The Use of Recombinant 31 kDa Antigens of Trichinella spiralis for Serodiagnosis of Experimental Trichinellosis.

BACKGROUND: We have previously reported that a 31 kDa protein was screened from the excretory-secretory (ES) proteins of Tichinella spiralis muscle larvae (ML) by immunoproteomics using early infection sera, and the gene encoding a 31 kDa protein from T. spiralis was cloned and expressed in an E. coli expression system. In this study, the recombinant 31 kDa antigens were used for detection of anti-Trichinella antibodies in serum of experimentally infected mice by ELISA.
METHODS: Anti-Trichinella IgG antibodies in sera of mice infected with Trichinella were assayed by ELISA with recombinant 31 kDa antigens, and its sensitivity and specificity were compared with ELISA with ES antigen.
RESULTS: The sensitivity and specificity of ELISA with recombinant antigens was 96.67% (29/30) and 96.87% (62/64), compared with 100% (30/30) and 98.44% (63/64) of ELISA with ES antigens was (P>0.05). In heavily, moderately and lightly infected mice (500, 300 and 100 larvae/mouse), anti-Trichinella antibodies were firstly detected by ELISA with recombinant antigens at 8, 12 and 14 dpi, respectively; then increased rapidly with a detection rate of 100% respectively at 28, 22 and 30 dpi. While the antibodies were firstly detected by ELISA with ES antigens at 10, 8 and 10 dpi, respectively, the antibody positive rate reached 100% at 14, 12 and 22 dpi, respectively.
CONCLUSION: The recombinant 31 kDa antigens of T. spirali had a good sensitivity and specificity for detecting anti-Trichinella antibodies and might be the potential diagnostic antigen for trichinellosis.

Introduction

Trichinellosis is an important zoonotic disease caused by the nematode parasite Trichinella. The parasite is infective to a wide range of hosts including bears, mice, rat, swine, horses and humans (1, 2). Human trichinellosis has been documented in 55 countries of the world, and is considered as an emerging/re-emerging disease (3, 4). From 2004 to 2009, 15 outbreaks of human trichinellosis, consisting of 1387 cases and 4 deaths, were reported in China (5). Therefore, trichinellosis is a major food-borne zoonosis with health, social, and economic impacts in China.

However, the diagnosis of trichinellosis is rather difficult, because its clinical manifestations are nonspecific. ELISA using excretory–secretory (ES) antigens of T. spiralis muscle larvae is the most commonly used serological method for diagnosis of trichinellosis (6, 7). But, the main disadvantage of detection of anti-Trichinella antibodies is the occurrence of a high rate of false negative results during the early stage of infection, and the cross-reaction between T. spiralis ES antigens and sera of patients with other parasitic diseases (e.g., paragonimiasis, schistosomiasis, clornorchiasis, cysticercosis, anisakiasis and so on) (8, 9). The IgG specific for Trichinella are not positive in pig and mice infected experimentally until 3–4 weeks after infection (10–12). When the synthetic tyvelose antigens were used in serodiagnosis of trichinellosis, the cross-reaction may occur with cases of anisakiasis, schistosomiasis, etc. (13, 14). Hence, there is an urgent need to develop the new specific antigens for serodiagnosis of trichinellosis.

A different approach for preparing diagnostic antigens was expression of T. spiralis antigens in heterologous systems such as a 53 kDa protein (15, 16), 49 kDa protein (17), 35.5 kDa protein (18) and 21 kDa protein (19). The recombinant Trichinella antigens had a low sensitivity or cross reaction with the sera of patients with paragonimiasis, cysticercosis and echinococcosis (16, 18, 19).

In our previous studies, two-dimensional electrophoresis (2-DE) and Western blot combined with MALDI-TOF/TOF-MS were used to screen the diagnostic antigens from the excretory–secretory (ES) proteins of T. spiralis muscle larvae by sera of infected mice at 18 days post infection (dpi), and a 31 kDa protein of T. spiralis (GenBank Accession No. AAA20539) was identified and found to have the obvious immunogenicity (20–22). The 31 kDa protein belongs to the trypsin-like serine protease superfamily and may be involved in a large number of processes and play pivotal roles of the developmental process of Trichinella larvae. The 31 kDa protein of T. spiralis was cloned and expressed in an E. coli expression system (23).

