Anti-Trichinella antibodies detected in chronically infected horses by IFA and Western blot, but not by ELISA

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1 Veterinary Parasitology 132 (2005) Anti-Trichinella antibodies detected in chronically infected horses by IFA and Western blot, but not by ELISA Lj. Sofronic-Milosavljevic a, *, N. Ilic a, M. Djordjevic b, M. Savic b, A. Gruden-Movsesijan a, K. Cuperlovic b, K.D. Murrell c a Institute for the Application of Nuclear Energy (INEP), Banatska 31b, Belgrade, Serbia and Montenegro b Institute of Meat Hygiene and Technology (IMHT), Kacanskog 13, Belgrade, Serbia and Montenegro c Danish Centre for Experimental Parasitology, Royal Veterinary and Agricultural University, Frederiksberg, Denmark Abstract In the Balkan countries, where trichinellosis is a re-emerging zoonosis, it is of great importance to determine Trichinella infection prevalence among the major hosts, including horses. One method for monitoring prevalence is serological surveillance; however, the validity of serological methods in horses is not well understood. The dynamics of anti-trichinella IgG production and circulating excretory/secretory (ES) antigens were investigated in three horses experimentally-infected with Trichinella spiralis. Horses were slaughtered at 32 week post infection (p.i.). Low worm burdens were found in all three animals. Anti- Trichinella IgG was detected up to 32 weeks p.i. by an indirect immunofluorescence assay (IFA) and by Western blot (Wb), but not by ELISA. The ELISA test detected antibodies for only a short period of time (up to 18 weeks p.i. using ES antigen or up to 20 weeks p.i. using tyvelose BSA antigen). The presence of circulating muscle larvae ES antigen in sera of infected horses was observed by dot blot from the 4th week p.i. up to the 32nd week p.i. # 2005 Elsevier B.V. All rights reserved. Keywords: Horses; Trichinellosis; Serology 1. Introduction Serbia, which has suffered a resurgence of trichinellosis over the past decade (Djordjevic et al., 2003), has been reported to be the source of a number of Trichinella infected horses exported to France and Italy (Boireau et al., 2000; Pozio, personal communication). Methods to assess the prevalence and epidemiology of Trichinella infection in live horses * Corresponding author. Tel.: x122. address: sofronic@inep.cp.yu (L. Sofronic-Milosavljevic). would be very useful; however, serological methods have proven unreliable for this purpose. Reasons for poor test performance might include the lack of sensitivity and specificity of the tests themselves and a tendency for horses to down regulate antibody production despite a continued presence of infective larvae (Pozio et al., 2002). There is, therefore, a need for reliable serological methods for ante-mortem surveillance and for a better understanding of horse s immune response to Trichinella infection. Studies involving experimental Trichinella infection in horses was recently conducted in Serbia in /$ see front matter # 2005 Elsevier B.V. All rights reserved. doi: /j.vetpar

2 108 L. Sofronic-Milosavljevic et al. / Veterinary Parasitology 132 (2005) as a part of an epidemiological study on how horses acquire a meat-borne parasite such as Trichinella spiralis (Murrell et al., 2004). This afforded an opportunity to investigate the antibody response of infected horses and to evaluate several serological methods. 2. Materials and methods 2.1. Experimental infection Three adult Yugoslavian domestic mountain breed (DMB) horses (designated as Horses I III, respectively) were infected with T. spiralis by feeding with raw ground meat balls prepared from a naturally infected pig. Each meat ball contained approximately 1100 T. spiralis muscle larvae (ML). Horses were slaughtered 32 weeks after exposure to infection (p.i.) Parasitological analyses Infective ML were recovered from all 3 horses by standard pepsin HCl digestion of 100 g of muscle tissue (Radix lingue) (Gamble et al., 2001). Worm burdens were reported as larvae per gram of muscle sample (lpg) Sera sampling and controls Experimentally-infected horses were bled at 2 week intervals starting from day 0 until the end of experiment. Sera from 50 Trichinella-free horses (no larvae found by