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Evaluation of an enzyme-linked immunosorbent assay to detect specific antibodies in pigs infested with the tick Ornithodoros erraticus (Argasidae)
Veterinary Parasitology, 37 (1990) 145-153 Elsevier Science Publishers B.V., Amsterdam
145
Evaluation of an enzyme-linked immunosorbent assay to detect specific antibodies in pigs infested with the tick Ornithodoros erraticus ( Argasidae ) A. Canals 1, A. Oleaga 2, R. P6rez 2, J. Dominguez 1, A. Encinas 2 and J.M. S~inchez-Vizcaino ~* ~Departamento de Sanidad Animal, INIA, Embajadores 68, 28012 Madrid (Spain) :Departamento de Parasitologia, Facultad de Farmacia, Universidad de Salamanca, 37007 Salamanca (Spain) (Accepted for publication 24 April 1990)
ABSTRACT Canals, A., Oleaga, A., P6rez, R., Dominguez, J., Encinas, A. and S~inchez-Vizcaino, J.M., 1990. Evaluation of an enzyme-linked immunosorbent assay to detect specific antibodies in pigs infested with the tick Ornithodoros erraticus (Argasidae). Vet. Parasitol., 37:145-153.
Ornithodoros erraticus is known to transmit the virus that causes the highly contagious disease, African Swine Fever, in Spain. As part of the disease eradication campaign, an ELISA test to detect specific antibodies against the tick was developed. The ELISA, using salivary gland preparations as an antigen, showed high sensitivity and was able to detect as few as 10 adult ticks. The specific antibodies were detected in the sera 6 weeks after the primary infestation and strongly increased after the challenge. The utility of this test under field conditions was also tested.
INTRODUCTION
African Swine Fever Virus (ASFV) causes a highly contagious disease of pigs. Since 1960 the virus has caused important economic losses in Spain. Soft ticks of the genus Ornithodoros act as biological vectors and reservoirs of the virus in the countries where the disease is endemic. In Spain, Ornithodoros erraticus is known to transmit the virus in the field (S~inchez Botija, 1963 ). Although the arthropod vector is not required for continued spread of the virus among domestic pigs, it is an important reservoir of the virus and greatly complicates eradication efforts (Hess et al., 1987 ). One of the approaches to eradicate the disease is control of the tick population. An important aspect of an effective ASFV eradication campaign would be serological diagnosis of the pigs bitten by these ticks. The systematic use of *Author to w h o m correspondence should be addressed.
0304-4017/90/$03.50
© 1 9 9 0 - Elsevier Science Publishers B.V.
146
A. CANALS ET AL.
this test in combination with different assays already available for the detection of antibodies against the virus (S~inchez-Vizcaino et al., 1982; Pastor et al., 1989) would help in understanding the epidemiology of the disease and in preventing outbreaks of the disease. Most of the studies on host i m m u n i t y to ticks have been conducted with ixodid ticks in laboratory animals. Antibodies against salivary gland antigens of Ornithodoros moubata have been described in rabbits using indirect immunofluorescence and ELISA techniques (Brossard et al., 1981; Centurier et al., 1981 ) but there is no information available on the humoral i m m u n e response of pigs to O. erraticus. A major complication in the development of assays for detection of tick reactive antibodies is the extremely small amount of antigen that can be isolated from these ticks. In the present study we developed an enzyme linked immunosorbent assay (ELISA) to detect specific antibodies against O. erraticus salivary gland antigens (SGA). We tested the sensitivity of the assay as the m i n i m u m number of specimens and infestations required to detect a response with our technique. Finally, we checked the assay in pig sera with a known ectoparasite population to assess the utility of the test in the field. MATERIAL AND METHODS
Antigen preparations The antigen used in the ELISA was an extract of salivary glands from unfed O. erraticus females and males. Briefly, salivary glands were excised, placed in phosphate buffered saline (PBS) pH 7.4, and washed with PBS several times. Extracts were further homogenized by hand in a glass tissue grinder at 4 ° C, following sonication for 6 min on ice with 40 W. The preparation was allowed to settle for 60 min at 4°C. Finally, extracts were centrifuged at 20 000 × g for 30 min, the supernatants were filtered, and the protein concentration determined by the m e t h o d of Lowry et al. (1951 ). They were then aliquoted and stored at - 2 0 ° C. Reference sera Positive and negative reference sera from infested and uninfested pigs were used to standardize the enzyme immunoassay. Three-month-old female outbred pigs, weighing between 15 and 20 kg were used. Pigs were obtained from a farm free of ectoparasites (tick, scabies mite, flea, hog louse), where they were bred under intensive farming conditions. The positive sera were obtained by infesting six pigs with O. erraticus larvae, nymphs or adults, weekly for 12 consecutive weeks. Blood was collected 7 days after each infestation by a small cut on the ear vein. The blood was allowed to clot for 2 h at room
EVALUATION OF ELISA TO DETECT ANTIBODIES AGAINST O. ERRATICUS
147
temperature and then placed overnight in the refrigerator at 4 ° C. The blood was then centrifuged at 8 0 0 × g for 10 min and the serum was extracted, aliquoted and kept a t - 2 0 ° C. For infestations, pigs were immobilized in a special trap exposing their ventral side and 200 adults, 500 nymphs or 1000 larvae held in an open cylinder were allowed to feed for 60 min.
