[The usefulness of ELISA test for early serological detection of Trichinella spp. infection in pigs].Wiad Parazytol. 2007; 53(2):149-51.WP
Trichinellosis is a parasitic zoonosis transmitted to humans through consumption of raw or undercooked meat from animals infected with nematodes of the Trichinella genus. Every year seropositive cases are found among the human population and thus trichinellosis still remains an epidemiologically important disease. The first step of the study was the optimization of a new ELISA method enabling an early and specific serological diagnosis of trichinellosis in pigs and wild boars using excretory-secretory (ES) antigens obtained from in vitro cultures of L1 T. spiralis. Serum samples were assayed for anti-T. spiralis IgG antibodies using the new ELISA protocol and a reference test--Standard manufactured by Institut Pourquier. The optimization involved the selection of suitable plates for antigen coating, dilution of sera and antibodies and their time of incubation. On the basis of the optimization a new ELISA procedure for the detection of IgG and IgM against T. spiralis was elaborated. Conventional, Iberian pigs and SPF (Specific Pathogen Free) pigs were infected with 200, 1000 and 20,000 muscle larvae of T. spiralis. Serum samples were obtained at 5 and 1 dbi (day before infection), and 5, 10, 15, 20, 25, 30, 40, 50, 60 dpi (day post infection) and screened for specific IgG antibodies against excretory-secretory L1 T. spiralis antigens. Serum samples were obtained from the EU project Trichiporse: "Safe pork and horse meat on EU markets: early and unbiased diagnostic tests for Trichinella". Field samples of conventional pigs (1474) and wild boars (1784) were obtained from slaughter houses in different parts of Poland. Pigs were examined for the presence of Trichinella spp. using the artificial digestion method. Only four pigs were naturally infected with T. spiralis, the remaining were Trichinella larvae free. ELISA was used to examine IgG levels against L1 T. spiralis in pig and wild boar sera. The usefulness of ELISA for anti-IgG detection in pigs is usually limited by the nature of the antigen. The antigens were prepared in different laboratories: in Germany--Ag ES L1 T. spiralis (N), Italy--Ag ES L1 T. spiralis (W)) and in Poland--Ag ES L1 T. spiralis. Cut-off values for ELISA along with the estimated sensitivity and specificity were calculated using different methods: S/P%, M+3SD and ROC (Receiver Operating Characteristic). In SPF and Iberian pigs inoculated with 200, 1000 and 20,000 L1 T. spiralis, specific antibodies were detected 40, 30 and 25 dpi, respectively, with the use of the Standard (reference test). The analysis of the two ELISA procedures demonstrated a high sensitivity and specificity for the newly elaborated test utilizing the Ag ES L1 T. spiralis. In conventional pigs infected with 20,000 L1 T. spiralis specific antibodies were detected from 20 dpi when employing the new protocol. Similar results for the Standard and new ELISA test were obtained for serum samples of conventional pigs infected with 200 and 1000 larvae, which became positive from 40 dpi and 30 dpi, respectively. The results showed that both: the Standard and new protocols were comparable, and based on this, the new test was applied for further research. Results obtained adopting the new protocol with three antigens showed that two of them: Ag ES L1 T. spiralis (W) and Ag ES L1 T. spiralis are similar. The specific IgG antibodies for infective doses of 200 and 1000 larvae for these antigens were detectable 40 and 30 dpi respectively. In pigs infected with the highest dose of T. spiralis larvae IgG antibodies were detectable from 20 dpi when Ag ES L1 T. spiralis was used. These results strongly indicate that in examined pigs, the specific IgG response to T. spiralis infection is dose dependant. Of 1474 examined pig sera only 0.99% gave a positive signal against ES L1 T. spiralis antigen. Of 1784 examined wild boars sera only 0.68 % gave positive results using the new ELISA protocol. ELISA is a useful method for detecting specific IgG antibodies in pigs experimentally infected with different doses of T. spiralis and naturally infected pigs. In pigs the specific IgG response is dose dependant. The Ag ES L1 T. spiralis increases the specifity of the method and reduces false positive results. Simultaneous use of both methods: digestion and ELISA for the diagnosis of Trichinella in naturally infected pigs and wild boars may increase the chances of eliminating meat infected with T. spiralis larvae.