Reactivity and Specificity of Trichinella spiralis Fractions in

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1 JOURNAL OF CUNICAL MICROBIOLOGY, Sept. 1977, p Copyright 1977 American Society for Microbiology Vol. 6, No. 3 Printed in U.S.A. Reactivity and Specificity of Trichinella spiralis Fractions in Cutaneous and Serological Tests OMAR 0. BARRIGA Department ofpathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania Received for publication 29 December 1976 Six diethylaminoethyl-cellulose fractions of a larval Trichinella spiralis extract, an Ascaris suum extract, and a nonrelated protein were used for cutaneous tests in guinea pigs with 8-, 14-, and 73-day-old T. spiralis infections, in guinea pigs with 13-day-old A. suum infections, and in normal guinea pigs. A selected T. spiralis fraction was used in hemagglutination (HA) tests with sera of 8 T. spiralis-infected rabbits, 41 sera of trichinellosis patients positive by bentonite agglutination tests, and 50 sera of clinically healthy persons. Immediate-type cutaneous reactions revealed extensive cross-reactivity between bothparasites, although the establishment of conventional limits for considering a reaction positive allowed the specific diagnosis of acute or chronic trichinellosis with different fractions. Delayed-type reactions were specific with all fractions except one, and different fractions reacted during either the acute or the chronic phase of trichinellosis. HA detected anti-trichinella antibodies in all the rabbits 9 to 10 days postinfection, in all trichinellosis patients, and in none of the healthy people. Correlation between HA and bentonite agglutination titers and other considerations suggest that HA with the selected fraction detects early antibodies. HA inhibition tests with A. suum extract suggest lack of HA crossreactivity between the A. suum- and T. spiralis-selected fractions. The use of different fractions in diverse tests for clinical or epidemiological studies is suggested. The prevalence and incidence of human and swine trichinellosis has declined steadily in the last decades in the U.S.A., but still million potentially infecting meals are produced every year, resulting in an estimated 150,000 to 300,000 actual human infections (16). Of these, only 152 (0.1 to 0.05%) clinical cases per year were reported during 1971 to 1975 (3), despite the fact that trichinellosis is a notifiable disease. It is the consensus among specialists that a major factor in explaining the difference between the number of actual infections and the number of clinical cases is the failure to diagnose the condition. Because of the nonspecific symptoms of the disease (5), laboratory confirmation is required for the proper diagnosis. The examination most favored by clinicians in the United States is the serological test, which was used in 590 (84%) of 705 cases for which information was available in 1971 to 1975, with the bentonite agglutination (BA) test being by far the most popular (82% of the serological tests in 1974 to 1975). Muscle biopsy was performed in 229 (34%) cases, and skin tests were used in only 75 instances (3). These three examinations, however, present serious drawbacks in their clinical application. The first positive serological reaction appears, on the average, about 30 days after the onset of the symptoms, which gives the reaction a rather academic value; muscle biopsy is disliked by the patient and misses about 22% of cases that are positive by serology; and the skin test detected only 58% of the few cases in which it was used. After a flare of research in the serology of trichinellosis in the late 1950s and the 1960s (reviewed by Kagan and Norman [8]), little has been done to improve the situation described above. Williams et al. (15) applied the radioactive iodine-labeled microprecipitin assay, which measured primary immunoglobulin E antibodies, and Ruitenberg et al. (13) used the enzyme-linked immunosorbent assay, which exhibited an exquisite sensitivity in swine. Regardless of the sensitivity of the test, I believe that an adequate test for trichinellosis must rely on the selection of antigens that are specific for the parasite, strong immunogens, released early in the infection, and (of preference for clinical diagnosis) transient. The im- 274