Enzyme immunoassay The working dilutions of antigen, test sera and conjugates were determined prior to use by checkerboard titration. ELISA assay plates (Costar, Cambridge, MA) were coated overnight at 4 °C with 2/tg of antigen per well, in 100 #1 of 0.1 M carbonate buffer (pH 9.6 ). The following day the plates were washed three times for 5 min each in saline solution (0.85% NaC1) containing 0.05% Tween 20. After the washes, 100 #1 of the test sera diluted 1/50 in PBS containing 0.05% Tween 20 were added and the plates incubated for 1 h at 37 °C. The plates were again washed three times and 100/tl of 1/4000 rabbit anti-porcine IgG ( H + L) conjugated with peroxidase (Bio Makor, Rehovoth, Israel) were added to the individual wells. After incubation at 37 °C for 1 h the plates were washed three times and 100/tl of the enzyme substrate were added to the wells. The substrate was prepared by dissolving 40 mg of O-phenylenediamine (Sigma, St. Louis, MO) in 1 ml of methanol; 100/tl of this solution was added in 10 ml of 0.1 M citrate buffer (pH 5.0). After 10 min the enzymatic reaction was stopped with 100/zl 3 N H2504 and the resulting colour in each well was determined by absorbance at 492 n m (O.D.492 nm).
Assessment of the assay sensitivity Six 3-month-old female pigs weighing between 15 and 20 kg were used to evaluate the sensitivity of the assay as the m i n i m u m number of ticks required to produce a measurable humoral response. Two of these pigs were infested with 10 adult Ornithodoros, two with 24 adults and the remaining two with 50 adults. In all the infestations equal numbers of males and females were used. Six weeks after the first infestation a similar challenge infestation was given to the pigs. Animals were bled weekly to test the antibody levels in the serum. Background levels were determined as the mean plus two standard deviations of the absorbance values seen for the sera of the six pigs at Day 0.
Field evaluation of the assay To evaluate the ELISA under field conditions two experiments were conducted. In the first experiment, one of the control pigs was kept for 24 h in a pen which had Ornithodoros erraticus, and it was challenged 4 weeks later in
148
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the same way. The animal was bled 1 week after infestation and 1 and 2 weeks after the challenge to evaluate the serum-specific antibody response. In the second experiment, sera from 27 pigs bred in the field at eight different farms were tested for specific O. erraticus antibodies. At the time of blood collection, soil samples from these farms were inspected for the presence of
O. erraticus. RESULTS
Antigen isolation Protein concentrations from the several antigen preparations yielded an average of 10/tg m l - ~ for each gland. To test the effect of feeding on the total amount of salivary gland proteins collected, some preparations were made with ticks that had already concluded the feeding process. Owing to the possibility that host proteins ingested by the ticks during feeding will interfere in the test, and because no differences in the protein concentration were found (data not shown ), unfed ticks were used for all the antigen preparations used on the ELISA.
ELISA Reference negative sera from the 10 uninfested pigs yielded a mean absorbance value (x) of 0.144 and a standard deviation ( s.d. ) o f 0.036. The mean plus two standard deviations (0.216) was considered as the background level, and values higher than this were considered positive. The kinetics of the O. erraticus-specific serum antibody response in the positive control animals are shown in Fig. 1. There was a sharp increase above background levels 2 weeks after the first infestation. This response reached a peak, almost eight times background levels, 8 weeks after the initial infestation, and was maintained above background values 10 weeks after the last infestation in most of the animals.
Assay sensitivity All the six pigs used to evaluate the sensitivity of the assay had specific antibody levels above background 1 week after the first infestation, reaching a peak 2 weeks after the first infestation and decreasing thereafter. Six weeks later, when the pigs were challenged, the levels still remained above background level (Fig. 2). One infestation was enough to develop a measurable response by the ELISA with just 10 ticks. After challenge infestation specific antibody levels showed a sharp increase of almost 10 times the background in some cases, which seems to point to a strong secondary antibody response.
