinococcosis and Bovine Cysticercosis in Hypo-Endemic Areas. Geospat. Health 2014, 8, 509–515. doi: 10.4081/gh.2014.40.

[4] Dupuy, C., Morlot, C., Gilot-Fromont, E., Mas, M., Grandmontagne, C., Gilli-Dunoyer, P., Gay, E., Callait-Cardinal, M.-P., 2014. Prevalence of Taenia saginata cysticercosis in French cattle in 2010. Vet. Parasitol. 203, 65–72. doi: 10.1016/j.vetpar.2014.02.054.

[5] Marshall LR, Prakashbabu BC, Ferreira JP, Buzdugan SN, Stärk KDC, Guitian J. Risk factors for Taenia saginata cysticercus infection in cattle in the United Kingdom: A farm-level case-control study and assessment of the role of movement history, age and sex. Prev Vet Med. 2016 Dec 1;135:1-8. doi: 10.1016/j.prevetmed.2016.10.015.

[6] Laranjo-González M, Devleesschauwer B, Jansen F, Dorny P, Dupuy C, Requena-Méndez A, Allepuz A. Epidemiology and economic impact of bovine cysticercosis and taeniosis caused by Taenia saginata in northeastern Spain (Catalonia). Parasit Vectors. 2018 Jun 28;11(1):376. doi: 10.1186/s13071-018-2931-4.

[7] Rubiola S, Moroni B, Carisio L, Rossi L, Chiesa F, Martano G, Cavallo E, Rambozzi L. Risk Factors for Bovine Cysticercosis in North-West Italy: A Multi-Year Case-Control Study. Animals (Basel). 2021 Oct 25;11(11):3049. doi: 10.3390/ani12050636.

[8] Abuseir S, Schicht S, Springer A, Nagel-Kohl U, Strube C. Genetic Characterization of Taenia saginata Cyst Isolates from Germany. Vector Borne Zoonotic Dis. 2018 Aug;18(8):433-439. doi: 10.1089/vbz.2017.2218.

[9] Sanpool O, Rodpai R, Intapan PM, Sadaow L, Thanchomnang T, Laymanivong S, Maleewong W, Yamasaki H. Genetic diversity of Taenia saginata (Cestoda: Cyclophyllidea) from Lao People's Democratic Republic and northeastern Thailand based on mitochondrial DNA. Parasit Vectors. 2017 Mar 11;10(1):141. doi: 10.1186/s13071-017-2079-7.

[10] Tran Thi G, Azzena I, Scarpa F, Cossu P, Danh Le C, Ton Nu PA, Chau Ngo TM, Sanna D, Casu M. Molecular Identification and Appraisal of the Genetic Variation of Taenia saginata in Central Regions of Vietnam. Life (Basel). 2022 Jan 4;12(1):70. doi: 10.3390/life12010070.

[11] Bilska-Zając E, Rosenthal B, Thompson P. Trich-tracker - a practical tool to trace Trichinella spiralis transmission based on rapid, cost-effective sampling of genome-wide genetic variation. Int J Parasitol. 2022 Feb;52(2-3):145-155. doi: 10.1016/j.ijpara.2021.08.002.

[12] Papaiakovou M, Fraija-Fernández N, James K, Briscoe AG, Hall A, Jenkins TP, Dunn J, Levecke B, Mekonnen Z, Cools P, Doyle SR, Cantacessi C, Littlewood DTJ. Evaluation of genome skimming to detect and characterise human and livestock helminths. Int J Parasitol. 2023 Feb;53(2):69-79. doi: 10.1016/j.ijpara.2022.12.002.

[13] Mora-Márquez F, García-Olivares V, Emerson BC, López de Heredia U. ddradseqtools: a software package for in silico simulation and testing of double-digest RADseq experiments. Mol Ecol Resour. 2017 Mar;17(2):230-246. doi: 10.1111/1755-0998.12550.