artificial digestion of 100 g of tongue muscle) were collected at a commercial slaughterhouse Damjanovic, Mladenovac, SCG. Serum isolated from an experimentally-infected horse, was generously supplied by van Knapen (Faculty of Veterinary Medicine Utrecht, the Netherlands) Indirect immunofluorescence assay (IFA) Histological sections of paraffin embedded, T. spiralis-infected rat tongue were used as antigens (Lalic et al., 1979). A serum sample was considered positive if a bright, apple green light emission was seen both on the cuticle and in the somatic tissues (especially the stichocytes) at a dilution of 1:20 or greater. Of the 50 Trichinella-free horse sera, three exhibited weak fluorescent staining on muscle larvae cuticle and stichocytes at a dilution of 1:20; one serum sample was reactive at a dilution of 1:40 and no sera reacted at a dilution of 1: ELISA using excretory/secretory (ES) or tyvelose BSA antigens The ELISA, using either ES or synthetic tyvelose BSA antigens (kindly supplied by Dr. E. Pozio from Istituto Superiore di Sanità, Rome, Italy), was performed as previously described (Gamble et al., 1983; Pozio et al., 2002). The optical density (OD) index was calculated as follows: OD index = (OD of sample OD of blank control)/(od of negative serum pool OD of blank control). The positive cut off OD index values were calculated as the average OD index + 5 S.D. for the 50 serum samples collected from Trichinella-free horses. The positive cut-off was set at 3.6 for ES and at 2.0 for tyvelose BSA antigens Western blot (Wb) and dot blot analyses Protein components of T. spiralis ES antigens were separated by SDS-PAGE, under reducing conditions, and transferred to a PVDF membrane (Gamble and Graham, 1984). Blots were incubated with positive or negative serum controls and experimental horse serum samples. As an additional control, monoclonal antibody (mab) 7C2C5 (Gamble and Graham, 1984), which recognizes the specific T. spiralis epitope present on the TSL-1 group of antigens (45, 49 and 53 kda), was used. A reaction was considered positive only if the triad of bands identical to that obtained with 7C2C5 mab was visualized. Dot blot tests for circulating T. spiralis ES antigen detection were performed as previously described (Ilic et al., 2004). 3. Results 3.1. Parasitological analyses Artificial digestion of tongue muscle, performed at 32 weeks p.i. revealed the following worm burdens:

3 L. Sofronic-Milosavljevic et al. / Veterinary Parasitology 132 (2005) Horse I, 0.97 lpg; Horse II, lpg; Horse III, 0.81 lpg IFA testing Serum from Horse III showed weak, non-specific reactivity at day 0 using a 1:20 serum dilution where low-density staining was observed on internal structures but not on the cuticle surface. Antibody conversion was detected in all three horses between weeks 2 and 6 p.i. Although the levels of antibody varied in IFA during the course of experiment (Fig. 1A), they remained detectable through the end of the infection period (32 weeks p.i.) ELISA testing The presence of anti-t.spiralis IgG in infected horses was detected by ELISA using ES antigen between weeks 4 and 18 p.i. and by ELISA using tyvelose BSA antigen between weeks 4 and 20 p.i. The OD index values peaked between weeks 4 and 12 p.i. Higher OD index values were observed with ES antigen as compared with tyvelose BSA (Fig. 1B,C). Non-specific reactions with tyvelose BSA in the ELISA test were observed at day 0 with sera from all three infected horses and at week 2 p.i. with serum of Horse III. No non-specific reactivity was observed with Trichinella-free control horse sera. Fig. 1. Detection of anti-trichinella IgG in three experimentally infected horses by IFA (A) and ELISA using ES antigens (B) or ELISA using tyvelose-bsa antigen (C). Fig. 2. Western blot analyses of sera from experimentally infected horses. (A) Lane 1 and 5: 7C2C5 mab; lanes 2 4: Horses I III at day 0; lanes 6 8: Horses I III, week 4 p.i.; lanes 9 11: Horses I III, week 6 p.i. (B) Lane 1: experimentally infected horse (van Knapen); lanes 5 and 6: 7C2C5 mab; lanes 2 4: Horses I III, week 8 p.i.; Lanes 7 9: Horses I III, week 32 p.i. Horse sera were considered anti-t. spiralis IgG positive if same antigen triad as with 7C2C5 mab was visible.