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Fig. 1. Kinetics of the O. erraticus-specific serum antibody responses in pigs infested with 200 adults (A), 500 nymphs (N) or 1000 larvae (L). Mean values from each of two pigs. Arrows show the infestations, and the discontinuous line the background level, as the mean plus two standard deviations of the 10 control pigs (error bars deleted to simplify graph).
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Fig. 2. Specific serum antibody responses in pigs infested with 10 ( A , • ), 24 (©, • ) and 50 (E3, • ) adult O. erraticus. Discontinuous line shows background levels as the mean plus two standard deviation of the six pigs at Day 0. Arrows show the infestation and challenge.
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Fig. 3. Ornithodoros erraticus-specific a n t i b o d y r e s p o n s e o f one pig infested u n d e r field conditions. Pig was i n t r o d u c e d for 24 h in a p e n with O. erraticus, a n d 4 weeks later it was challenged in the s a m e way. TABLE 1 Detection of O. erraticus specific antibodies in extensive cultured pigs Sera
Farm
Place
Presence of
O. erraticus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Blank + 2 s.d.
A A A B B C C C D D D D D E E E E F F F F G G G H H H
Villanueva del Fresno Villanueva del Fresno Villanueva del Fresno Ventifio Ventifio Rosal de la Frontera Rosal de la Frontera Rosal de la Frontera Jerez de los Caballeros Jerez de los Caballeros Jerez de los Caballeros Jerez de los Caballeros Jerez de los Caballeros Aroche Aroche Aroche Aroche Segura de Le6n Segura de Le6n Segura de Leon Segura de Le6n Painogo Painogo Painogo Bodonal Bodonal Bodonal
-
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+
Absorbance values 0.705 0.501 0.884 0.858 0.421 0.844 0.740 0.693 0.406 0.341 0.401 0.561 0.520 1.038 0.624 0.610 0.510 0.718 0.379 0.232 0.366 0.728 0.478 0.865 2.918 2.718 3.000 0.164
EVALUATIONOF ELISATO DETECT ANTIBODIESAGAINST O. ERRATICUS
151
Field evaluation
The antibody levels developed in the pig introduced in the O. erraticus infested pen are shown in Fig. 3. The response was above background level 1 week after the infestation, reaching a peak 2 weeks after the challenge. Table 1 show the results of the screening of field sera; in only one of the eight farms which were investigated could we detect the presence of O. erraticus. When analysed by ELISA, the sera of pigs from this farm had high absorbance values, close to 3.0 O . D . 4 9 2 n m . However, the rest of the sera were also positive giving absorbance values clearly higher than background although lower than 1.10 0 . 0 . 4 9 2 r i m . DISCUSSION
Antibodies reactive with tick extracts have been described by several researchers (Willadsen, 1980; Wikel, 1982, 1984). Immunoelectrophoresis, immunofluorescence, ELISA and more recently DOT-ELISA (Brossard, 1976; Brossard et al., 1981; Centurier et al., 1981; Wikel and Osburn, 1982; Whelen et al., 1984) have been used to examine serological responses against ticks. All of these studies have been primarily focused towards an immunological tick-control program based on the development of protection against the respective arthropods. Nevertheless, our purpose in this study was to determine for the first time if an antibody response against O. erraticus was generated after infestation, and to develop an ELISA test to measure the response. Our results showed that this humoral response, like that in the case of Ornithodoros moubata in rabbits, (Brossard et al., 1981; Centurier et al., 1981 ) does develop and that the ELISA described is sensitive enough to be useful as a diagnostic tool in the ASFV-eradication campaign. The choice of salivary gland preparations as a source of antigen was based on the evidence supporting the opinion that the antigens responsible for the induction and elicitation of host i m m u n e responses to ticks following feeding are salivary gland derived (Wikel, 1981; Brown et al., 1984). Salivary gland antigens have been used to study the humoral and cellular responses to ixodid ticks (Whelen et al., 1984; Wikel and Whelen, 1986) and they are involved in the acquisition of resistance (Brown and Askenase, 1986). The results of the standardization of the technique show that SGA are extremely immunogenic preparations, showing strong reactivity with specific antibodies. Our protein concentration yield of 10/~g per gland seems to agree with other estimations for unfed ticks (McSwain et al., 1982 ). The present test was able to detect an infestation as low as 10 specimens, giving one of the pigs a three-fold increase above background. This was achieved with just one infestation and the level of antibodies remained above background until the challenge 6 weeks later. Some studies of the ecology of
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O. erraticus in Spain showed that during the cold months, from November to March, the number of bites on parasitized pigs ranged from 5 to 159 per animal (Oleaga et al., 1990). With this field situation the sensitivity of our test will allow us to detect low level infestations, which are difficult to detect in a rapid visual check. The good sensitivity of the assay was corroborated with the high anti-O. erraticus response developed in the pig introduced into the infested pen. The results of the screening of field sera was promising, showing a difference in absorbance around 2 O.D.492 nm between animals from infested and uninfested farms. The high absorbance values in animals from farms where O. erraticus was not found could be the result of the existence of cross-reactivity with other ectoparasites or just reflect residual O. erraticus antibodies from former infestations. Current investigations are aimed at the characterization of the O. erraticus immunogens and their cross-reactivity with other pig ectoparasites.
