RESEARCHNOTE ANTIBODY
RESPONSES AGAINST NATURAL 1”AENIA HYllA TIGENA INFECTION IN DOGS IN KENYA D. J. JENKINS,* R. B. GASSER,~ T. ROMIG~ and E. ZEYHLE$
*South-East New South Wales and Australian Capital Territory Hydatid Control Campaign, P.O. Box 112, Queanbeyan, New South Wales 2620, Australia tuniversity of Melbourne, Veterinary Clinical Centre, Werribee, Victoria 3030, Australia $African Medical Research Foundation (AMREF), P.O. Box 30125, Nairobi, Kenya (Received9 October 1990; accepted 30 November 1990) ASSET&-JENKINS
D. J., GASSERR. B., ROMIG T. and ZEYHLE E. 1991. Antibody responses against natural Taenia hydatigena infection in dogs in Kenya. International Journalfor Parasitology 21: 251-253. Antibody responses (IgG) against Tuenia hydatigena infection in dogs in Kenya were analysed in ELISA using exc~tory/~retory products of 7: hydutige~u scokces derived from goat cysticercus cysts. Helming infections of individual dogs were confirmed at autopsy. T. hy~tjge~ worms were found in 89.5% of I43 dogs, and positive anti-r hydatigena antibody levels were detected in 58.7% of infected dogs. Positive antiscolex antibody levels were detected in 40.0% of Turkana dogs uninfected with T. hydatigena, suggesting previous infection. Antibody was not detected in 34.4% of infected dogs. There was no relationship between individual T. hydutigena worm burdens and absorbance values for sera in ELISA. It was not possible to distinguish between sera from T. hydntigena-infected and uninfected dogs. INDEX KEY WORDS: Taenia hydatigena; dogs; antibody responses; Kenya.
EGGS of taeniid cestodes are morphologically indistinguishable by light microscopy. Diagnosis of taeniid cestode infections in the definitive host species (dog) is usually performed by purging with arecoline hydrobromide and examining purge sampies for the species of worms recovered. This technique is time consuming, and some dogs suffer from drastic sideeffects of arecoline. Recently, alternative techniques have been developed for diagnosis of taeniid cestode infections in dogs (Jenkins & Rickard, 1985; Heath, Lawrence & Oudemans, lP88; Craig, Macpherson, Watson-Jones & Nelson, 1988; Gasser, Lightowlers, Obendorf, Jenkins & Rickard, 1988). Immunodetection of antigens in dog faeces has been reported for diagnosis of T. hydatigena infection in dogs (Deplazes, Gottstein, Stingelin & Eckert, 1990) and a previous study showed much promise toward developing a practical test for serodiagnosis of naturally acquired infection (Jenkins & Rickard, 1985). Using an enzymelinked immunosorbent assay (ELISA), specific anti-T. hydutigena antibody responses (IgG) were detected in experimental dogs with monospecific T. hydutigena infection. In contrast to detection of antigens or eggs in faeces, a serological test system has the potential to diagnose both ‘current’ and ‘past’ infections. The aims of this study were to analyse antibody responses in dogs against naturally acquired T. hydutigena infection in an endemic region of Kenya and evaluate sensitivity and specificity of an ELISA for detection of infected dogs.
Dogs (Canis familiaris) shot in north-western Turkana (Oropoi, Nanam, Lokichoggio, Lakankai, Kalobayei, Kakuma) were examined. Blood samples for serum separation were collected, and helminth burdens of individual dogs were recorded at autopsy. Taenia worms were classified according to Verster (1969). Sera were tested in ELBA according to the method of Gasser et al. (1988). Antigen for the ELBA was prepared from excretory/secretory products of T. hydatigena scoleces (E/S) according to the method of Jenkins & Rickard (1985). Metacestodes from goats naturally infected with T. h~d~r~gen~ were collected in a Nairobi abattoir. An absorbance cut-off value in ELISA above which sera were regarded as having ‘positive’ reactions was determined (mean absorbance value + 2 S.D. = 0.23) using 16 sera from Turkana dogs raised and maintained helminth-free according to Jenkins & Rickard (1985).