2 VOL. 6, 1977 mune responses elicited by such antigens should be specific and intense enough to be detectable by conventional methods, and should appear early in the course of the infection and vanish soon after the acute episode. Ideally, these antigens should be appropriate for use in simple assays, like skin tests, that can be performed at a physician's office, or be able to withstand prolonged storage in the hospital laboratory. The dependency of the test results on the antigen used has been illustrated by Kozar et al. (11), Kagan (6), and Bessonov and Volfson (2), who recommended that several tests be performed on the same serum to maximize the chances of success. Since clinical trichinellosis is not an everyday event for most clinicians, any diagnostic test that is very complex or requires repeated investigation is likely to add to the pool of cases that go unrecognized. With these purposes in mind, I decided to test the sensitivity and specificity of different antigenic fractions of the parasite for detecting skin reactivity in infected guinea pigs and to investigate the serological reactivity of a cathodic antigen that was the first and strongest to react in gel precipitation tests and that seemed to be identical to the band found by Van Peenen (cited by Kent [10]), Dymovska et al. (4), and Kagan (7) with sera of human patients. MATERIAL AND METHODS The skin tests were performed on g female guinea pigs. Six of them were infected per os with 1,000 viable Trichinella spiralis larvae; two were infected with 4,0 embryonatedascaris suum eggs; and two were normal controls. Serological tests were carried out with the sera of eight young adult rabbits (ca. 3 k) previously infected with four viable T. spiralis larvae per gram of body weight and taken at different times of infection, with 41 sera of human patients positive to T. spiralis by the BA test (donated by I. G. Kagan, Center for Disease Control, and the late E. H. Sadun, Walter Reed Army Institute of Research), and with 50 sera of clinically healthy people (donated by R. J. Martin, Illinois Department of Public Health). T. spiralis for infection and for the preparation of extracts was obtained by hydrochloride-pepsin digestion of carcasses of Swiss mice infected 30 to days previously. A. suum parasites were obtained from naturally infected pigs, and eggs were taken from the uteri of females and embryonated in 2% sulfuric acid. Extract of T. spiralis was prepared by homogenization of clean larvae in a VirTis 45 apparatus (Research Equipment, Gardiner, N.Y.) in 0.08 M phosphate-buffered saline at 45,000 rpm for 10 1-min periods to avoid heating, followed by sonic treatment three bursts of s each) in a Branson S-125 REACTIVITY AND SPECIFICITY OF T. SPIRALIS 275 Sonifier (Heat Systems Co., Long Island, N.Y.) and centrifugation at 28,000 x g for 30 min. All the procedures were carried out in an ice bath or at 2 C. The supernatant was collected, and its protein content was measured by the biuret technique, using crystallized bovine serum albumin (BSA; Pentex, Kankakee, Ill.) as standard. Extracts of A. suum were obtained in the same manner, using equivalent volumes of male and female worms. The uteri of the females had been removed because they seem to be a major source of cross-reacting antigens (14). Fractionation of the T. spiralis extract was done by stepwise elution in a diethylaminoethyl-cellulose column with phosphate-buffered saline (ph 6.8) at , 0.07, 0.1, 0.14, 0.2, and 0.3 M (fractions I, II, III, IV, V, and VI, respectively). The protein in the effluent was monitored by optical absorption at 2 nm; the portion of each eluate that exhibited optical activity was concentrated by lyophilization, and the protein content was estimated by the Lowry technique (12), using BSA as a standard. The skin tests were performed by injecting,ug of protein of each T. spiralis fraction intradermally in 0.1 ml of fluid in various predetermined places on the backs of guinea pigs that had been shaved 18 h before. The same amount of proteint of A. suum extract and of BSA was inoculated in separate places. Evans blue (2.5%; 0.5 ml) was injected intravenously into each guinea pig about 0.5 h before the skin testing to make the immediate-type response more evident. Two guinea pigs were tested 8, 14, or 73 days after the T. spiralis infection, and two were tested 13 days after the A. suum infection. Skin reactions were read within 30 min of inoculation (immediate-type reaction) and after 24 h (delayedtype reaction). The immediate-type reaction was evaluated by measuring the area of the blue spot produced. It was considered positive when the spot was larger than 0.6 cm2 and coincided with a negligible blue spot at the site of BSA inoculation. Areas over 1.0 cm2 were recorded as 2+; areas between 0.6 and 0.4 cm2 were considered suspicious. The delayed-type reaction was detected by palpation, and the thickness of the skin at the site of injection was measured with a Starret gauge 1015 MB (Athol, Mass.). It was considered positive when an increase of more than 15% over the value before the injection coincided with negligible increase at the site of BSA inoculation; an increase between 10 and 15% was considered suspicious. In the few cases in which the reactions in two animals differed, the results were averaged. Serological tests were performed by hemagglutination (HA) with sheep erythrocytes (SRBC) sensitized with fraction II by the glutaraldehyde method (1), using serum of a rabbit infected with T. spiralis and hyperimmunized with the same fraction as positive control and pooled rabbit sera taken before the infection as negative controls. Inhibition of the HA was attempted with fractions I, II, V, and VI and a combination of fractions III and IV (III-IV), using 16 HA units of the positive control serum. Serum from the infected rabbits was taken before and 5 to 6, 9 to 10, 14 to 15, and 30 days after infection. The BA tests with human sera were per-

