Serological Diagnosis of Cysticercosis by an Enzyme-linked Immunosorbent Assay in Experimentally Infected Cattle H.J. Smith, K.E. Snowdon and R.C. Finlay ABSTRACT
ete prileves chez tous les sujets durant une periode de 210 jours et ont ete analyses par un essai d'enzymoimmunocaptation (ELISA) utilisant comme antigene une portion du liquide du kyste T. hydatigena au stade larvaire pour la presence d'anticorps (IgG) anti-T. saginata. A l'abatage, la langue, les masseters, le diaphragme, le myocarde et le foie ont ete examin6s pour la presence de cysticerques. Une correlation entre la quantite d'oeufs ingeres et le nombre de cysticerques a ete retrouvee lors de l'autopsie. Une correlation a aussi ete trouvee entre les niveaux d'anticorps et la quantite d'oeufs ingeres. Une seroconversion au 25me jour apres l'infection lors d'infections severes et plus tard dans les cas benins a ete observee. Les titres maximums d'anticorps se sont situes entre 40 et 60 jours postinfection. Un deuxieme pic d'anticorps est survenu entre 160 et 200 jours apres l'infection, lequel serait cause par le relachement d'antigene apres la mort des cysticerques. Les faibles concentrations d'anticorps circulants lors d'infections legeres peuvent nous amener a deduire des resultats faux positifs ou negatifs dependamment du seuil choisi. (Traduit par Dr Yves Larouche)
Taenia saginata infections were established in four groups of calves by administering doses of 10, 102, 103 and 104 infective eggs respectively by gavage. A fifth group remained as uninfected controls. Sera were collected from all calves over a period of 210 days. The sera were examined by an enzyme-linked immunosorbent assay (ELISA) with a fraction of larval Taenia hydatigena cyst fluid as antigen for the presence of antiT. saginata IgG antibodies. At slaughter, the tongue, masseter, diaphragm and cardiac muscles and liver were examined for cysticerci. The higher dose rates of T. saginata eggs were reflected in higher numbers of cysticerci found in the calves at necropsy. There was also a correlation between higher levels of antibodies produced as measured by the ELISA and the numbers of eggs given. Seroconversion was first detected about 25 days postinfection in heavy infections and later in the lighter infections. Maximal levels of antibody occurred between 40 and 60 days postinfection, followed by a gradual decrease in levels of antibody. A secondary increase in antibody occurred between 160 and 200 days postinfection which might INTRODUCTION have been due to release of antigen after death of the cysticerci. The low Traditionally, diagnosis of cysticerlevel of circulating antibodies in light infections may result in false positive cosis in cattle is done at slaughter by direct inspection but is notoriously or false negative diagnoses depending insensitive (1). Consequently, efforts upon the selection of the cut-off point. to develop more reliable diagnostic RESUME techniques, including antemortem serological methods, have been underDes doses infectantes de 0, 10, 102, taken. Rhoads and colleagues showed 103, et 104 oeufs de Taenia saginata that a fraction of larval Taenia furent administrees par voie orale a hydatigena cyst fluid (ThFAS antigen) cinq groupes de veaux. Des serums ont has sensitivity and specificity in the
enzyme-linked immunosorbent assay (ELISA) for the detection of bovine anti-Taenia saginata antibodies (2,3). In experimental calves this test detected IgG and IgM antibody levels by the third week until slaughter at 26 weeks (3). On the other hand, Smith et al (4) observed a high rate of false negative and false positive reactions when the ELISA with the ThFAS antigen was used in an outbreak of cysticercosis in feedlot cattle with light infections. To determine if the rate and development of anti-T. saginata antibodies are related to the challenge dose of infective eggs and the number of the lesions produced, experimental infections of varying magnitudes were established in cattle. MATERIALS AND METHODS ANIMALS
Fifteen four month old Hereford (3/4) x Angus (1/4) calves were used in the study. They were maintained in a feedlot situation on a concrete pad with bunk feeders. The ration was alfalfa cubes and wheat straw. The Canadian Council on Animal Care guidelines outlined in the "Guide to the Care and Use of Experimental Animals, Volume I" were followed. TAENIA SAGINA TA
Infective T. saginata eggs were obtained from the Parasitology and Tropical Medicine Institute, Toronto, Ontario. Fecal material, fluids and tapeworm segments were collected from an infected patient following treatment and purging. The eggs were freed from the fecal material and debris and concentrated by repeated washing and sedimentation with warm water.
