Experimental study in wild boars infected with Trichinella patagoniensis, T. pseudospiralis and T. spiralis

BESSI, C; ERCOLE, M; FARIÑA, F; RIBICICH, M.; BONBONI, A; MONTALVO, F; ACERBO, M.; KRIVOKAPICH, S.; PASQUALETTI, M

Conferencia; 15th International Conference on Trichinellosis; 2019

1. Introduction. Trichinellosis is a relevant disease for the publichealth in Argentina, essentially because of the hunt of wild animals, unofficialslaughterhouses and commercialization of meat products without previousdiagnosis of this parasite. In order to determine another method to controland reduce the impact of this disease, the effectiveness to inactivateTrichinella muscle larvae (ML) with irradiation is put under study. InArgentina the present species of Trichinella found are T. spiralis, T.pseudospiralis, T. britovi, and T. patagoniensis. This last specie mentioned wasonly found in mountain cougars in this country and, limited information of thisspecie is known. For this reason, this study aims to determine thesusceptibility, serological response, larvae distribution and irradiation effectsof wild boars infected with T. patagoniensis, T. spiralis and T. pseudospiralisand compare them all.2. Materials and methods. 2.1. Experimental design. Eighteen wildboars (Sus scrofa), 60 days of age, were used. Each wild boar was inoculatedper os with 20000 larvae using a stomach tube. The genotypes employed wereT. patagoniensis (ISS2311, from a mountain cougar), T. pseudospiralis (Krivokapich et al., 2015 from a domestic pig), and T. spiralis (ISS1097, hybridpig, Landrace x Yorkshire). The parasites were maintained in CF1 mice, andrecovered by artificial digestion. Each Trichinella genotype was inoculatedinto 5 wild boars, and additionally, three animals served as uninfected control.The animals were sacrificed 19 weeks post inoculation (pi).2.2. Larval distribution. To determine larval distribution from eachwild boar, 9 muscles or muscle groups were analysed by artificial digestion.Twenty grams of muscle samples were used from: tongue, masseters, bostonbutt, oesophagus muscle, diaphragm, intercostal muscles, tenderloin, upperforeleg and upper hindlimb. For wild boars inoculated with T. patagoniensis,100 gram samples were used. All muscles were freed from fascia and tendons,and digested using artificial digestion (Gamble et al., 2000). Recovered larvaeof each muscle sample were expressed as larvae per gram (lpg).2.3. Serology. Blood samples were weekly collected with EDTA, byjugular venepuncture. This procedure was done at weeks 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 11, 13, 15, and 19 pi. The serums were stored at -80° C until used. Serumsamples were evaluated using the ELISA Kit, PrioCHECK Trichinella Ab.2.4. Muscle Juice. After euthanasia, tissue samples from tongue,diaphragm, upper foreleg and upper hindlimb were collected in conicalcontainers and frozen at -20°C for 24 hours in order to obtain the musclejuices. Afterwards the samples were let to thaw at 4°C for 18-24 h. Musclejuice samples were maintained frozen at -20°C until immunoassay wasperformed using an ELISA Kit, PrioCHECK Trichinella Ab.2.5. Irradiation. Wild boar infected meat with T. spiralis and T.pseudospiralis were used to determine the effectiveness of irradiation toinactivate muscle larvae. Two hundred and fifty grams of muscle samples ofintercostal muscles, boston butt and upper foreleg were used. These werevacuum packed and sent for irradiation treatment at the Atomic Centre ofEzeiza (CNEA ? Argentine National Commission of Atomic Energy) with analanine dosimeter with a minimum and maximum dose of 0.32 ? 0.41 kGy.From each treated sample 20 g of its center were taken 24 h, 7, 14 and 21 dayspost-irradiation and artificially digested so as to obtain muscle larvae (L1)which were afterwards inoculated in 72 CF1 mice. All mice were inoculatedper os with 300 L1, and 42 days pi were sacrificed and their carcasses wereindividually digested. Furthermore, three mice were inoculated with 600 L1,obtained from the irradiated and digested muscle samples, to recover and count adult worms. They were sacrificed 72 h pi, and their intestine wasremoved, longitudinally opened, and cut in smaller pieces of 5 cm. This wasplaced in 50 ml Falcon tubes with 0.9% NaCl saline solution, to incubate at37°C for 5 h.2.6 Statistical Analysis. Muscle larvae recovery was analysed byKruskal Wallis analysis of variance. ELISA readings from sera and muscle juicewere compared using the Spearman rank correlation test. A repeatedmeasures design in time was applied for the evaluation of the antibodykinetics with the three Trichinella species (p-value