REFERENCES Brossard, M., 1976. Relations immunologiques entre bovins et tiques, plus particulierment entre bovins et Boophilus microplus. Acta Trop., 33:15-36. Brossard, M., Fivaz, V., Aeschlimann, A. and Moret, J., 1981. Response immunologique du lapin aux infestations repet6es de femelles d'Ornithodoros moubata Murray: son influencc sur la biologie des tiques. Acta Trop., 38: 55-70. Brown, S.J. and Askenase, P.W., 1986. Amblyomma americanum: Physicochemical isolation of a salivary gland derived protein capable of immunizing guinea pigs. Exp. Parasitol., 62: 4050. Brown, S.J., Shapiro, S.Z. and Askenase, P.W., 1984. Characterization of tick antigens inducing host immune resistance. I. Immunization of guinea pigs with Amblyomma americanum derived salivary gland extracts and identification of an important salivary gland protein antigen with guinea pig anti-tick antibodies. J. Immunol., 133:3319-3325. Centurier, V.C., Weiland, G. and Seubert, S., 198 I. Immunobiologische Untersuchungen zum Wirt-Parasit-Verh~iltnis zwischen Kaninchen und der Lederzecke Ornithodoros moubata. Berl. Muench. Tierarztl. Wochenschr., 94:238-241. Hess, W.R., Endris, G.R., Haslett, T.M., Monahan, M.J. and McCoy, J.P., 1987. Potential arthropod vectors of African Swine Virus in North America and the caribbean basin. Vet. Parasitol., 26:145-155. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. McSwain, J.L., Essenberg, R.C. and Sauer, J.R., 1982. Protein changes in the salivary glands of the female Lone Star tick, Amblyomma americanum. J. Parasitol., 68:100-106. Oleaga, A., P6rez, R. and Encinas, A., 1990. Distribution and biology of Ornithodoros erraticus in the African Swine Fever enzootic area of Spain. Vet. Rec., 126: 32-37. Pastor, M.J., Laviada, M.D., Sfinchez-Vizcaino, J.M. and Escribano, J.M., 1989. Serodiagnosis of African Swine Fever by immunoblotting assay. Can. J. Vet. Res., 53:105-107. Sanchez Botija, C., 1963. Reservorios del virus de la Peste Porcina Africana. Investigaci6n del
EVALUATION OF ELISA TO DETECT ANTIBODIES AGAINST O. ERRATICUS
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virus de la P.P.A. en los artropodos mediante la prueba de la hemoadsorci6n. Bull. Off. Int. Epizoot., 60: 895-899. S~nchez-Vizcaino, J.M., Tabares, E., Salvador, E. and Ordas, A., 1982. Semipurified structural viral protein for the detection of ASF antibodies by the indirect ELISA. Curr. Top. Vet. Med. Anim. Sci., 22: 214-222. Whelen, S.K., Gerorge, J.E., Osburn, R.L. and Wikel, S.K., 1984. Dot-ELISA to examine serologic responses of Bos taurus and Bos indicus to ixodid tick infestations. Fed. Proc., 43: 1628. Wikel, S.K., 1981. The induction of host resistance to tick infestation with salivary gland antigen. Am. J. Trop. Med. Hyg., 30: 284-288. Wikel, S.K., 1982. Immune responses to arthropods and their product. Annu. Rev. Entomol., 27:21-48. Wikel, S.K. and Osburn, R.L., 1982. Immune responsiveness of the bovine host to repeated low level infestations with Dermacentor andersoni. Ann. Trop. Med. Parasitol., 76:405-414. Wikel, S.K., 1984. Immunomodulation of host responses to ectoparasite infestation - an overview. Vet. Parasitol., 14: 321-339. Wikel, S.K. and Whelen, A.C., 1986. Ixodid host immune interaction. Identification and characterization of relevant antigens and tick-induced host immunosuppression. Vet. Parasitol., 20: 149-174. Willadsen, P., 1980. Immunity to ticks. Adv. Parasitol., 18:293-313.