The aim of this study was to detect anti-Trichinella IgG antibodies in sera of mice infected with Trichinella by ELISA with recombinant 31 kDa antigens, and its sensitivity and specificity were compared with ELISA with ES antigen.

Materials and Methods

Parasites and experimental animals

T. spiralis isolate (T1, ISS534) used in this study was obtained from a domestic pig in Nanyang City of Henan Province, China. The reference Trichinella isolates used in this study were T. nativa (T2, ISS10), T. britovi (T3, ISS100), T. pseudospiralis (T4, ISS13) and T. nelsoni (T7, ISS29), obtained from International Trichinella Reference Centre (ITRC; Rome, Italy). All of the Trichinella isolates were maintained by serial passages in Kunming mice in our laboratory. Specific pathogen free (SPF) female BALB/c mice aged 6 weeks were purchased from the Experimental Animal Center of Henan province (Zhengzhou, China).

All procedures of animal experiment of this study were approved by the Life Science Ethics Committee of Zhengzhou University.

Serum samples

Mouse infection sera were obtained from BALB/c mice infected with 300 larvae of T. spiralis, T. nativa, T. britovi, T. pseudospiralis and T. nelsoni at 42 dpi. Serum samples of mice infected with three spargana of Spirometra mansoni were collected at 30 dpi in our department. Serum samples of mice infected with Toxoplasma gondii were gifted by Prof. GR Yin of Shanxi Medical University.

Thirty female BALB/c mice were randomly divided into 3 groups (10 mice/group): heavily infected group (500 larvae/mouse), moderate infected group (300 larvae/mouse), and lightly infected group (100 larvae/mouse). The mice were orally inoculated with of T. spiralis ML, and about 50 μl of tail blood was collected on alternate days during 2–42 dpi (24). All of the serum samples had been stored at −80 °C until used.

Collection of muscle larvae and preparation of ES antigens

T. spiralis muscle larvae were recovered from the infected mice at 42 days post-infection (dpi) by artificial digestion as described previously (25, 26). The ES antigens of the muscle larvae were prepared as previously described (27, 28). Briefly, after being washed thoroughly in sterile saline and serum-free RPMI-1640 medium supplemented with 100 U penicillin/ml and 100 μg streptomycin/ml, the larvae were incubated in the same medium at 5 000 worms/ml for 18 h at 37 °C in 5% CO2. After incubation, the media containing the ES proteins were poured into 50-ml conical tubes and the larvae were allowed to settle for 20 min. The supernatant containing the ES products was filtered through a 0.2 μm membrane. The ES products were dialyzed and then lyophilized by a vacuum concentration and freeze-drying (Heto Mxi-Dry-Lyo, Denmark). The protein concentration (2.5 μg/ml) was determined by the Bradford assay (29).

The recombinant 31 kDa antigens of T. spiralis

The recombinant 31 kDa antigens of T. spiralis were expressed in an E. coli expression system and characterized in our laboratory. The purified fusion protein consisted of the 31 kDa proteins and the N-terminal maltose-binding protein. The immune serum against recombinant 31 kDa antigens recognized the native 31 kDa protein by Western blotting of muscle larval crude or ES antigens. An immunolocalization analysis identified 31 kDa proteins in the cuticle and stichocytes of the parasite, demonstrating the 31 kDa antigens came from the ES products of T. spiralis muscle larvae (23).

ELISA for detection of anti-Trichinella antibodies

ELISA was performed as previously described (23, 30). In brief, 96-well ELISA plates (Corning, USA) were coated with purified recombinant antigens (2.5 μg/ml) or ES antigens (2.5 μg/ml) in 100 μl of bicarbonate buffer (pH 9.6) overnight at 4°C. After being blocked in PBS-0.1% Tween 20 (PBST) containing 5% skimmed milk for 2 h at 37°C, the following reagents were sequentially added and incubated for 1 h at 37°C: (1) mouse sera diluted at 1:100 in PBST, and (2) HRP-conjugated anti-mouse IgG (Sigma, USA) diluted at 1: 5 000. The reactions were detected by addition of the substrate o-phenylenediamine dihydrochloride (OPD; Sigma, USA) plus H2O2 and stopped with 50 μl/well of 2M H2SO4. Optical density (OD) values at 490 nm were measured with a microplate reader (TECAN, Austria). All samples were run in duplicate. The ratio <2.1 of samples to be tested/negative sample (OD values of the samples to be tested divided by OD of the negative, S/N < 2.1) were regarded as negative, and S/N ≥2.1 as positive (31). The cut-off values of ELISA with recombinant antigens and ELISA with ES antigens for detection of mouse sera were 0.26 and 0.21, respectively.

Statistical analysis

All statistical analyses of data were done with SPSS for Windows, version 17.0 (SPSS Inc., Chicago, IL). Chi-square test and repeated measures of analysis of variance (ANOVA) were used to determine the difference between antibody levels at various periods post-infection, the difference between the heavily, moderately, and lightly infected group, and the difference at different time points in the same group with respect to the detection rate and serum level antibody. P < 0.05 was considered statistically significant.

Results

Detection of specific antibodies in mice infected with Trichinella and other parasites

The sensitivity of both ELISA with recombinant antigens and ELISA with ES antigens for detecting anti-Trichinella antibodies was 96.67% (29/30) and 100% (30/30) (χ=1.017, P>0.05) when the sera of mice experimentally infected with T. spiralis (Table 1). No cross-reactions of both ELISA were observed with sera of mice infected T. gondii and normal mice. When the serum samples of mice infected with S. mansoni were determined, the cross-reaction rate was 7.14% (2/28) by ELISA with recombinant antigens ELISA and 3.57% (1/28) by ELISA with ES antigens (χ=0.352, P>0.05). The specificity of both ELISA with recombinant antigens and ELISA with ES antigens for detecting anti-Trichinella antibodies was 96.87% (62/64) and 98.44% (63/64) (χ=0.341, P>0.05).

Table 1:

Detection of serum anti-Trichinella IgG antibodies in mice infected with other parasites by ELISA

Sera of mice infected with	No. of serum samples	ELISA with recombinant antigens		ELISA with ES antigens
		OD value	No. of positive serum samples (%)	OD value	No. of positive serum samples (%)
Trichinella spiralis	30	0.50±0.11	29 (96.67)	0.80±0.05	30 (100)
Spirometra mansoni	28	0.17±0.05	2 (7.14)	0.16±0.04	1 (3.57)
Toxoplasma gondii	6	0.15±0.02	0	0.14±0.01	0
Normal mice	30	0.12±0.01	0	0.10±0.02	0

The specific serum anti-Trichinella IgG antibodies in mice infected with other species of Trichinella were also assayed by ELISA, and the results are shown in Table 2. The detection rate of anti-Trichinella antibodies by ELISA with recombinant antigens and ELISA with ES antigens was no significant difference among the mice infected with T. spiralis, T. nativa, T. britovi and T. nelsoni (χT1=1.017, χT2=3.158, χT3=1.034, χ2T7=1.32, P>0.05). However, the antibody detection rate (40.91%) of mice infected with T. pseudospiralis by ELISA with recombinant antigens was obviously lower than 90.91% by ELISA with ES antigens (χ=12.239, P<0.05).

Table 2:

Detection of serum anti-Trichinella IgG antibodies in mice infected with other species of Trichinella by ELISA

Sera of mice infected with	No. of serum samples	ELISA with recombinant antigens		ELISA with ES antigens
		OD value	No. of positive serum samples (%)	OD value	No. of positive serum samples (%)
T. spiralis	30	0.50±0.11	29 (96.67)	0.80±0.05	30 (100)
T. nativa	30	0.43±0.15	27 (90.00)	0.72±0.13	30 (100)
T. britovi	15	0.40±0.17	14 (93.33)	0.79±0.08	15 (100)
T. pseudospiralis	22	0.22±0.16	9 (40.91)	0.52±0.21	20 (90.91)
T. nelsoni	16	0.40±0.16	15 (93.75)	0.76±0.08	16 (100)

Serum anti-Trichinella antibody dynamics in mice experimentally infected with different-level infections

The levels of anti-Trichinella antibodies in sera from 3 groups of infected mice at different time points after infection were determined by ELISA with recombinant antigens and ELISA with ES antigens (Fig. 1). In heavily, moderately and lightly infected mice, anti-Trichinella antibodies were firstly detected by ELISA with recombinant antigens at 8, 12 and 14 dpi, respectively; the antibody positive rate reached 100% at 28, 22 and 30 dpi, respectively.