Agriculture Canada, Health of Animals Laboratory, P.O. Box 1410, Sackville, New Brunswick EOA 3C0 (Smith, Snowdon) and Agriculture Canada, Animal Diseases Research Institute, P.O. Box 640, Lethbridge, Alberta TIJ 3Z4 (Finlay). Submitted October 1, 1990.
274
Can J Vet Res 1991; 55: 274-276
EXPERIMENTAL DESIGN
The calves were divided into five groups of three animals each. Group 1 calves were uninoculated controls. Groups 2, 3, 4 and 5 calves were given 10, 102, 103 and 104 T. saginata eggs respectively by gavage. Sera from all calves were examined on days 0, 7, 9, 11, 15, 16, 18, 21, 25, 28, 31, 35, 39, 42, 56, 70, 98, 126, 154, 182 and 210 postinfection for the presence of anti-T. saginata antibodies. At the conclusion of the trial the calves were slaughtered and selected organs were examined for the presence of cysticerci.
1.5
0-0 Control 0o-0 10
0
/ Os
1.2 -
~A-A
\
LC)
0.9
-
0.6
-
0.3
-
Un 0
nn0
C')
A
NECROPSY TECHNIQUES
The organs examined for the presof cysticerci were the tongue, masseter muscles, diaphragm, crural muscles, liver and the heart. The tongue was transversely sectioned at approximately 5 mm intervals from the anterior to the posterior extremities. The masseter muscles on both sides of the face were sliced from front to back parallel to the zygomatic ridge beginning just beneath it and proceeding downward. The diaphragm was removed by cutting it away from its attachment to the parietal pleura internal to the ribs. The crura were left attached to the diaphragm and were also removed. All muscle tissues were sliced at 5 mm intervals. The liver was similarly sliced throughout.
io2
v-v 103
A
I--,
48
0.0' 40
0
80
160
120
200
ence
Days postinfection Fig. 1. Mean absorbance by ELISA using ThFAS antigen of sera of five groups of cattle challenged with 0, 10, 102, 103 and 104 infective Taenia saginata larvae and examined from 0 to 210 days postinfection.
RESULTS GROSS LESIONS
Three types of lesions were found. Type 1 lesions were designated "normal" or presumably infective cysts. These cysts were approximately 0.7 cm in diameter, clear and translucent with a scolex visible through the cyst wall. These cysts were not common and were found in masseter and tongue muscles. Type 2 lesions were not discernible as cysts but were round or spherical with sand-like granules inside LABORATORY PROCEDURES the cyst. The cyst walls were Examination of sera for the presence still collapsed so that the granules transparent of anti-T. saginata IgG antibodies was could be clearly seen. The were carried out in duplicate by the ELISA up to 1 cm in diameter. lesions These were triple antibody procedure using the seen most commonly on the epicarThFAS antigen of Rhoads et al (2) and dium. Type 3 lesions were smaller in rabbit antibovine IgG (heavy and light diameter than either of the above chains specific) antisera (Organon (up to 4 mm). When cut, they Teknika Corporation, Scarborough, lesions had caseous or calcified centers and Ontario), goat antirabbit IgG horse- were the predominant lesion within radish peroxidase conjugate (Organon cardiac muscle. No lesions were found Teknika Corporation, Scarborough, in the control calves. Lesions were Ontario) and 2,2 '-azino-bis (3- found in all calves challenged with ethylbenzothiazoline-6-sulfonic acid) T. saginata eggs except for one calf in diammonium salt (ABTS) substrate group 2. The mean number of lesions (Sigma Chemical Co., St. Louis, found in the five groups of calves was Missouri). An absorbance reading at as follows: 1 0, group 2 2, 405 nm 3x mean of three negative group 3 Group 12 and group 2, group 4 (preinfection) sera was selected as posi- 5 56. tive for anti-Taenia antibodies. Sera from all groups of steers collected on SEROLOGICAL FINDINGS each bleeding date were examined on The mean absorbance of sera from the same plate. the various groups of calves examined
by ELISA using the ThFAS antigen is given in Fig. 1. The level of antiT. saginata IgG antibodies developed correlated with the challenge doses of infective eggs. Maximal peaks of antibody occurred in all groups except group 2 between 50 and 60 days postinfection. In calves receiving 104 T. saginata eggs, seroconversion was initially detected about 25 days postinfection and later in groups receiving lighter infections. Antibody levels tended to decrease gradually after peaking until a secondary rise was detected between 160 and 200 days postinfection. The mean optical density readings (absorbance) of sera from the control calves was 0.023. DISCUSSION This work demonstrates that the level of anti-T. saginata IgG antibodies was directly related to challenge dose of infective eggs and the number of cysts established in cattle. Even though the amount of antibody as indicated by absorbance was dose related, a clearly defined rise in level of detectable IgG occurred in most groups of calves between 40 and 60 days postinfection. In heavy infections, an initial rise of antibody was first detected at about 25 days postinfection. Following the peak in antibody levels between 40 and 60 days postinfection, a gradual 275
decline in antibody levels occurred until a secondary rise occurred between 160 and 200 days postinfection. This latter rise is possibly due to the death, caseation, and calcification of cysts, with release of antigens. Two peaks of antibody have been reported previously for taeniosis in cattle using the indirect hemagglutination test but the peaks were between 18-24 days and 81-101 days (5). The results of this study support previous findings (2,3) that the ELISA using the ThFAS antigen is sensitive for anti-Taenia antibodies. However the low levels of antibody produced in light infections pose difficulties in selecting a positive cut-off absorbance value and in interpreting the findings. Using the selected criterion of 2 3x mean of the optical density readings (absorbance) of negative sera as positive would likely result in a few false positives particularly of individual samples considering the mean absorbance of control sera in the study was 0.023. On the other hand, increasing the cut off to 2 4 or 5 x mean of negative sera would probably result in some false negatives, in animals with light infections. The difficulties in using this assay are not with its sensitivity or specificity but with the low levels of antibody produced in cattle by very light T. saginata infections. The absorbance of negative sera from the cattle of this study is typical of that seen in older cattle and higher than that of sera from very young calves.
276
(Smith, H.J. unpublished data). Geerts et al (6) demonstrated false positive reactions with ELISA in serial serum samples of experimental calves which they suggested were caused through transfer of colostral antibodies and which waned with increasing age of the
calves. The mean number of cysts found in the various groups of cattle was found to be dose-related but did not indicate the total number that may have been present since all of the musculature of each carcass was not examined. The muscles examined were those that are routinely examined during postmortem inspection in the abattoir. The ELISA using the ThFAS antigen should not be used to detect cattle infected with larval T. saginata because low levels of antibody appear to be produced in light infections. Therefore the usefulness of the ELISA is perhaps limited to epidemiological studies. It should not be used to certify beef and beef products free of cysticercosis. ACKNOWLEDGMENTS The authors are indebted to Dr. D. Gregory, Agriculture Canada, Ottawa, Ontario for making the arrangements and to Dr. Keystone at the Parasitology and Tropical Medicine Institute, Toronto, Ontario for collecting and providing the T. saginata infected material used to infect the experimental cattle.
The assistance of the animal attendants at ADRI, Lethbridge in the collection of specimens and the care of the experimental cattle is much appreciated. The authors are grateful to Dr. P.H.G. Stockdale, Animal Diseases Research Institute, Lethbridge, Alberta, for the postmortem examination of the carcasses and classifying the lesions.
REFERENCES 1. MURRELL KD, FAYER R, DUBEY JP. Parasitic organisms. Advances in Meat Research 1986; 2: 311-376. 2. RHOADS ML, MURRELL KD, DILLING GW, WONG MM, BAKER NF. A potential diagnostic reagent for bovine cysticercosis. J Parasitol 1985; 71: 779-787. 3. KAMANGA-SOLLO EIP, RHOADS ML, MURRELL KD. Evaluation of an antigenic fraction of Taenia hydatigena metacestode cyst fluid for immunodiagnosis of bovine cysticercosis. Am J Vet Res 1987; 48: 1206-1210. 4. SMITH HJ, SNOWDON KE, GREGORY D, FINLEY GG. Assessment of an enzymelinked immunosorbent assay using a Taenia hydatigena fraction antigen in the diagnosis of cysticercosis in cattle. Can J Vet Res 1990; 54: 299-300. 5. PAWLOWSKI Z, SCHULTZ MG. Taeniosis and cysticercosis (Taenia saginata). In: Dawes B, ed. Advances in Parasitology. New York: Academic Press, 1972; 10: 269-343. 6. GEERTS S, KUMAR V, AERTS N, CEUCEMANS F. Comparative evaluation of immunoelectrophoresis, counterimmunoelectrophoresis and enzyme linked immunosorbent assay for the diagnosis of Taenia saginata cysticercosis. Vet Parasitol 1981; 8: 299-307.
ete prileves chez tous les sujets durant une periode de 210 jours et ont ete analyses par un essai d'enzymoimmunocaptation (ELISA) utilisant comme antigene une portion du liquide du kyste T. hydatigena au stade larvaire pour la presence d'anticorps (IgG) anti-T. saginata. A l'abatage, la langue, les masseters, le diaphragme, le myocarde et le foie ont ete examin6s pour la presence de cysticerques. Une correlation entre la quantite d'oeufs ingeres et le nombre de cysticerques a ete retrouvee lors de l'autopsie. Une correlation a aussi ete trouvee entre les niveaux d'anticorps et la quantite d'oeufs ingeres. Une seroconversion au 25me jour apres l'infection lors d'infections severes et plus tard dans les cas benins a ete observee. Les titres maximums d'anticorps se sont situes entre 40 et 60 jours postinfection. Un deuxieme pic d'anticorps est survenu entre 160 et 200 jours apres l'infection, lequel serait cause par le relachement d'antigene apres la mort des cysticerques. Les faibles concentrations d'anticorps circulants lors d'infections legeres peuvent nous amener a deduire des resultats faux positifs ou negatifs dependamment du seuil choisi. (Traduit par Dr Yves Larouche)
Taenia saginata infections were established in four groups of calves by administering doses of 10, 102, 103 and 104 infective eggs respectively by gavage. A fifth group remained as uninfected controls. Sera were collected from all calves over a period of 210 days. The sera were examined by an enzyme-linked immunosorbent assay (ELISA) with a fraction of larval Taenia hydatigena cyst fluid as antigen for the presence of antiT. saginata IgG antibodies. At slaughter, the tongue, masseter, diaphragm and cardiac muscles and liver were examined for cysticerci. The higher dose rates of T. saginata eggs were reflected in higher numbers of cysticerci found in the calves at necropsy. There was also a correlation between higher levels of antibodies produced as measured by the ELISA and the numbers of eggs given. Seroconversion was first detected about 25 days postinfection in heavy infections and later in the lighter infections. Maximal levels of antibody occurred between 40 and 60 days postinfection, followed by a gradual decrease in levels of antibody. A secondary increase in antibody occurred between 160 and 200 days postinfection which might INTRODUCTION have been due to release of antigen after death of the cysticerci. The low Traditionally, diagnosis of cysticerlevel of circulating antibodies in light infections may result in false positive cosis in cattle is done at slaughter by direct inspection but is notoriously or false negative diagnoses depending insensitive (1). Consequently, efforts upon the selection of the cut-off point. to develop more reliable diagnostic RESUME techniques, including antemortem serological methods, have been underDes doses infectantes de 0, 10, 102, taken. Rhoads and colleagues showed 103, et 104 oeufs de Taenia saginata that a fraction of larval Taenia furent administrees par voie orale a hydatigena cyst fluid (ThFAS antigen) cinq groupes de veaux. Des serums ont has sensitivity and specificity in the
enzyme-linked immunosorbent assay (ELISA) for the detection of bovine anti-Taenia saginata antibodies (2,3). In experimental calves this test detected IgG and IgM antibody levels by the third week until slaughter at 26 weeks (3). On the other hand, Smith et al (4) observed a high rate of false negative and false positive reactions when the ELISA with the ThFAS antigen was used in an outbreak of cysticercosis in feedlot cattle with light infections. To determine if the rate and development of anti-T. saginata antibodies are related to the challenge dose of infective eggs and the number of the lesions produced, experimental infections of varying magnitudes were established in cattle. MATERIALS AND METHODS ANIMALS
Fifteen four month old Hereford (3/4) x Angus (1/4) calves were used in the study. They were maintained in a feedlot situation on a concrete pad with bunk feeders. The ration was alfalfa cubes and wheat straw. The Canadian Council on Animal Care guidelines outlined in the "Guide to the Care and Use of Experimental Animals, Volume I" were followed. TAENIA SAGINA TA
Infective T. saginata eggs were obtained from the Parasitology and Tropical Medicine Institute, Toronto, Ontario. Fecal material, fluids and tapeworm segments were collected from an infected patient following treatment and purging. The eggs were freed from the fecal material and debris and concentrated by repeated washing and sedimentation with warm water.