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Evaluation of an enzyme-linked immunosorbent assay to detect specific antibodies in pigs infested with the tick Ornithodoros erraticus ( Argasidae ) A. Canals 1, A. Oleaga 2, R. P6rez 2, J. Dominguez 1, A. Encinas 2 and J.M. S~inchez-Vizcaino ~* ~Departamento de Sanidad Animal, INIA, Embajadores 68, 28012 Madrid (Spain) :Departamento de Parasitologia, Facultad de Farmacia, Universidad de Salamanca, 37007 Salamanca (Spain) (Accepted for publication 24 April 1990)
ABSTRACT Canals, A., Oleaga, A., P6rez, R., Dominguez, J., Encinas, A. and S~inchez-Vizcaino, J.M., 1990. Evaluation of an enzyme-linked immunosorbent assay to detect specific antibodies in pigs infested with the tick Ornithodoros erraticus (Argasidae). Vet. Parasitol., 37:145-153.
Ornithodoros erraticus is known to transmit the virus that causes the highly contagious disease, African Swine Fever, in Spain. As part of the disease eradication campaign, an ELISA test to detect specific antibodies against the tick was developed. The ELISA, using salivary gland preparations as an antigen, showed high sensitivity and was able to detect as few as 10 adult ticks. The specific antibodies were detected in the sera 6 weeks after the primary infestation and strongly increased after the challenge. The utility of this test under field conditions was also tested.
INTRODUCTION
African Swine Fever Virus (ASFV) causes a highly contagious disease of pigs. Since 1960 the virus has caused important economic losses in Spain. Soft ticks of the genus Ornithodoros act as biological vectors and reservoirs of the virus in the countries where the disease is endemic. In Spain, Ornithodoros erraticus is known to transmit the virus in the field (S~inchez Botija, 1963 ). Although the arthropod vector is not required for continued spread of the virus among domestic pigs, it is an important reservoir of the virus and greatly complicates eradication efforts (Hess et al., 1987 ). One of the approaches to eradicate the disease is control of the tick population. An important aspect of an effective ASFV eradication campaign would be serological diagnosis of the pigs bitten by these ticks. The systematic use of *Author to w h o m correspondence should be addressed.
0304-4017/90/$03.50
© 1 9 9 0 - Elsevier Science Publishers B.V.
146
A. CANALS ET AL.
this test in combination with different assays already available for the detection of antibodies against the virus (S~inchez-Vizcaino et al., 1982; Pastor et al., 1989) would help in understanding the epidemiology of the disease and in preventing outbreaks of the disease. Most of the studies on host i m m u n i t y to ticks have been conducted with ixodid ticks in laboratory animals. Antibodies against salivary gland antigens of Ornithodoros moubata have been described in rabbits using indirect immunofluorescence and ELISA techniques (Brossard et al., 1981; Centurier et al., 1981 ) but there is no information available on the humoral i m m u n e response of pigs to O. erraticus. A major complication in the development of assays for detection of tick reactive antibodies is the extremely small amount of antigen that can be isolated from these ticks. In the present study we developed an enzyme linked immunosorbent assay (ELISA) to detect specific antibodies against O. erraticus salivary gland antigens (SGA). We tested the sensitivity of the assay as the m i n i m u m number of specimens and infestations required to detect a response with our technique. Finally, we checked the assay in pig sera with a known ectoparasite population to assess the utility of the test in the field. MATERIAL AND METHODS
Antigen preparations The antigen used in the ELISA was an extract of salivary glands from unfed O. erraticus females and males. Briefly, salivary glands were excised, placed in phosphate buffered saline (PBS) pH 7.4, and washed with PBS several times. Extracts were further homogenized by hand in a glass tissue grinder at 4 ° C, following sonication for 6 min on ice with 40 W. The preparation was allowed to settle for 60 min at 4°C. Finally, extracts were centrifuged at 20 000 × g for 30 min, the supernatants were filtered, and the protein concentration determined by the m e t h o d of Lowry et al. (1951 ). They were then aliquoted and stored at - 2 0 ° C. Reference sera Positive and negative reference sera from infested and uninfested pigs were used to standardize the enzyme immunoassay. Three-month-old female outbred pigs, weighing between 15 and 20 kg were used. Pigs were obtained from a farm free of ectoparasites (tick, scabies mite, flea, hog louse), where they were bred under intensive farming conditions. The positive sera were obtained by infesting six pigs with O. erraticus larvae, nymphs or adults, weekly for 12 consecutive weeks. Blood was collected 7 days after each infestation by a small cut on the ear vein. The blood was allowed to clot for 2 h at room
EVALUATION OF ELISA TO DETECT ANTIBODIES AGAINST O. ERRATICUS
147
temperature and then placed overnight in the refrigerator at 4 ° C. The blood was then centrifuged at 8 0 0 × g for 10 min and the serum was extracted, aliquoted and kept a t - 2 0 ° C. For infestations, pigs were immobilized in a special trap exposing their ventral side and 200 adults, 500 nymphs or 1000 larvae held in an open cylinder were allowed to feed for 60 min.
Enzyme immunoassay The working dilutions of antigen, test sera and conjugates were determined prior to use by checkerboard titration. ELISA assay plates (Costar, Cambridge, MA) were coated overnight at 4 °C with 2/tg of antigen per well, in 100 #1 of 0.1 M carbonate buffer (pH 9.6 ). The following day the plates were washed three times for 5 min each in saline solution (0.85% NaC1) containing 0.05% Tween 20. After the washes, 100 #1 of the test sera diluted 1/50 in PBS containing 0.05% Tween 20 were added and the plates incubated for 1 h at 37 °C. The plates were again washed three times and 100/tl of 1/4000 rabbit anti-porcine IgG ( H + L) conjugated with peroxidase (Bio Makor, Rehovoth, Israel) were added to the individual wells. After incubation at 37 °C for 1 h the plates were washed three times and 100/tl of the enzyme substrate were added to the wells. The substrate was prepared by dissolving 40 mg of O-phenylenediamine (Sigma, St. Louis, MO) in 1 ml of methanol; 100/tl of this solution was added in 10 ml of 0.1 M citrate buffer (pH 5.0). After 10 min the enzymatic reaction was stopped with 100/zl 3 N H2504 and the resulting colour in each well was determined by absorbance at 492 n m (O.D.492 nm).
Assessment of the assay sensitivity Six 3-month-old female pigs weighing between 15 and 20 kg were used to evaluate the sensitivity of the assay as the m i n i m u m number of ticks required to produce a measurable humoral response. Two of these pigs were infested with 10 adult Ornithodoros, two with 24 adults and the remaining two with 50 adults. In all the infestations equal numbers of males and females were used. Six weeks after the first infestation a similar challenge infestation was given to the pigs. Animals were bled weekly to test the antibody levels in the serum. Background levels were determined as the mean plus two standard deviations of the absorbance values seen for the sera of the six pigs at Day 0.
Field evaluation of the assay To evaluate the ELISA under field conditions two experiments were conducted. In the first experiment, one of the control pigs was kept for 24 h in a pen which had Ornithodoros erraticus, and it was challenged 4 weeks later in
148
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the same way. The animal was bled 1 week after infestation and 1 and 2 weeks after the challenge to evaluate the serum-specific antibody response. In the second experiment, sera from 27 pigs bred in the field at eight different farms were tested for specific O. erraticus antibodies. At the time of blood collection, soil samples from these farms were inspected for the presence of
O. erraticus. RESULTS
Antigen isolation Protein concentrations from the several antigen preparations yielded an average of 10/tg m l - ~ for each gland. To test the effect of feeding on the total amount of salivary gland proteins collected, some preparations were made with ticks that had already concluded the feeding process. Owing to the possibility that host proteins ingested by the ticks during feeding will interfere in the test, and because no differences in the protein concentration were found (data not shown ), unfed ticks were used for all the antigen preparations used on the ELISA.
ELISA Reference negative sera from the 10 uninfested pigs yielded a mean absorbance value (x) of 0.144 and a standard deviation ( s.d. ) o f 0.036. The mean plus two standard deviations (0.216) was considered as the background level, and values higher than this were considered positive. The kinetics of the O. erraticus-specific serum antibody response in the positive control animals are shown in Fig. 1. There was a sharp increase above background levels 2 weeks after the first infestation. This response reached a peak, almost eight times background levels, 8 weeks after the initial infestation, and was maintained above background values 10 weeks after the last infestation in most of the animals.
Assay sensitivity All the six pigs used to evaluate the sensitivity of the assay had specific antibody levels above background 1 week after the first infestation, reaching a peak 2 weeks after the first infestation and decreasing thereafter. Six weeks later, when the pigs were challenged, the levels still remained above background level (Fig. 2). One infestation was enough to develop a measurable response by the ELISA with just 10 ticks. After challenge infestation specific antibody levels showed a sharp increase of almost 10 times the background in some cases, which seems to point to a strong secondary antibody response.
149
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Fig. 1. Kinetics of the O. erraticus-specific serum antibody responses in pigs infested with 200 adults (A), 500 nymphs (N) or 1000 larvae (L). Mean values from each of two pigs. Arrows show the infestations, and the discontinuous line the background level, as the mean plus two standard deviations of the 10 control pigs (error bars deleted to simplify graph).
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Fig. 2. Specific serum antibody responses in pigs infested with 10 ( A , • ), 24 (©, • ) and 50 (E3, • ) adult O. erraticus. Discontinuous line shows background levels as the mean plus two standard deviation of the six pigs at Day 0. Arrows show the infestation and challenge.
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ch
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Fig. 3. Ornithodoros erraticus-specific a n t i b o d y r e s p o n s e o f one pig infested u n d e r field conditions. Pig was i n t r o d u c e d for 24 h in a p e n with O. erraticus, a n d 4 weeks later it was challenged in the s a m e way. TABLE 1 Detection of O. erraticus specific antibodies in extensive cultured pigs Sera
Farm
Place
Presence of
O. erraticus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Blank + 2 s.d.
A A A B B C C C D D D D D E E E E F F F F G G G H H H
Villanueva del Fresno Villanueva del Fresno Villanueva del Fresno Ventifio Ventifio Rosal de la Frontera Rosal de la Frontera Rosal de la Frontera Jerez de los Caballeros Jerez de los Caballeros Jerez de los Caballeros Jerez de los Caballeros Jerez de los Caballeros Aroche Aroche Aroche Aroche Segura de Le6n Segura de Le6n Segura de Leon Segura de Le6n Painogo Painogo Painogo Bodonal Bodonal Bodonal
-
-
-
-
-
-
+
Absorbance values 0.705 0.501 0.884 0.858 0.421 0.844 0.740 0.693 0.406 0.341 0.401 0.561 0.520 1.038 0.624 0.610 0.510 0.718 0.379 0.232 0.366 0.728 0.478 0.865 2.918 2.718 3.000 0.164
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Field evaluation
The antibody levels developed in the pig introduced in the O. erraticus infested pen are shown in Fig. 3. The response was above background level 1 week after the infestation, reaching a peak 2 weeks after the challenge. Table 1 show the results of the screening of field sera; in only one of the eight farms which were investigated could we detect the presence of O. erraticus. When analysed by ELISA, the sera of pigs from this farm had high absorbance values, close to 3.0 O . D . 4 9 2 n m . However, the rest of the sera were also positive giving absorbance values clearly higher than background although lower than 1.10 0 . 0 . 4 9 2 r i m . DISCUSSION
Antibodies reactive with tick extracts have been described by several researchers (Willadsen, 1980; Wikel, 1982, 1984). Immunoelectrophoresis, immunofluorescence, ELISA and more recently DOT-ELISA (Brossard, 1976; Brossard et al., 1981; Centurier et al., 1981; Wikel and Osburn, 1982; Whelen et al., 1984) have been used to examine serological responses against ticks. All of these studies have been primarily focused towards an immunological tick-control program based on the development of protection against the respective arthropods. Nevertheless, our purpose in this study was to determine for the first time if an antibody response against O. erraticus was generated after infestation, and to develop an ELISA test to measure the response. Our results showed that this humoral response, like that in the case of Ornithodoros moubata in rabbits, (Brossard et al., 1981; Centurier et al., 1981 ) does develop and that the ELISA described is sensitive enough to be useful as a diagnostic tool in the ASFV-eradication campaign. The choice of salivary gland preparations as a source of antigen was based on the evidence supporting the opinion that the antigens responsible for the induction and elicitation of host i m m u n e responses to ticks following feeding are salivary gland derived (Wikel, 1981; Brown et al., 1984). Salivary gland antigens have been used to study the humoral and cellular responses to ixodid ticks (Whelen et al., 1984; Wikel and Whelen, 1986) and they are involved in the acquisition of resistance (Brown and Askenase, 1986). The results of the standardization of the technique show that SGA are extremely immunogenic preparations, showing strong reactivity with specific antibodies. Our protein concentration yield of 10/~g per gland seems to agree with other estimations for unfed ticks (McSwain et al., 1982 ). The present test was able to detect an infestation as low as 10 specimens, giving one of the pigs a three-fold increase above background. This was achieved with just one infestation and the level of antibodies remained above background until the challenge 6 weeks later. Some studies of the ecology of
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O. erraticus in Spain showed that during the cold months, from November to March, the number of bites on parasitized pigs ranged from 5 to 159 per animal (Oleaga et al., 1990). With this field situation the sensitivity of our test will allow us to detect low level infestations, which are difficult to detect in a rapid visual check. The good sensitivity of the assay was corroborated with the high anti-O. erraticus response developed in the pig introduced into the infested pen. The results of the screening of field sera was promising, showing a difference in absorbance around 2 O.D.492 nm between animals from infested and uninfested farms. The high absorbance values in animals from farms where O. erraticus was not found could be the result of the existence of cross-reactivity with other ectoparasites or just reflect residual O. erraticus antibodies from former infestations. Current investigations are aimed at the characterization of the O. erraticus immunogens and their cross-reactivity with other pig ectoparasites.