3 276 BARRIGA formed in the Center for Disease Control according to their standard procedure (9). Portions of sensitized SRBC were lyophilized and stored at 4 C. After 30, 60, and 90 days, some of them were reconstituted to the original volume with distilled water and tested for T. spiralis reactivity with the same anti-t. spiralis serum. T. spiralis infections in guinea pigs were verified by observation of adults in the intestine or of larvae in the muscles after the experiments. The infection in rabbits was quantified by counting the number of larvae in 0.5 g of diaphragm on day 30 of infection. RESULTS The relative protein distribution among the fractions appears in Fig. 1. T. spiralis fraction I yielded a positive immediate-type response in guinea pigs with infections 8, 14, or 73 days old, the response being stronger during the early phase of the infection. Fraction II produced an intense reaction only with 14- and 73-day-old infections. Fractions III and IV yielded a strong reaction during the early infection that tended to wane later on, becoming only suspicious or negative by day 73. Fractions V and VI were negative or suspicious at all times. Inoculations of BSA were always negative, but A. suum extracts were positive in animals with 8- and 14-day-old infections. Guinea pigs with 13-dayold A. suum infection reacted strongly to the homologous extract, but yielded weaker positive responses to all T. spiralis fractions except fraction I. BSA inoculations were negative. Normal guinea pigs showed only a suspicious reaction to the A. suum extract (Table 1). Delayed-type reaction was suspicious in 73- z I 0 I11 II, IV V MOLAR ITY FIG. 1. Relative protein content, by the method of Lowry et al. (12), ofsix fractions of T. spiralis extract obtained by elution of a diethylaminoethyl-cellulose column with phosphate-buffered saline (ph 6.8) at different molarities. TABLE 1. Immediate skin reactivity to fractions of T. spiralis extract, BSA, and A. suum extract in T. spiralis-infected, A. suum-infected, and normal guinea pigsa T. spiralis infection A suu Nor- Antigens (days) infec- man ani tion mals Fraction I Fraction II Fraction III Fraction IV Fraction V Fraction VI BSA A. suum a 2+, >1.0 cm2; +, 0.6 to 1.0 cm2; ±, 0.4 to 0.6 cm2; -, no reaction. J. CLIN. MICROBIOL. day-old T. spiralis infections with fraction I and positive with fraction II; all other animals yielded negative results with these fractions. Fraction III yielded positive reactions only in animals with 14- and 73-day-old T. spiralis infections. Fraction IV elicited a positive reaction in all animals infected with T. spiralis or A. suum and a suspicious reaction in normal animals. Fractions V and VI induced a suspicious or positive reaction only in guinea pigs with an 8-day-old T. spiralis infection. Inoculations of BSA were negative in all animals, and A. suum extract showed a positive response only with the homologous infection (Table 2). HA tests with fraction II became positive in two of eight rabbits by day 5 to 6 after infection and in all rabbits by day 9 to 10. All titers remained positive and rising to day 30 postinfection (Table 3). There was no evidence of correlation between the number of larvae observed in the diaphragm and the antibody titers. All of the 41 human sera that were positive by the BA test were also positive by the HA test, but at almost four twofold dilutions higher, on the average (Table 4). The correlation coefflcient between the titers of the two tests was However, it was only 0.23 when only the 23 sera with BA titers of or lower were considered and 0.44 when only the 18 sera with BA titers of or higher were studied. All of the sera from the 50 clinically healthy persons were negative. Inhibition of the HA using 16 HA units of the positive rabbit serum (serum from a T. spiralis-infected rabbit boosted with inoculations of fraction II) was achieved with 2 ng of whole T. spiralis extract, 5.4 ng offraction II, 7,600 ng of fraction III-IV, 22,000 ng of fraction V, and 50,000 ng of fraction VI. Inhibition was not achieved with 75,000 ng of fraction I or with 100,000 ng ofa. suum extract or BSA (Table 5).

