These days, in Europe, trichinellosis is rarely reported in association with the consumption of pork from conventionally raised pigs [1]. An EU regulation [2] directs inspection of T. spiralis in each pig carcass at slaughter by direct parasitological methods. This regulation also states that serological tests may be implemented as a supplement for monitoring purposes.

Unlike T. spiralis, no such regulations exist for T. gondii, although the prevalence of this parasite in pigs is higher and health consequences of toxoplasmosis can be, like those of trichinellosis, rather serious. For example, in a Dutch survey in 2004, T. gondii infection was found in 2.6% of the studied pigs [3], while in that year none of the over 13 million slaughtered pigs were found T. spiralis positive [4].

Consumption of raw or undercooked T. gondii infected pork may cause toxoplasmosis in humans. Obviously, determination of the T. gondii status of the meat producing pigs, subsequently followed by precautionary methods, like freezing of pork to kill the parasite or altogether removal of this meat from the food chain, could contribute to fewer infections in humans. A Dutch study which assessed the epidemiology and impact of, amongst others, T. gondii infections in humans indicated that this parasite is one of the major contributors of disease through zoonotic transmission [5]. Similarly, an American study indicated that T. gondii in pork ranked second on the list of the zoonotic micro-organisms with the greatest impact on annual disease burden in that country [6] and was only surpassed by Campylobacter in chicken. In a scientifically opinion to the European Food Safety Authority (EFSA) it is recommended that standardized methods should be used on T. gondii pre-harvest monitoring of, amongst others, pigs [7].

Like T. spiralis, the T. gondii infection status of animals can be examined by serological tests in order to produce T. gondii controlled pork. Testing serum samples of finisher pigs requires an automated and easy to perform test method with a high sensitivity (Se) and specificity (Sp). Enzyme-linked immunosorbent assays (ELISAs) are such test methods which are commonly used.

Bead-based assays (BBA) are a new dimension in the determination of specific antibody responses. The test is performed on beads which are available in different sizes and levels. During flow cytometric analysis individual beads are distinguishable by size and intrinsic fluorescence intensity level. The bead surface is carboxylate modified, which allows covalent coupling of protein. The great advantage of these tests over ELISA is the possibility of simultaneous detection of specific responses against multiple antigens per single serum sample. More specifically, by individual coupling of antigens to specific bead levels, and combination of these bead levels per test sample, a multitude of specific responses can be determined simultaneously per sample. Furthermore, by the use of a non-coupled bead, non-specific binding (NSB) can be monitored and corrected for. The use of T. gondii and T. spiralis antigens on two bead levels in a combined bead-based test to determine the serological status of swine would provide a new innovative assay which could be used as an alternative to ELISA in a T. gondii and T. spiralis monitoring system.

The results of T. gondii and T. spiralis negative field sera illustrate that the response of the reference beads could not directly be used as a measure for non-specific binding. The log linear relations between responses of reference beads and T. gondii or T. spiralis beads are depicted in Figures ​Figures11 and ​and2,2, and the relations were expressed as:

The slopes of the two regression lines indicate that T. gondii beads are subject to less NSB as compared to T. spiralis beads. This finding may be explained by, for example, variable concentration of antigens on the bead, differences in antigen molecule structures and orientation on the bead surface and/or the affinity between non-specific antibodies and the unoccupied bead surface or coupled antigens. A variety in antigen composition between biologically produced batches can therefore be of importance in relation to NSB. To test whether the correction factor to calculate bead correlated NSB is stable between batches of antigen, further evaluation is necessary. Subtraction of uncorrected responses of reference beads, according to formula 1, would lead to an underestimation or overestimation of NSB for responses below and above 53,480 and 183,110 for T. gondii and T. spiralis, respectively.

Results of ROC calculations, presented in Table ​Table2,2, showed that the AUC, a measure of agreement between specific responses and the experimental infection status of the animals, of all indirect tests ranged between 0.837 and 0.930 for T. gondii and 0.855 and 0.879 for T. spiralis. These values indicate that there is a good relation between the responses of all indirect tests and the infection status of the animals.

A perfect test is a test in which the responses correspond 100%, i.e. an AUC of 1.000, with the values of the test to which it is compared. The imperfect AUC values (< 1.000) found in our study (Table ​(Table3)3) can partly be explained by a late immunological development of antibodies, which is associated to the time course of parasite antigen expression and the immune response of infected animals. Evidence from earlier studies showed that muscle larvae, depending on the infection dose, can be found in pork by digestion or trichinoscopy as early as 17 days p.i. [13]. Other studies showed that T. spiralis ES could be measured within the developing muscle larvae and its cuticular surface as early as 14 days p.i. [14] and in the surrounding tissue around 15 days p.i. [15]. Consequently, the response time, i.e. the time of development of antibodies against the antigen used in the indirect T. spiralis assays, is affected by this late production. Porcine IgG antibodies against ES are developed approximately 3 to 4 weeks after infection with 5,000 muscle larvae [16,17]. Porcine IgG antibodies against T. gondii tachyzoites are produced much earlier in time and can be detectable after one to two weeks of infection [18]. In our study, the sera used for ROC calculations originated from animals which were collected on a weekly basis [8]. Samples drawn 5 days after T. gondii inoculation and 5, 12 and 19 days after T. spiralis inoculation would produce a false negative result when compared to the experimental infection status, resulting in lower AUC values. Calculations of ROC curves without these sera resulted in notably higher AUC values of 0.995, 0.999, 0.998 and 0.999 for BBA-TOX, E3-TOX, BBA-TRI and E-TRI, respectively (Figures ​(Figures33 and ​and44).

According to data of test performances and inter test agreement, presented in Table ​Table3,3, the T. gondii and T. spiralis bead assays agree excellently with their respective highest scoring tests, i.e. with the E-TOX3 and E-TRI, respectively. Marginal homogeneity by McNemars test, a test which determines the equality between positive and negative test proportions of one test compared to the other, indicated that there is a balance between BBA-TOX and E-TOX3.

The potential use of the bead based test is prevention of T. gondii and/or T. spiralis infections in pork to enter the human food chain. Nonetheless, direct parasitological testing, like T. spiralis artificial digestion, are more reliable methods to test for present infection in meat. Existing T. gondii direct tests are either laborious, e.g. due to the need of pathogen extraction in PCR [19], or are undesirable because of utilization of experimental animals, like in cat and mice bioassays [20], to determine the infectious status of meat. In the case of T. gondii infections, serological testing is the next best option to perform on large scale. Unfortunately, due to the time window between infection and development of specific antibody responses, serological tests are less reliable for detection on individual scale; however, they can be used for monitoring purposes on herd level [21].

To prevent human T. gondii and T. spiralis infections through consumption of infected pork by serological monitoring of pig herds, a high sensitivity of 99% [22], and an approximation of AP to TP (Table ​(Table4)4) is desired. None of the assays used for this paper met this requirement. However, when ROC calculations were restricted to serum samples in which antibody responses were to be expected, as was described above, the sensitivity was 97% and 99% for BBA-TOX and BBA-TRI, respectively (data not presented). Subsequent calculations for true and apparent prevalence resulted in an overall T. gondii TP of 52.7% and AP's of 51.5% and 53.3% for BBA and ELISA, respectively. The overall TP of T. spiralis was 44.4% while the APs were 44.1% and 44.3% for BBA and ELISA, respectively (data not presented). These data would indicate that the combined bead-based assay is applicable for serological monitoring purposes.