REFERENCES Brossard, M., 1976. Relations immunologiques entre bovins et tiques, plus particulierment entre bovins et Boophilus microplus. Acta Trop., 33:15-36. Brossard, M., Fivaz, V., Aeschlimann, A. and Moret, J., 1981. Response immunologique du lapin aux infestations repet6es de femelles d'Ornithodoros moubata Murray: son influencc sur la biologie des tiques. Acta Trop., 38: 55-70. Brown, S.J. and Askenase, P.W., 1986. Amblyomma americanum: Physicochemical isolation of a salivary gland derived protein capable of immunizing guinea pigs. Exp. Parasitol., 62: 4050. Brown, S.J., Shapiro, S.Z. and Askenase, P.W., 1984. Characterization of tick antigens inducing host immune resistance. I. Immunization of guinea pigs with Amblyomma americanum derived salivary gland extracts and identification of an important salivary gland protein antigen with guinea pig anti-tick antibodies. J. Immunol., 133:3319-3325. Centurier, V.C., Weiland, G. and Seubert, S., 198 I. Immunobiologische Untersuchungen zum Wirt-Parasit-Verh~iltnis zwischen Kaninchen und der Lederzecke Ornithodoros moubata. Berl. Muench. Tierarztl. Wochenschr., 94:238-241. Hess, W.R., Endris, G.R., Haslett, T.M., Monahan, M.J. and McCoy, J.P., 1987. Potential arthropod vectors of African Swine Virus in North America and the caribbean basin. Vet. Parasitol., 26:145-155. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. McSwain, J.L., Essenberg, R.C. and Sauer, J.R., 1982. Protein changes in the salivary glands of the female Lone Star tick, Amblyomma americanum. J. Parasitol., 68:100-106. Oleaga, A., P6rez, R. and Encinas, A., 1990. Distribution and biology of Ornithodoros erraticus in the African Swine Fever enzootic area of Spain. Vet. Rec., 126: 32-37. Pastor, M.J., Laviada, M.D., Sfinchez-Vizcaino, J.M. and Escribano, J.M., 1989. Serodiagnosis of African Swine Fever by immunoblotting assay. Can. J. Vet. Res., 53:105-107. Sanchez Botija, C., 1963. Reservorios del virus de la Peste Porcina Africana. Investigaci6n del
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virus de la P.P.A. en los artropodos mediante la prueba de la hemoadsorci6n. Bull. Off. Int. Epizoot., 60: 895-899. S~nchez-Vizcaino, J.M., Tabares, E., Salvador, E. and Ordas, A., 1982. Semipurified structural viral protein for the detection of ASF antibodies by the indirect ELISA. Curr. Top. Vet. Med. Anim. Sci., 22: 214-222. Whelen, S.K., Gerorge, J.E., Osburn, R.L. and Wikel, S.K., 1984. Dot-ELISA to examine serologic responses of Bos taurus and Bos indicus to ixodid tick infestations. Fed. Proc., 43: 1628. Wikel, S.K., 1981. The induction of host resistance to tick infestation with salivary gland antigen. Am. J. Trop. Med. Hyg., 30: 284-288. Wikel, S.K., 1982. Immune responses to arthropods and their product. Annu. Rev. Entomol., 27:21-48. Wikel, S.K. and Osburn, R.L., 1982. Immune responsiveness of the bovine host to repeated low level infestations with Dermacentor andersoni. Ann. Trop. Med. Parasitol., 76:405-414. Wikel, S.K., 1984. Immunomodulation of host responses to ectoparasite infestation - an overview. Vet. Parasitol., 14: 321-339. Wikel, S.K. and Whelen, A.C., 1986. Ixodid host immune interaction. Identification and characterization of relevant antigens and tick-induced host immunosuppression. Vet. Parasitol., 20: 149-174. Willadsen, P., 1980. Immunity to ticks. Adv. Parasitol., 18:293-313.