4 VOL. 6, 1977 TABLE 2. Delayed skin reactivity to fractions of T. spiralis extract, BSA, and A. suum extract in T. spiralis-infected, A. suum-infected, and normal guinea pigsa T. spiralis infection A: suum Nor- Antigens (ays) infec mal tion ani mals Fraction I Fraction II Fraction III Fraction IV ± Fraction V Fraction VI BSA - - A. suum a +, >15% increase in skin thickness; +, 10 to 15% increase; -, <10% increase. TABLE 3. Parasitic load and HA titersa in eight rabbits at different times after T. spiralis infection No. of lar- HA titer Rabbit vae/g of diaphragm 5-6b , c 6 2, , , ,000 0 a Reciprocals of the end-point serum dilutions. b Days postinfection. e Died before day 30 from causes not related to infection. Sensitized SRBC retained their specific Trichinella activity, as tested by HA with the positive rabbit serum, when they were lyophilized and stored at 4 C for at least 3 months. DISCUSSION From the results reported above, it is evident that some fractions oft. spiralis are more effective in detecting immediate-type hypersensitivity during early infection (fractions I, Ill, IV; Table 1), whereas others are more efficacious in late infection (fraction II). The cross-reactivity with A. suum was extensive; however, practically all T. spiralis fractions reacted in A. suum-infected guinea pigs, and A. suum extract yielded positive results in animals with 8- and 14-day-old T. spiralis infection. If only reactions with an area >1.0 cm2 are considered positive, fractions I, III, and IV are useful in detecting early T. spiralis infection and fraction II is useful in detecting late infection, without interfering with the presence ofa. suum. REACTIVITY AND SPECIFICITY OF T. SPIRALIS 277 Because the early-reacting fractions would be suitable for the clinical diagnosis of acute disease, whereas the late-reacting fraction would be valuable in epidemiological studies, it seems important to investigate their reactivity in humans and pigs infected with T. spiralis and with Ascaris spp. and to establish a conventional limit between positive and cross-reacting responses. Delayed-type reactivity in early T. spiralis infection was shown only by fractions IV and VI, but the former exhibited too much crossreactivity to be of practical value (Table 2). Fraction III yielded positive results in either 14- or 73-day-old infection, and fraction II reacted TABLE 4. Anti-Trichinella titers a of 41 sera from human patients, tested by BA and HA a Serum no Lot Lot BA titer HA titer 10,2,4 81,9 10,2 10,2 10,2,960 10,2,4 10,2 6 10,2 Reciprocals of the end-point serum dilutions.

5 278 BARRIGA TABLE 5. Inhibition of HA of anti-trichinella hyperimmune serum against fraction II, with homologous and heterologous preparations Antigen ng of hibit protein 16 HA needed units to in- T. spiralis extract 2 Fraction I >75,000 Fraction II 5.4 Fraction III-IV 7,600 Fraction V 22,000 Fraction VI 50,000 BSA >100,000 A. suum extract >100,000 only in late infection. As opposed to the immediate-type reaction, the delayed reaction revealed a remarkable degree of specificity: A. suum extract was consistently negative in T. spiralis-infected guinea pigs, and it yielded a positive response with the homologous infection. If the same results are found in human and swine trichinellosis, fraction VI should be useful for identifying overt disease, and fraction II should be useful for detecting chronic infection. Fraction III could be used in either case, since it yielded positive delayed responses at least from day 14 to 73 of infection. HA tests with fraction II, chosen for its early and strong reaction in gel precipitation tests and its probable reactivity with sera of human patients (see above), became positive in all rabbits by 9 to 10 days after infection (Table 3), which is well below the average time of onset of the symptoms in humans and much earlier than the appearance of BA titers (3). Although the correlation coefficient between the HA and the BA titers in sera of human patients was fairly high on the average, the lower correlation between low-titered BA sera and their corresponding HA titers as compared with hightitered BA sera and the corresponding HA titers suggests that the two tests detect different antibodies when sera