Universidad de Buenos Aires Facultad de Ciencias Veterinarias ...
Trichinellosis is a zoonotic disease transmitted by the consumption of foods infected with Trichinella spp. In Argentina the disease is endemic and mainly transmitted by pigs, reemerging in the period 1990/2005. The diagnosis can be made through direct and indirect methods. The artificial digestion...
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| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Ciencias Veterinarias
2024
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7380 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7380.dir/7380.PDF |
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| Sumario: | Trichinellosis is a zoonotic disease transmitted by the consumption of foods infected with Trichinella spp. In Argentina the disease is endemic and mainly transmitted by pigs, reemerging in the period 1990/2005. The diagnosis can be made through direct and indirect methods. The artificial digestion and molecular techniques are the method of \nchoice for the direct observation of the parasite. While the indirect ones detect the host's \nresponse to the parasite, particularly in pigs, the ELISA technique is used and it is confirmed by means of a Western Blot. These methodologies are carried out either to develop prevalence studies or direct diagnoses of this disease. The aim of this work was to study the acute and chronic infection of the disease, the species susceptibility and the \nimmune response of wild boars experimentally infected with T. patagoniensis and T.\npseudospiralis. As well as to evaluate the detection capacity of newborn larvae during the window period in sera from wild boars and pigs infected with T. patagoniensis, T. \npseudospiralis and T. spiralis by means of the real-time PCR technique. For this study, \n17 wild boars (Sus scrofa) and 6 pigs (Sus scrofa domestica) were used. The animals were inoculated with 20,000 larvae of T. patagoniensis (5 wild boars and 1 pig), with \n20,000 larvae of T. pseudospiralis (5 wild boars and 2 pigs), and with 20,000 larvae of T. spiralis (4 wild boars and 2 pigs). These animals were divided into groups based on \ntheir animal species and the Trichinella species inoculated. The rest of the animals were kept as control (3 wild boars and 1 pig). These animals were checked periodically, and during the acute phase of the disease they were weighed weekly. Blood samples were taken weekly throughout the study. These samples were used for various purposes. On one hand, this was used to evaluate the relative eosinophil count in wild boars infected with T. patagoniensis and T. pseudospiralis, by means of blood smears. Moreover, the \nsera obtained from the blood samples of wild boars infected with T. patagoniensis and \nT. pseudospiralis was also used to determine the antibody titer using the ELISA technique, applying a commercial kit named PrioCHECK Trichinella Ab. On the other hand, the sera of all infected animals was used to evaluate the presence of newborn larvae (NBL) using the real-time PCR technique. This was measured in wild boars serum at 7- and 14-days post infection (d.p.i.), in pigs infected with T. spiralis and T. \npseudospiralis at 2, 5, 9, 12 and 15 d.p.i., and in pigs infected with T. patagoniensis at \n2, 6, 10, 12 and 15 d.p.i. In order to evaluate the distribution pattern of the muscle larvae, \nsamples of muscles of parasitological and commercial interest were taken during the \nnecropsy of wild boars infected with T. patagoniensis and T. pseudospiralis. These \nsamples were: tongue, diaphragm, masseter, intercostals, muscular portion of the esophagus, upper forelegs, upper hind limbs, pork shoulders and sirloin. These were analyzed using the artificial digestion technique. At the same time, samples of pork shoulders, upper foreleg and upper hind limbs were also taken from wild boars infected with T. pseudospiralis to evaluate the tolerance of muscle larvae (ML) to freezing. These muscles were subjected to temperatures of -18ºC for 14 days. The sampling dates were 2, 4, 7, 9, 11 and 14 days, and on each date a sample of each muscle was digested, by artificial digestion, in order to recover the ML there, and then inoculate them in BALB/c mice to determine the Reproductive Capacity Index (ICR). No animal presented clinical signs related to this disease. During the acute phase, no differences in weight gain were evident between infected wild boars and controls. An increase in the relative eosinophil count was seen immediately after infection in wild boars infected with T. pseudospiralis (1 week p.i.) and at week 2 p.i. in wild boars infected with T. patagoniensis. Regarding antibody dynamics, wild boars inoculated with T. patagoniensis sero-converted between \n2 4 weeks p.i., while wild boars inoculated with T. pseudospiralis at 2 weeks p.i. All animals, except controls, remained above the cut-off value throughout the experience (19 weeks). Statistically significant differences were observed in optical density (OD) values between wild boars inoculated with T. pseudospiralis and those inoculated with T. patagoniensis at 2 weeks p.i. (P<0.050). The early detection of NBL showed variable results depending on the animal species and the Trichinella species under study. \nSamples taken at 7 d.p.i. of wild boars infected with T. spiralis were positive when \nanalyzed with the Rep primer. These positive samples presented a Ct range of 29.26 ± \n0.16 for one sample and up to 33.37 ± 0.45 for another. and the average Ct (of the \npositive samples) was 31.91 ± 1.70. At 14 d.p.i. NBL DNA was not detected in the \nanalyzed sera. Regarding pigs infected with T. spiralis, the Rep primer failed to detect circulating DNA in serum on any sampled date. The 18S primer failed to detect NBL DNA in wild boars infected with T. pseudospiralis or T. patagoniensis on the sampled dates (7 \nand 14 d.p.i.). With this primer, DNA was only detected in a sample at 7 d.p.i. in a single \nwild boar infected with T. spiralis when 1 l of DNA was used, when increasing to 5 l of DNA the other 3 samples were also detected as positive. The sample made with 1 l of DNA presented an average Ct value of 37.32 ± 0.45, while the samples analyzed with 5 l presented a Ct range of 35.13 ± 1.02 to 36.80 ± 0 .29, with an average Ct of 36.21 ± 1.02. The muscle distribution of the larvae of T. pseudospiralis and T. patagoniensis gave the following results. T. patagoniensis had a larval density considerably lower than T. pseudospiralis. Both Trichinella species showed a predilection mainly for the tongue and diaphragm. Followed by the upper forelegs for T. patagoniensis, and the sirloins and \npork shoulders for T. pseudospiralis. The highest larval loads were: for T. pseudospiralis\n134.3 larvae per gram (lpg) (in the tongue and intercostal muscles) and for T. \npatagoniensis 0.087 lpg (in the tongue). The T. pseudospiralis larvae recovered from the \nmuscles subjected to freezing treatment (-18 ºC) remained viable until 48 hours after being placed in the proposed medium. From day 4 onwards, the RCI value was equal to 0 in all muscle groups studied. These results allow us to evaluate how T. patagoniensis behaves when it infects wild boars. This study allows us to understand how these species develop when they affect wild boars, as well as allowing us to explore their biological capabilities. Using various diagnostic methods not only allowed us to delve into how this disease develops, but also allowed us to begin to build new diagnostic strategies to reach its early diagnosis and treatment or even be able to prevent this disease |
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