4 110 L. Sofronic-Milosavljevic et al. / Veterinary Parasitology 132 (2005) Western blot (Wb) and dot blot analyses Serum samples from infected horses were negative at day 0 in Wb (Fig. 2A). Antibodies recognizing the characteristic band triad were detected beginning at week 4 p.i. (Horse I) and week 6 p.i. (Horse III). The antibodies were detected in serum samples from all three horses at week 8 p.i. and persisted to the end of the experiment (32 weeks p.i.) (Fig. 2B). The band triad was not seen in Wb with Trichinella-free horse serum samples. The results of the dot blot test showed that circulating T.spiralis ES antigens were not present either in the sera at experimental horses collected at day 0 of the experiment or in the serum of T. spiralis free horses (data not shown); however, ES antigens were detected between weeks 4 and 32 p.i. in the sera of all three infected horses. 4. Discussion Dot blot analyses showed that ES antigens can be detected in the serum of Trichinella-infected horses, even with very low worm burdens. Although the detection of antigen in sera or body fluids offers definitive evidence of active infection, its absence may not necessarily indicate a lack of infection with Trichinella spp. In the dot blot test, the monoclonal antibody 7C2C5 was utilized to capture antigen. Recent data shows that this monoclonal antibody (mab) provides the most specific reaction relative to other mab investigated (Li and Co, 2001). The IFA test demonstrated the presence of specific anti-trichinella IgG antibodies in all three lightlyinfected horses and their persistence to the end of the experiment. These results were unexpected since Soulé et al. (1989) reported that at an infection dose of ML no specific IgG was detected by IFA. Although we were successful in antibody detection in the three experimentally-infected horses at serum dilutions of 1:20 and greater, a potential problem in determining a cut-off dilution for horses sampled in the field still exists. We found that 6% of horse serum samples from Trichinella-negative slaughterhouse horses produced non-specific staining by IFA if a dilution of 1:20 was used, and about 2% of samples displayed background staining if a dilution of 1:40 was used. However, no samples displayed background staining at a 1:80 dilution. These results could be due to the presence of other animal and plant parasitic nematodes in the horse (cross-reactivity), as suggested by Pozio et al. (2002). We cannot recommend an IFA cut-off dilution of 1:80 for horses sampled in the field until more research is conducted on this method. Similar to the results of others, the ELISA did not prove satisfactory for the detection of Trichinella infection in horses. Our results confirm previous findings that antibody detection in infected horses could be followed by ELISA for short periods of time depending on the specific antigen used and the Trichinella species detected (Soulé et al., 1989; Boireau et al., 2000; Pozio et al., 2002). Western blot analyses appear to be promising as a confirmatory test for anti-trichinella IgG, especially if equivocal results were obtained by IFA or ELISA. Our results indicated that a characteristic antigen triad was observed with experimentally-infected horse sera. The specificity of this interaction was confirmed by complete inhibition of an ES 7C2C5 mab interaction with anti-trichinella IgG from experimentally-infected horse sera (unpublished results). Our investigations on swine, dogs and human Trichinella antibody responses, in ether experimentally- or naturally-infected hosts using Wb also demonstrated the appearance of this antigen triad (Sofronic-Milosavljevic et al., 1997, 2001). These results suggest that the presence of the 45, 49 and 53 kda band triad can be valuable for detecting specific anti-t. spiralis antibodies in the sera of infected hosts. In conclusion, the results obtained in this study indicate that both parasite antigen and anti- Trichinella IgG can be detected up to 8 months following infection in horses where live T. spiralis larvae persist in muscle tissue. Further, the detection of specific antibody varied based on the type of test used (after 8 months p.i., antibodies were revealed by IFA and Wb, but not by ELISA). For investigations on equine trichinellosis, simultaneous examination for the presence of circulating ES antigen and specific antibody (by IFA), as well as the appearance of the specific band triad in Western blots may overcome some of the inconsistencies encountered in detecting antibody by serological means.

5 L. Sofronic-Milosavljevic et al. / Veterinary Parasitology 132 (2005) Acknowledgements This study received support from research project No. 1507, entitled Components of molecular communication between host and parasite, of the Serbian Ministry of Science and Environmental Protection, Serbia and Montenegro, and from the Danish Centre for Experimental Parasitology, Denmark. We also wish to thank Dr. E. Pozio (Istituto Superiore di Sanità, Rome, Italy) for valuable advice. References Boireau, P., Vallee, I., Roman, T., Perret, C., Mingyuan, L., Gamble, H.R., Gajadhar, A., Trichinella in horses: a low frequency infection with high human risk. Vet. Parasitol. 93, Djordjevic, M., Bacic, M., Petricevic, M., Cuperlovic, K., Malakauskas, A., Kapel, C.M., Murrell, K.D., Social, political, and economic factors responsible for the reemergence of trichinellosis in Serbia: a case study. J. Parasitol. 89, Gamble, H.R., Anderson, W.R., Graham, C.E., Murrell, K.D., Diagnosis of swine trichinosis by enzyme-linked immunosorbent assay (ELISA) using an excretory/secretory antigen. Vet. Parasitol. 13, Gamble, H.R., Bessonov, A.S., Cuperlovic, K., Gajadhar, A.A., van Knapen, F., Noeckler, K., Schenone, H., Zhu, X., International commission on trichinellosis: recommendations on methods for the control of Trichinella in domestic and wild animals intended for human consumption. Vet. Parasitol. 93, Gamble, H.R., Graham, C.E., Monoclonal antibody-purified antigen for the immunodiagnosis of trichinosis. Am. J. Vet. Res. 45, Ilic, N., Petrovic, M., Djordjevic, M., Sofronic-Milosavljevic, Lj., A novel dot blot test for Trichinella spiralis antigen detection. Acta Vet. (Belgrade) 54, Lalic, R., Movsesijan, M., Cuperlovic, K., Petrovic, M., Antibodies during infection with T. spiralis. Period. Biol. 81, Li, C.K., Co, R.C., The detection and occurrence of circulating antigens of Trichinella spiralis during worm development. Parasitol. Res. 87, Murrell, K.D., Djordjevic, M., Cuperlovic, K., Sofronic, Lj., Savic, M., Djordjevic, M., Damjanovic, S., Epidemiology of Trichinella infection in the horse: the risk from animal product feeding practices. Vet. Parasitol. 123, Pozio, E., Sofronic Milosavljevic, Lj., Gomez Morales, M.A., Boireau, P., Nockler, K., Evaluation of ELISA and Western Blot analysis using three antigens to detect anti-trichinella IgG in horses. Vet. Parasitol. 108, Sofronic-Milosavljevic, Lj., Cuperlovic, K., Cokic, Z., Tumor originated antibodies from tumor patients recognize Trichinella spiralis antigens. In: Ortega-Pierres, G., Gamble, H.R., van Knapen, F., Wakelin, D. (Eds.), Trichinellosis, vol. 9. Centro de Investigacion y Estudios Avanzados IPN, D.F. Mexico, Mexico, pp Sofronic-Milosavljevic, Lj., Petrovic, M., Miloradovic, M., Immunodiagnosis of trichinellosis by application of specific Trichinella antigens. In: Proceedings of the Scientific Meeting on Tissue Helminthoses of Man and Animals, Serbian Academy of Sciences and Arts, Belgrade, Department of Medical Sciences and Yugoslav Association of Parasitologists, Pasteur Institute, Novi Sad, pp Soulé, C., Dupouy-Camet, J., Georges, P., Ancelle, T., Gillet, J.P., Vaissaire, J., Delvigne, A., Plareau, E., Experimental trichinellosis in horses: biological and parasitological evaluation. Vet. Parasitol. 31,