Agriculture Canada, Health of Animals Laboratory, P.O. Box 1410, Sackville, New Brunswick EOA 3C0 (Smith, Snowdon) and Agriculture Canada, Animal Diseases Research Institute, P.O. Box 640, Lethbridge, Alberta TIJ 3Z4 (Finlay). Submitted October 1, 1990.
274
Can J Vet Res 1991; 55: 274-276
EXPERIMENTAL DESIGN
The calves were divided into five groups of three animals each. Group 1 calves were uninoculated controls. Groups 2, 3, 4 and 5 calves were given 10, 102, 103 and 104 T. saginata eggs respectively by gavage. Sera from all calves were examined on days 0, 7, 9, 11, 15, 16, 18, 21, 25, 28, 31, 35, 39, 42, 56, 70, 98, 126, 154, 182 and 210 postinfection for the presence of anti-T. saginata antibodies. At the conclusion of the trial the calves were slaughtered and selected organs were examined for the presence of cysticerci.
1.5
0-0 Control 0o-0 10
0
/ Os
1.2 -
~A-A
\
LC)
0.9
-
0.6
-
0.3
-
Un 0
nn0
C')
A
NECROPSY TECHNIQUES
The organs examined for the presof cysticerci were the tongue, masseter muscles, diaphragm, crural muscles, liver and the heart. The tongue was transversely sectioned at approximately 5 mm intervals from the anterior to the posterior extremities. The masseter muscles on both sides of the face were sliced from front to back parallel to the zygomatic ridge beginning just beneath it and proceeding downward. The diaphragm was removed by cutting it away from its attachment to the parietal pleura internal to the ribs. The crura were left attached to the diaphragm and were also removed. All muscle tissues were sliced at 5 mm intervals. The liver was similarly sliced throughout.
io2
v-v 103
A
I--,
48
0.0' 40
0
80
160
120
200
ence
Days postinfection Fig. 1. Mean absorbance by ELISA using ThFAS antigen of sera of five groups of cattle challenged with 0, 10, 102, 103 and 104 infective Taenia saginata larvae and examined from 0 to 210 days postinfection.
RESULTS GROSS LESIONS
Three types of lesions were found. Type 1 lesions were designated "normal" or presumably infective cysts. These cysts were approximately 0.7 cm in diameter, clear and translucent with a scolex visible through the cyst wall. These cysts were not common and were found in masseter and tongue muscles. Type 2 lesions were not discernible as cysts but were round or spherical with sand-like granules inside LABORATORY PROCEDURES the cyst. The cyst walls were Examination of sera for the presence still collapsed so that the granules transparent of anti-T. saginata IgG antibodies was could be clearly seen. The were carried out in duplicate by the ELISA up to 1 cm in diameter. lesions These were triple antibody procedure using the seen most commonly on the epicarThFAS antigen of Rhoads et al (2) and dium. Type 3 lesions were smaller in rabbit antibovine IgG (heavy and light diameter than either of the above chains specific) antisera (Organon (up to 4 mm). When cut, they Teknika Corporation, Scarborough, lesions had caseous or calcified centers and Ontario), goat antirabbit IgG horse- were the predominant lesion within radish peroxidase conjugate (Organon cardiac muscle. No lesions were found Teknika Corporation, Scarborough, in the control calves. Lesions were Ontario) and 2,2 '-azino-bis (3- found in all calves challenged with ethylbenzothiazoline-6-sulfonic acid) T. saginata eggs except for one calf in diammonium salt (ABTS) substrate group 2. The mean number of lesions (Sigma Chemical Co., St. Louis, found in the five groups of calves was Missouri). An absorbance reading at as follows: 1 0, group 2 2, 405 nm 3x mean of three negative group 3 Group 12 and group 2, group 4 (preinfection) sera was selected as posi- 5 56. tive for anti-Taenia antibodies. Sera from all groups of steers collected on SEROLOGICAL FINDINGS each bleeding date were examined on The mean absorbance of sera from the same plate. the various groups of calves examined
by ELISA using the ThFAS antigen is given in Fig. 1. The level of antiT. saginata IgG antibodies developed correlated with the challenge doses of infective eggs. Maximal peaks of antibody occurred in all groups except group 2 between 50 and 60 days postinfection. In calves receiving 104 T. saginata eggs, seroconversion was initially detected about 25 days postinfection and later in groups receiving lighter infections. Antibody levels tended to decrease gradually after peaking until a secondary rise was detected between 160 and 200 days postinfection. The mean optical density readings (absorbance) of sera from the control calves was 0.023. DISCUSSION This work demonstrates that the level of anti-T. saginata IgG antibodies was directly related to challenge dose of infective eggs and the number of cysts established in cattle. Even though the amount of antibody as indicated by absorbance was dose related, a clearly defined rise in level of detectable IgG occurred in most groups of calves between 40 and 60 days postinfection. In heavy infections, an initial rise of antibody was first detected at about 25 days postinfection. Following the peak in antibody levels between 40 and 60 days postinfection, a gradual 275
decline in antibody levels occurred until a secondary rise occurred between 160 and 200 days postinfection. This latter rise is possibly due to the death, caseation, and calcification of cysts, with release of antigens. Two peaks of antibody have been reported previously for taeniosis in cattle using the indirect hemagglutination test but the peaks were between 18-24 days and 81-101 days (5). The results of this study support previous findings (2,3) that the ELISA using the ThFAS antigen is sensitive for anti-Taenia antibodies. However the low levels of antibody produced in light infections pose difficulties in selecting a positive cut-off absorbance value and in interpreting the findings. Using the selected criterion of 2 3x mean of the optical density readings (absorbance) of negative sera as positive would likely result in a few false positives particularly of individual samples considering the mean absorbance of control sera in the study was 0.023. On the other hand, increasing the cut off to 2 4 or 5 x mean of negative sera would probably result in some false negatives, in animals with light infections. The difficulties in using this assay are not with its sensitivity or specificity but with the low levels of antibody produced in cattle by very light T. saginata infections. The absorbance of negative sera from the cattle of this study is typical of that seen in older cattle and higher than that of sera from very young calves.
276
(Smith, H.J. unpublished data). Geerts et al (6) demonstrated false positive reactions with ELISA in serial serum samples of experimental calves which they suggested were caused through transfer of colostral antibodies and which waned with increasing age of the
calves. The mean number of cysts found in the various groups of cattle was found to be dose-related but did not indicate the total number that may have been present since all of the musculature of each carcass was not examined. The muscles examined were those that are routinely examined during postmortem inspection in the abattoir. The ELISA using the ThFAS antigen should not be used to detect cattle infected with larval T. saginata because low levels of antibody appear to be produced in light infections. Therefore the usefulness of the ELISA is perhaps limited to epidemiological studies. It should not be used to certify beef and beef products free of cysticercosis. ACKNOWLEDGMENTS The authors are indebted to Dr. D. Gregory, Agriculture Canada, Ottawa, Ontario for making the arrangements and to Dr. Keystone at the Parasitology and Tropical Medicine Institute, Toronto, Ontario for collecting and providing the T. saginata infected material used to infect the experimental cattle.
The assistance of the animal attendants at ADRI, Lethbridge in the collection of specimens and the care of the experimental cattle is much appreciated. The authors are grateful to Dr. P.H.G. Stockdale, Animal Diseases Research Institute, Lethbridge, Alberta, for the postmortem examination of the carcasses and classifying the lesions.
REFERENCES 1. MURRELL KD, FAYER R, DUBEY JP. Parasitic organisms. Advances in Meat Research 1986; 2: 311-376. 2. RHOADS ML, MURRELL KD, DILLING GW, WONG MM, BAKER NF. A potential diagnostic reagent for bovine cysticercosis. J Parasitol 1985; 71: 779-787. 3. KAMANGA-SOLLO EIP, RHOADS ML, MURRELL KD. Evaluation of an antigenic fraction of Taenia hydatigena metacestode cyst fluid for immunodiagnosis of bovine cysticercosis. Am J Vet Res 1987; 48: 1206-1210. 4. SMITH HJ, SNOWDON KE, GREGORY D, FINLEY GG. Assessment of an enzymelinked immunosorbent assay using a Taenia hydatigena fraction antigen in the diagnosis of cysticercosis in cattle. Can J Vet Res 1990; 54: 299-300. 5. PAWLOWSKI Z, SCHULTZ MG. Taeniosis and cysticercosis (Taenia saginata). In: Dawes B, ed. Advances in Parasitology. New York: Academic Press, 1972; 10: 269-343. 6. GEERTS S, KUMAR V, AERTS N, CEUCEMANS F. Comparative evaluation of immunoelectrophoresis, counterimmunoelectrophoresis and enzyme linked immunosorbent assay for the diagnosis of Taenia saginata cysticercosis. Vet Parasitol 1981; 8: 299-307.