with low BA titers are used. Since it is logical to assume that most BA low-titered sera in patients with overt disease correspond to the early stages of infection, the HA tests probably detected earlier antibodies than the BA tests did; this was supported by the HA results with rabbit sera. One of the major drawbacks of tests as sensitive as HA is that they are able to detect minute amounts of crossreacting antibodies in healthy populations, producing a number of falsely positive tests. This seems not to be the case with fraction II, since 50 sera from clinically healthy people were all negative. A further advantage of the HA test as performed here is that the sensitized SRBC may be lyophilized and stored at 4 C for at least J. CLIN. MICROBIOL. 3 months without losing their antigenic potency. The HA inhibition tests with serum of a rabbit infected with T. spiralis and boosted with fraction II revealed that this fraction was almost 45 times more efficient in detecting anti- Trichinella antibodies (based on protein content of the inhibitory preparation) than the whole T. spiralis extract and more than 1,0 times more effective than the next most active preparation (fraction III-IV). A. suum protein (0.1 mg) was unable to inhibit the reaction, which suggests that there is no cross-reactivity between T. spiralis fraction II and A. suum extracts as detected by HA. In conclusion, we believe that the rational use of selected T. spiralis fractions in cutaneous or serological tests may be a valuable tool in the diagnosis or acute or chronic trichinellosis. ACKNOWLEDGMENTS This work was supported by Biomedical Sciences support grant RRO I am grateful to N. D. Levine and D. Segre, College of Veterinary Medicine, University of Illinois, for their kind assistance during part of the work. LITERATURE CITED 1. Avrameas, S., B. Taudon, and S. Chuiton Glutaraldehyde, cyanuric chloride and tetraazotized-odianisidine as coupling reagents in the passive hemagglutination test. Immunochemistry 6: Bessonov, A. S., and A. G. Volfson The use of an immunological survey method of the Chukotsk population for trichinellosis studies. Wiad. Parazytol. 16: (In Russian; English summary) 3. Center for Disease Control Trichinosis surveillance, animal summaries U.S. Department of Health, Education and Welfare, publication no. (CDC) Center for Disease Control, Atlanta, Ga. 4. Dymowska, Z., J. Aleksandrowicz, and A. Zakrzewska Antigens of Trichinella spiralis. II. Serological tests. Acta Parasitol. Pol. 15: Gould, S. E Clinical manifestations. A. Symptomatology, p In S. E. Gould (ed.), Trichinosis in man and animals. Charles C Thomas, Publisher, Springfield, Ill. 6. Kagan, I. G Evaluation of routine serologic testing for parasitic diseases. Am. J. Public Health 11: Kagan, I. G Characterization of parasite antigens, p In Immunological aspects of parasitic diseases, scientific publication no Pan American Health Organization, Washington, D.C. 8. Kagan, I. G., and L. G. Norman The serology of trichinosis, p In S. E. Gould (ed.), Trichinosis in man and animal. Charles C Thomas, Publisher, Springfield, Ill. 9. Kagan, I. G., and L. G. Norman Serodiagnosis of parasitic diseases, p In N. R. Rose and H. Friedman (ed.), Manual of clinical immunology. American Society for Microbiology, Washington, D.C. 10. Kent, N. H Fractionation, isolation and definition of antigens from parasitic helminths. Am. J.

6 VOL. 6, 1977 Hyg. 22: Kozar, M., A. Kozar, and K. Karmanska The comparative evaluation of some agglutination tests in the diagnosis of trichinellosis. Wiad. Parazytol. 10: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Ruitepberg, E. J., P. A. Steerenberg, B. J. M. Brosi, REACTIVITY AND SPECIFICITY OF T. SPIRALIS 279 and J. Buys Serodiagnosis of Trichinella spiralis infection in pigs by enzyme-linked immunosorbent assays. Bull. W.H.O. 51: Torres, T., and Barriga Intra- and interspecific antigenic relationship among some Ascaroidea. Acta Parasitol. Pol. 23: Williams, J. S., R. W. Gore, and E. H. Sadum Trichinella spiralis: antigen-antibody interaction assayed by radioactive iodinated antigen. Exp. Parasitol. 31: Zimmermann, W. J., J. H. Steele, and I. G. Kagan Trichinosis in the United States population, Public Health Serv. Rep. 88: