Estudio integral de la trichinellosis silvestre en Extremadura

Abstract

In this research work a comprehensive analysis of wild trichinellosis in Extremadura (Spain) has been carried out. This study can be divided into two large blocks. The first part is constituted by an experimental assay in wild boar, one of the main hosts of Trichinella (not only in Extremadura, but also Spain and even Europe) but still poorly studied in the context of this disease. The second part of this work is composed by an epidemiological study of trichinellosis in our region, that analyzes not only usual (pig, wild boar and fox) but also other potential hosts (birds and wild mammalian carnivores), and which has allowed to perform an epidemiological map of Trichinella in Extremadura and a Combat and Control Plan against this zoonosis. The indirect double-antibody micro-ELISA (Enzyme Linked Immunosorbent Assay) technique has been tested as diagnosis tool for the disease, and its efficacy has been compared to trichinelloscopy and artificial digestion. Western Blot technique has been also conducted as complementary diagnosis technique, as well as histopathological lesions produced by T. spiralis and T. britovi in wild boar. These data allow extending the knowledge for the understanding of the pathogenesis of trichinellosis. Finally, the muscle distribution and parasite load in experimental wild boars have been analyzed, comparing these results with those obtained in the direct diagnosis of positive cases referred by Public Health veterinarians. For experimental study, 40 males and females wild boars from several hunting reserves of Caceres province were employed. Animals were between 2 and 18 months old at the beginning of the experiment; their initial weights ranged from 3.95 kg to 60 kg. These 40 wild boars were grouped into two experimental assays: 22 animals were used for a short experimental infection (euthanized at 125 DPI); and the remaining 18 animals were maintained for a longer infection period and euthanized between 201 and 957 DPI. In the first group (short infection period) four animals were selected as controls free from Trichinella; the remaining 18 animals were divided into two lots and infected with different doses (200, 1,000 and 20,000 L1 per animal) of T. spiralis and T britovi. Blood serum samples were obtained on -5, 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 90, 105 and 125 DPI from each wild boar. In the second group (prolonged infection) two animals were used as controls and the remaining 16 were infected with 10,000 or 20,000 L1 of T. spiralis or T. britovi; blood samples were taken monthly. All animals were euthanized using a captive gun bolt before a complete necropsy was carried out. Parasite infection was determined by trichinelloscopy and artificial digestion in diaphragm muscle samples of each animal. A larvae distribution study was also carried out in diaphragm, tongue base, masseter, upper forelimb, lower forelimb, intercostals, abdomen and upper hindlimb muscles. Control animals were confirmed as negative after digestion of at least 500 g of muscle. A relationship between administered infecting dose and infective larvae per gram obtained could be observed. Wild boars infected with T. spiralis presented higher parasite load than those animals infected with T. britovi. Furthermore, highest larvae densities were found in the diaphragm followed by sublingual and masseter muscles. The lowest larval tropism was observed in the intercostals. More irregular muscle distribution was reported in animals with low parasite loads. An indirect double-antibody micro-ELISA method was used in order to detect circulating anti EBL and ES antibodies in wild boars sera. 96 well Micro-ELISA flat botommed polystyrene plates of 300 �Êl capacity were used. 100 �Êl of 3 �Êg/ml antigen were placed per well and then were incubated at 37oC for 4 hours. Serum was diluted 1/1,000 and conjugate (anti-pig IgG) was diluted 1/25,000. Finally, substrate was added (0.01% TMB in a citrate buffer, also adding 3 �Êl/ml of H2O2). Reaction was stopped at 8- 10 minutes by applying 50 �Êl of H2SO4 3N per well. Optical density obtained in spectrophotometer (450nm) was expressed in percentage of reactivity respect to the positive and negative control in each plate, in order to reduce the variability between the results from different assays. The 90% and 99% confidence limits or �gcut-off�h for each antigen were established employing the results of 1,844 Trichinella spp. negative (by artificial digestion of at least 25 grams of diaphragm) wild boars from Extremadura region hunts, considering three different categories: negative, doubtful and positive. In the first group of experimentally-challenged wild boars (with short infection period) a slight increase in the percentage of reactivity against some antigens could be observed in several animals at 10 DPI, although the first significant increases are evident around day 15-20 PI. In animals infected with 200 L1, in which the muscle parasitism was very low, a delayed antibody production could be observed, with a peak about 40-50 DPI for animals infected with T. britovi and about 90-125 DPI for those infected with T. spiralis. The peak of antibody response could be observed at the same time in both animals with high parasite loads (infected with 20,000 L1 doses) an in those infected with low parasite loads (200 L1), but the percentage of reactivity was higher in the first ones, with 120% observed in some cases. Two different types of kinetics depending on Trichinella species used for challenge could be noticed the wild boar. Thus, in animals infected with T. britovi, a clear peak in the production of antibodies around day 30-50 PI was observed, decreasing later until the end of experiment. In contrast, wild boars infected with T. spiralis presented a constant antibody increase from the first week postinfection until the end of the study. In wild boars with prolonged infection period, the persistence of antibodies in the last days of the experiment is strongly correlated with the individual characteristics of each animal. Thus, antibodies could be detected in