Fig. 1:

Kinetics of anti-Trichinella antibodies in sera of heavily (infected with 500 T. spiralis larvae), moderately (infected with 300 T. spiralis larvae), and lightly (infected with 100 T. spiralis larvae) infected mice. The anti-Trichinella IgG antibodies were detected by ELISA with recombinant antigens (A) and ELISA with ES antigens (B), and cut-off value is represented by the dotted line

While the antibodies were firstly detected by ELISA with ES antigens at 10, 8 and 10 dpi, respectively; the antibody positive rate reached 100% at 14, 12 and 22 dpi, respectively (Fig. 2). The anti-Trichinella antibody levels in 3 groups of infected mice increased rapidly at 8 dpi and reached their peak at 32 dpi by both ELISA. When ELISA with recombinant antigens was used, the antibody levels in 3 groups of infected mice was not significant different (F=2.049, P>0.05), but the antibody levels at different time points after infection was statistically significant (F=219.924, P<0.05); there was interaction between detection time and inoculation dose (F=3.311, P<0.05).

Fig. 2:

Comparison of detection rate of anti-Trichinella IgG antibodies in sera from heavily (A), moderately (B), and lightly (C) infected with T. spiralis at different time intervals post-infection by ELISA with recombinant antigens (▪) and ELISA with ES anigens (□)

Discussion

The ES antigens of T. spiralis muscle larvae is the most commonly used serodiagnostic antigens for trichinellosis recommended by the International Commission on Trichinellosis (ICT) (11). However, the reparation of ES antigens requires collection of muscle larvae from experimentally infected laboratory animals, which is practically inconvenient in terms of cost, labor, and time. Recombinant proteins are a good alternative to the ES antigens as they can be produced easily in large amounts by using a bacterial expression system and can be used as an antigen in a sensitive, specific, and standardized ELISA for serodiagnosis of trichinellosis. Therefore, development of sensitive and specific recombinant Trichinella antigens will improve the serodiagnosis of this disease. In the present study, the sensitivity and specificity of ELISA with recombinant antigens for detecting anti-Trichinella antibodies in serum of experimentally infected mice was 96.67% (29/30) and 96.87% (62/64), respectively; the results were similar with that of ELISA with ES antigens, suggesting that the recombinant 31 kDa antigens of T. spiralis could be the alter-native antigens of ES antigens for diagnosis of trichinellosis.

Moreover, there was no significant difference in the antibody detection rates in sera of mice infected with T. nativa, T. britovi, and T. nelsoni by ELISA with recombinant antigens and ELISA with ES antigens (P>0.05). But, the antibody detection rate (40.91%) of mice infected with T. pseudospiralis by ELISA with recombinant antigens was obviously lower than 90.91% by ELISA with ES antigens (P<0.05). The 31 kDa proteins might expressed at high levels in encapsulated species (such as T. spiralis, T. nativa, T. britovi, and T. nelsoni), but at a low level in non-encapsulated species (T. pseudospiralis) (32, 33). The results suggested that the most epitopes of 31 kDa antigens recognized by mouse infection sera were common to the encapsulated species of Trichinella (34).

Our ELISA results also showed the antigenic epitopes of 31 kDa proteins were recognized by infection sera at 8–14 dpi, suggesting that the 31 kDa proteins might be secreted by the parasite into the peripheral blood circulation of host at early infection stage, induced an early antibody response continuing to the muscle larval stage. In the present study, the specific anti-Trichinella IgG antibodies were firstly detected at 8, 12 and 14 dpi in heavily, moderately and lightly infected mice, and continued to grow up to the end of this experiment (42 dpi). Similarly, other some recombinant proteins (such as the recombinant 53 kDa protein, rTs21, rTsDAF-21) were firstly recognized by the sera of mice infected with T. spiralis at 8–14 dpi (19, 35, 36). However, the levels of anti-Trichinella IgG antibodies did not increased proportionally with the different infected doses. Similar observation regarding the kinetics of anti-T. spiralis newborn larva antibodies and the infection dose effects has also been observed in experimentally infected rats (37).

Conclusion

The recombinant 31-kDa antigens of T. spirali had a good sensitivity and specificity for detecting anti-Trichinella antibodies in serum of experimentally infected mice, and might be a potential diagnostic antigen for trichinellosis.