TABLEI-ELISA RESULTS OF 143 SERAFROMTURKANA DOGS TESTED USINGTaenia hydatigena SCOLEX EXCRETORY/SECRETORY ANTIGEN
ELISA result Positive Negative Total 2.51
No. of dogs Infected Not infected with Tuenia hydatigerw 84 44 128
6 9 15
Total 90 53 143
D. J. JENKINS,R. B.
252
GASSER,
T. ROMIGand E. ZEYHLE
TABLE2-HELMINTHOLOGICAL DATA AND MEAN ABSORBANCE VALUES OF SERA FROM143 TURKANA DOGS (GROUPS A AND BITESTED INELISA USING Taenia hydatigena SCOLEX EXCRE~ORY~ECRETORY ANTIGEN Dog sera
No. of sera tested
Mean absorbance values in ELISA (S.D)
A. Dogs with hehninth infections
T. hydatigena* T. hydatigena and Echinococcus eranulosust E. granulosus ” B. Dogs without helminth infections Total C. Control dogs Positive Negative Helminth-free dogs
82
0.45 (0.35)
46 4
0.52 (0.31) 0.45 (0.43j
11 143
0.28 (0.32)
I 1 16
0.86 0.03 0.07 (0.08)
* Three dogs also infected with Ancyfostoma caninum. t One dog also infected with A. caninum and another with Dipylidium can&urn.
Results of 143 dog sera tested in ELISA are shown in Table 1. Sensitivity and specificity of the test were calculated (Thorner & Remein, 1967) to be 65.6 and 60.0%, respectively. Helminthological data of the dogs and mean absorbance values of individual serum groups are shown in Table 2. T. hydatigena was found in 89.5% of 143 dogs. Infections with ~c~~nococc~~ granulosus, ~ipy~idi~m caninum and Ancyfostoma caninum were present in 35.0,0.7 and 2.0% of all dogs, respectively. Mixed infections of T. hydatigena and E. granulosus were found in 32.2% of the 143 dogs. There was no correlation between T. hydatigena worm burdens in individual dogs and absorbance values of sera in ELBA (data not presented). Anti-T. hy~~igena E/S antibodies were detected in 62.9% of 143 dog sera tested in ELISA. However, it was not possible to distinguish between sera from T. hydatigena infected dogs and sera from dogs found not to harbour the parasite at autopsy. Positive anti-E/S antibody levels were measured in 40.0% of Turkana dogs uninfected with T. hy~~igena, suggesting that they had had previous infection. Due to the hyperendemic situation of T. hydatigena cysticercosis in Turkana, it is likely that dogs frequently become reinfected. Also, dogs in this region were observed frequently to ingest faeces from other dogs (D. J. Jenkins, unpublished observation), and ingestion of faeces from T. hy~~igena infected dogs could lead to the absorption of antigens through the gut, resulting in positive serological reactions. It has been postulated that antigens released from any part of an adult tapeworm could be absorbed through the mucosa and generate systemic antibody responses in the definitive host (Rickard, 1983). Recent studies have demonstrated that anti-Taetria antibody levels can persist for several weeks after removal of worms by an~elmintic treatment (Jenkins & Rickard, 1985; Heath et al., 1988). Some sera (34.4%) from T. hydafigena-infected Turkana dogs did not contain detectable levels of anti-
scolex antibodies. This lack of systemic antibody responses in some dogs has been demonstrated in other recent studies in dogs with E. granulosus infections (Gasser et al., 1988; Jenkins, Gasser, Zeyhle, Romig & Macpherson, 1990) and may be due to either host factors (genetic constitution, nutritional deficiency, presence of other infectious diseases or absence of systemic immune induction via the gut) or parasite factors (strain variation, immune evasion or number of repeated infections). It is also possible that some dogs absorb relatively large amounts of parasite antigens through the intestinal wall, and that the circulating antigens bind antibody thus resulting in ‘false negatives’ in ELISA. The present study demonstrated that the ELISA using excretory/secretory scolex antigens could not be used to reliably diagnose currently infected dogs. The use of well-defined diagnostic antigens may improve specificity and sensitivity of a serodiagnostic test, and these could be purified by immunoabsorption affinity chromato~aphy techniques (Gottstein, Eckert & Fey, 1983) or produced in vitro using recombinant DNA technology (Gasser, Lightowlers & Rickard, 1990). Acknowledgements-The authors are indebted to Dr C. N. L. Macpherson (Swiss Tropical Institute Field Laboratory, Ifakara, Tanzania), Dr T. Arap Siongok (Ministry of Health, Kenya) and all officials for their support during this project. Sincere thanks to Dr C. Wood (AMREF), Dr R. Gray (ILRAD) and Dr M. J. Howell (Australian National University) for comments on the manuscript and to A. Jones, E. Lundu and Z. Sigira for technical assistance. Thanks also to the International Committee of the Red Cross for their support. Financial support was provided by the Australian Counterpart Wheat Fund, made available to AMREF by the Kenyan government. REFERENCES CRAIG P. S., MACPHERSON C. N., WATSON-JONES D. L. & NELSONG. S. 1988. Immunodetection ofEchinococcuseggs from naturally infected dogs and from environmental
Research Note contamination sites in settlements in Turkana, Kenya. Transactions of the Royal Society of Tropical Medicine and Hygiene 82: 268-214. DEP&ZESP., GOT~STEIN B., STINCELIN Y. & ECKERTJ. 1990. Detection of Taenia hydatigena copro-antigens by ELISA in dogs. Veterinary Parasitology 36: 91-139. GASSERR. B., LIGHTOWLER~ M. W., OBENDORF D. L., JENKINS D. J. & RICKARDM. D. 1988. Evaluation of a serological test system for the diagnosis of natural Echinococcus grantdosus infection in dogs using E. granulosus protoscolex and oncosphere antigens. Australian Veterinary Journal 65: 369-373. GASSERR. B., LIGHTOWLERS M. W. & RICKARD M. D. 1990. A recombinant antigen with potential for serodiagnosis of Echinococcus granulosus infection in dogs. International Journal for Parasitology 20: 943-950. GOTTSTEIN B., ECKERTJ. & FEY H. 1983. Serological differentiation between Echinococcus granulosus and E. multilocukaris infections in man. Zeitschrtft fur Parasitenkunde 69: 347-356. HEATHD. D., LAWRENCE S. B. & OUDEMANS G. 1988. A
253
blind test of the serological response of dogs to infection with Taenia ovis. New Zealand Veterinary Journal 36: 143145. JENKINSD. J. & RICKARDM. D. 1985. Specific antibody responses to Taenia hydatigena, Taenia pistformis and Echinococcus granulosus infection in dogs. Australian Veterinary Journal 62: 72-78. JENKINSD. J., GA~SER R. B., ZEYHLE E., ROMIG T. & MACPHERSON C. N. L. 1990. Assessment of a serological test for diagnosis of Echinococcus granulosus infection in dogs in Kenya. Acta Tropica 47: 245-248. RICKARDM. D. 1983. Immunity. In: Biology of the Eucestoda, Vol. 2 (Edited by PAPPASP. & ARMESC.), pp. 539-579. Academic Press, London. THORNERR. M. & REMEINQ. R. 1967. Principles and procedures in the evaluation of screening for disease. Public Health Service Publication No. 846, pp. l-24. U.S. Department of Health, Education and Welfare, Virginia. VERSTER A. 1969. A taxonomic revision of the genus Taenia Linnaeus, 1758 s. str. Onderstepoort Journal of Veterinary Research 36: 3-58.
RESPONSES AGAINST NATURAL 1”AENIA HYllA TIGENA INFECTION IN DOGS IN KENYA D. J. JENKINS,* R. B. GASSER,~ T. ROMIG~ and E. ZEYHLE$
*South-East New South Wales and Australian Capital Territory Hydatid Control Campaign, P.O. Box 112, Queanbeyan, New South Wales 2620, Australia tuniversity of Melbourne, Veterinary Clinical Centre, Werribee, Victoria 3030, Australia $African Medical Research Foundation (AMREF), P.O. Box 30125, Nairobi, Kenya (Received9 October 1990; accepted 30 November 1990) ASSET&-JENKINS
D. J., GASSERR. B., ROMIG T. and ZEYHLE E. 1991. Antibody responses against natural Taenia hydatigena infection in dogs in Kenya. International Journalfor Parasitology 21: 251-253. Antibody responses (IgG) against Tuenia hydatigena infection in dogs in Kenya were analysed in ELISA using exc~tory/~retory products of 7: hydutige~u scokces derived from goat cysticercus cysts. Helming infections of individual dogs were confirmed at autopsy. T. hy~tjge~ worms were found in 89.5% of I43 dogs, and positive anti-r hydatigena antibody levels were detected in 58.7% of infected dogs. Positive antiscolex antibody levels were detected in 40.0% of Turkana dogs uninfected with T. hydatigena, suggesting previous infection. Antibody was not detected in 34.4% of infected dogs. There was no relationship between individual T. hydutigena worm burdens and absorbance values for sera in ELISA. It was not possible to distinguish between sera from T. hydntigena-infected and uninfected dogs. INDEX KEY WORDS: Taenia hydatigena; dogs; antibody responses; Kenya.
EGGS of taeniid cestodes are morphologically indistinguishable by light microscopy. Diagnosis of taeniid cestode infections in the definitive host species (dog) is usually performed by purging with arecoline hydrobromide and examining purge sampies for the species of worms recovered. This technique is time consuming, and some dogs suffer from drastic sideeffects of arecoline. Recently, alternative techniques have been developed for diagnosis of taeniid cestode infections in dogs (Jenkins & Rickard, 1985; Heath, Lawrence & Oudemans, lP88; Craig, Macpherson, Watson-Jones & Nelson, 1988; Gasser, Lightowlers, Obendorf, Jenkins & Rickard, 1988). Immunodetection of antigens in dog faeces has been reported for diagnosis of T. hydatigena infection in dogs (Deplazes, Gottstein, Stingelin & Eckert, 1990) and a previous study showed much promise toward developing a practical test for serodiagnosis of naturally acquired infection (Jenkins & Rickard, 1985). Using an enzymelinked immunosorbent assay (ELISA), specific anti-T. hydutigena antibody responses (IgG) were detected in experimental dogs with monospecific T. hydutigena infection. In contrast to detection of antigens or eggs in faeces, a serological test system has the potential to diagnose both ‘current’ and ‘past’ infections. The aims of this study were to analyse antibody responses in dogs against naturally acquired T. hydutigena infection in an endemic region of Kenya and evaluate sensitivity and specificity of an ELISA for detection of infected dogs.
Dogs (Canis familiaris) shot in north-western Turkana (Oropoi, Nanam, Lokichoggio, Lakankai, Kalobayei, Kakuma) were examined. Blood samples for serum separation were collected, and helminth burdens of individual dogs were recorded at autopsy. Taenia worms were classified according to Verster (1969). Sera were tested in ELBA according to the method of Gasser et al. (1988). Antigen for the ELBA was prepared from excretory/secretory products of T. hydatigena scoleces (E/S) according to the method of Jenkins & Rickard (1985). Metacestodes from goats naturally infected with T. h~d~r~gen~ were collected in a Nairobi abattoir. An absorbance cut-off value in ELISA above which sera were regarded as having ‘positive’ reactions was determined (mean absorbance value + 2 S.D. = 0.23) using 16 sera from Turkana dogs raised and maintained helminth-free according to Jenkins & Rickard (1985).