animals with calcified larvae, and in the other hand absent humoral response was observed in wild boars that still had viable larvae in the musculature. In these wild boars a mixed antigen using two species of Trichinella was employed in ELISA technique with the purpose of improving in the diagnosis, but false negatives were not properly detected even with the use this antigenic combination. The presence of infection by protozoa (Balantidium coli and Sarcocystis miescheriana), acanthocephala (Macracanthorhynchus hirudinaceus) and nematodes (Metastrongylus spp., Strongylida spp., Ascarops strongylina and Oesophagostomum dentatum) did not affect the final result of the ELISA technique, so no cross reactivity was found. Western Blot was performed using the serum of animals identified as 23, 25, 30, 32, 34, 36, 37 and Control E, employing both crude and excretory-secretory antigens. Besides, some samples of hunted wild boars that presented a negative result by artificial digestion but high reactivity by ELISA were also analyzed by Western Blot technique. 1,000 �Êg of EBL antigen or 600 �Êg of ES antigen for SDS-PAGE were used. Antigens were diluted 1:4 with Sample buffer. Serum samples were diluted 1:8 in TBS-Tw20 3% casein. Anti-pig IgG conjugate was diluted 1/500 in TBS-Tw20 3% casein. Cloronaftol + H2O2 were used as substrate in a solution of methanol and TBS. The reaction was finnally stopped by washing with distilled water. It could be appreciated an evident analogy between this method and the humoral evolution observed in all wild boars by ELISA. This fact demonstrates the reliability of the results obtained by both techniques. Although the there is not a uniform pattern recognition in WB, it could be highlighted a series of bands apparently homologous to the group of bands referenced by other authors as TSL-1 For the Ag EBL T1, apparent molecular weights detected in these bands have been approximately 75, 72, 62, 52 and 47 kDa. On the other hand, apparent molecular weights of 70, 60, 49, 45 and 42 kDa were calculated for these bands by using EBL Ag T3. Given the accuracy of the calibration curve, it is possible that both groups of bands were partially homologous, and the low kDa differences observed were more apparent than real in some cases. Apart from this prominent banding that appears in all wild boars, there were also different bands of heterogeneous development. The use of Ag ES reveals a pattern of lower antigen recognition. At 125 DPI, this group of medium molecular weight bands was detected in most wild boars. Sera from hunted wild boars with high reactivity by ELISA, but negative by 25 g of muscle tissue artificial digestion, reacted against different antigenic bands by WB. The pattern observed in many cases was comparable to that observed in experimentally infected wild boars, suggesting that they may provide a specific response against Trichinella although infection is not detectable by direct methods. In general, the banding obtained in most hunted wild boards had common bands with the antigenic pattern observed in some experimentally infected animals. Histopathological study revealed the lesions derived from Trichinella infection in wild boar. The alterations were mainly observed in skeletal muscle; in the other organs studied (heart, liver, lung, kidney, spleen, lymph node and intestine) they were slightly higher in wild boars infected with T. spiralis. In muscle (diaphragm), inflammatory infiltrates (lymphocytes and plasma cells) were observed around the larvae. This infiltrate is slightly higher around T. britovi, causing the presence of calcifications in these wild boars. In general, cellular response tended to decrease over time. The epidemiological study has been performed on different hosts during 2001- 2010 period. An important part of this study was the searching of Trichinella parasite in foxes. A prevalence of 2.86% in these animals has been detected. For diagnosis purpose, lower forelimb muscles were used, and an average of 25 g per animal was digested. The presence of T. britovi in foxes is higher than in pigs or wild boars, so they are natural reservoir of T. britovi. However, the presence of T. spiralis in this host is increasing and replacing T. britovi. The presence of Trichinella in other carnivores and birds has also been studied. Samples proceeded from "Los Hornos" wildlife recovery center (Sierra de Fuentes, Caceres, Spain). A T. britovi positive stone marten (Martes foina) with 2.72 LPG parasite load in forelimb, and also a tawny owl (Strix aluco) infected with unidentified Trichinella (with a parasite load of 0.898 LPG in pectoral muscle) could be found. Therefore prevalences of 2.63% in wild mammals (excluding the fox) and 0.14% in birds have been detected. In hunted wild boars, 13 positives cases (0.43% of the 3,055 animals tested) were found; T. spiralis was isolated in 76.92% of these cases. Data from all positive cases detected by Regional Health Service official veterinarians had also been taken into account for the establishment of 0.18% prevalence in wild boar, 0.0047% in domestic pigs sacrificed at home and 0.00011% in industrial pigs. T. spiralis was more present in both hosts, being the most common parasite loads between 1 and 10 LPG. Muscles with more larval tropism were the diaphragm, sublingual and masseter. In naturally-infected wild boars high larval tropism in upper hindlimb, upper forelimb and back was detected, being the muscular distribution more irregular in cases with low parasite load. Mixed infections (T1+T3) were only found in wild boars (2.3%). There was an increase of T. spiralis compared to T. britovi, which has been mainly isolated in mountain areas. Most prevalent zones were Valle del Jerte, Sierra de Gata and La Vera, and the higher casuistics were found in Las Villuercas, Los Ibores and Monfrague regions. In all these areas the Combat and Control Plan for this disease must be very strict, involving pork/wild boar final consumers, hunters, hunting managers, farmers, veterinarians, government and researchers. The objective of this plan is the coexistence with the parasite under control levels to ensure food safety.