Natural history of Trichinella britovi in the neighboring Mediterranean islands of Corsica and Sardinia
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1 Workshop of National Reference Laboratories for Parasites Istituto Superiore di Sanità, Rome, Italy, May, 2018 Natural history of Trichinella britovi in the neighboring Mediterranean islands of Corsica and Sardinia Gianluca Marucci 1, Giuseppe La Rosa 1, Isabelle Vallée 2, François Casabianca 3, Pascal Boireau 2, Ennio Bandino 3, Fabio Galati 1, Edoardo Pozio 1 1 Istituto Superiore di Sanità, Rome, Italy 2 ANSES, INRA, ENVA, Université Paris Est, Laboratory for Animal Health, Maisons-Alfort, France 3 INRA, Corte, France 4 Istituto Zooprofilattico Sperimentale della Sardegna, Nuoro, Italy
2 Introduction In 2004 and 2005, two foci of Trichinella britovi appeared in free ranging pigs of two restricted areas of the Mediterranean islands of Corsica (France) and Sardinia (Italy), considered until then to be Trichinella free CORSICA (FR) SARDINIA (IT) Orgosolo 2005 Cozzano 2004 An epidemiological link between the two foci was suspected due to their geographic proximity and illegal animal trade between the two islands
3 Aim of the work To investigate the hypothesis of a common origin of the Corsican and Sardinian T. britovi foci by multilocus genotype analyses using microsatellites Microsatellites or Simple Sequence Repeats (SSR) are stretches of DNA consisting of tandemly repeated short units of 1-6 base pairs in length Commonly used in population genetic studies because the number of repeats is highly variable among individuals (high level of polymorphism)
4 Trichinella britovi tested isolates 63 isolates (21.7 average number of individuals per isolate) were analysed No. isolates Host/source Locality of origin (region) Country domestic pigs 11 red foxes 7 domestic pig 1 red fox Orgosolo (Sardinia) Cozzano (Corsica) Italy France 7 domestic pigs Vallée de Gravona (Corsica) France domestic pig Bastelica (Corsica) France domestic pig Aullène (Corsica) France 2 Figatelli+sausage Aullène (Corsica) France wolf Berceto (Emilia Romagna) Italy wolf Andria (Apulia) Italy wild boar Arquata del Tronto (Marche) Italy No. isolates Host/source Locality of origin (region) Country red fox Entraigues (Provence) France red fox Entemont le vieux (Haute Savoie) France red fox Chignin (Haute Savoie) France red fox Rimaucourt (Marne) France red fox (Iozère) France wild boar wolf (Var) France wild boar (Ariège) France wolf La combe du lars (Haute Savoie) France wolf Péone (Alpes Maritimes) France 9 wolf Monte S. Angelo (Apulia) Italy wolf Teramo (Abruzzo) Italy red fox Villavallelonga (Abruzzo) Italy wild boar Jarandilla (Extremadura) Spain wild boar Deleitosa (Extremadura) Spain wild boar Tornavacas (Extremadura) Spain wolf Tufara (Molise) Italy wild boar Monte S. Angelo (Apulia) Italy red fox (Isère) France 8 wild boar Fresnedoso de Ibor (Extremadura) Spain wild boar Boltana (Aragon) Spain wild boar Hontanares (Castile and León) Spain 14 red fox Saint Pierre d'allevard (Isère) France red fox Chichilianne (Isère) France wild boar Beraton (Castile and León) Spain wild boar Sabinanigo (Aragon) Spain
5 Microsatellite Analysis DNA purification from single larva (DNA IQ System, Promega) Multiplex PCR to confirm species (Multiplex PCR Kit, Qiagen) PCR with primers designed for microsatellites (Type-it microsatellite PCR kit, Qiagen) Run on capillary electrophoresis system (Qiaxcel with high resolution cartrige, Qiagen) Sequencing of each different band for size confirmation Microsatellite size deduced by comparison with fragments of known lenght
6 Microsatellite loci Locus Repeat motif N of alleles TS10.10b (AT) n 5 TS1380 (TGA) n N 6 (TGA) n 6 TB1019 (TAA) n 5 TB922 (TA) n 8
7 Genetic variability Total No. Samples: 1424 Total No. Pops. 63 Level of genetic variability for geographical area (average values) Area Pop N Na Ho F is %Pl Corsica Sardinia Italy France Spain Corsica isolates: lowest level of genetic variability Sardinia isolates: highest level of genetic variability
8 TB1019 Allelic frequencies Alleles frequencies observed at the four microsatellite loci for the 63 isolates TS1380 TS1010B TB922 Populations from Corsica and Sardinia are more homogeneous than the others and show peculiar allelic pattern
9 Genetic differentiation by F ST index Investigated areas Corsica Continental (number of tested isolates) Sardinia all foci Italy France Spain Sardinia (13) Corsica all foci (19) Continental Italy (9) Continental France (14) Continental Spain (8) (average values)
10 x-axis Principal Coordinates Analysis y-axis A = Corsica isolates (Vallée de Gravona) B = Corsica isolates (Cozzano) C = Sardinia isolates x-axis D = Corsica isolates (all) E = Sardinia isolates z-axis
11 Bayesian clustering algorithm
12 Bayesian clustering algorithm
13 Trichinella britovi outbreak In 2015 an outbreak of trichinellosis occurred in the region of Nice (South of France) for the consumption of raw sausages (Figatelli) ordered from a delicatessen based in Aullène, (Corsica) Investigation carried out by the Regional Health Agency and ANSES revealed that the sausages were prepared from pigs bred in Aullène, illegally slaughtered and not controlled for Trichinella infection
14 Multilocus genotype analysis confirmed outbreak investigation x-axis z-axis y-axis T. britovi larvae isolated from figatelli (isolate No. 30) show an identical genetic structure to that of larvae isolated from a sausage (isolate No. 31) collected at the breeder-butcher of Aullène and to an isolate from a domestic pig (isolate No. 32) reared in Aullène x-axis
15 Conclusions The multilocus genotype analysis suggests that: T. britovi was introduced to Sardinia and Corsica by two or more independent events and the populations of the two island do not derive from each other Gene flow among the isolates of Corsica is very limited and restricted to foci present in the deep valleys (Cozzano and Vallée de Gravona) The MLG analysis may support epidemiological investigations during outbreaks, whereas its use at the continental level could be more difficult due to the limited amount of data
16 Thanks for your attention
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