TABLEI-ELISA RESULTS OF 143 SERAFROMTURKANA DOGS TESTED USINGTaenia hydatigena SCOLEX EXCRETORY/SECRETORY ANTIGEN
ELISA result Positive Negative Total 2.51
No. of dogs Infected Not infected with Tuenia hydatigerw 84 44 128
6 9 15
Total 90 53 143
D. J. JENKINS,R. B.
252
GASSER,
T. ROMIGand E. ZEYHLE
TABLE2-HELMINTHOLOGICAL DATA AND MEAN ABSORBANCE VALUES OF SERA FROM143 TURKANA DOGS (GROUPS A AND BITESTED INELISA USING Taenia hydatigena SCOLEX EXCRE~ORY~ECRETORY ANTIGEN Dog sera
No. of sera tested
Mean absorbance values in ELISA (S.D)
A. Dogs with hehninth infections
T. hydatigena* T. hydatigena and Echinococcus eranulosust E. granulosus ” B. Dogs without helminth infections Total C. Control dogs Positive Negative Helminth-free dogs
82
0.45 (0.35)
46 4
0.52 (0.31) 0.45 (0.43j
11 143
0.28 (0.32)
I 1 16
0.86 0.03 0.07 (0.08)
* Three dogs also infected with Ancyfostoma caninum. t One dog also infected with A. caninum and another with Dipylidium can&urn.
Results of 143 dog sera tested in ELISA are shown in Table 1. Sensitivity and specificity of the test were calculated (Thorner & Remein, 1967) to be 65.6 and 60.0%, respectively. Helminthological data of the dogs and mean absorbance values of individual serum groups are shown in Table 2. T. hydatigena was found in 89.5% of 143 dogs. Infections with ~c~~nococc~~ granulosus, ~ipy~idi~m caninum and Ancyfostoma caninum were present in 35.0,0.7 and 2.0% of all dogs, respectively. Mixed infections of T. hydatigena and E. granulosus were found in 32.2% of the 143 dogs. There was no correlation between T. hydatigena worm burdens in individual dogs and absorbance values of sera in ELBA (data not presented). Anti-T. hy~~igena E/S antibodies were detected in 62.9% of 143 dog sera tested in ELISA. However, it was not possible to distinguish between sera from T. hydatigena infected dogs and sera from dogs found not to harbour the parasite at autopsy. Positive anti-E/S antibody levels were measured in 40.0% of Turkana dogs uninfected with T. hy~~igena, suggesting that they had had previous infection. Due to the hyperendemic situation of T. hydatigena cysticercosis in Turkana, it is likely that dogs frequently become reinfected. Also, dogs in this region were observed frequently to ingest faeces from other dogs (D. J. Jenkins, unpublished observation), and ingestion of faeces from T. hy~~igena infected dogs could lead to the absorption of antigens through the gut, resulting in positive serological reactions. It has been postulated that antigens released from any part of an adult tapeworm could be absorbed through the mucosa and generate systemic antibody responses in the definitive host (Rickard, 1983). Recent studies have demonstrated that anti-Taetria antibody levels can persist for several weeks after removal of worms by an~elmintic treatment (Jenkins & Rickard, 1985; Heath et al., 1988). Some sera (34.4%) from T. hydafigena-infected Turkana dogs did not contain detectable levels of anti-
scolex antibodies. This lack of systemic antibody responses in some dogs has been demonstrated in other recent studies in dogs with E. granulosus infections (Gasser et al., 1988; Jenkins, Gasser, Zeyhle, Romig & Macpherson, 1990) and may be due to either host factors (genetic constitution, nutritional deficiency, presence of other infectious diseases or absence of systemic immune induction via the gut) or parasite factors (strain variation, immune evasion or number of repeated infections). It is also possible that some dogs absorb relatively large amounts of parasite antigens through the intestinal wall, and that the circulating antigens bind antibody thus resulting in ‘false negatives’ in ELISA. The present study demonstrated that the ELISA using excretory/secretory scolex antigens could not be used to reliably diagnose currently infected dogs. The use of well-defined diagnostic antigens may improve specificity and sensitivity of a serodiagnostic test, and these could be purified by immunoabsorption affinity chromato~aphy techniques (Gottstein, Eckert & Fey, 1983) or produced in vitro using recombinant DNA technology (Gasser, Lightowlers & Rickard, 1990). Acknowledgements-The authors are indebted to Dr C. N. L. Macpherson (Swiss Tropical Institute Field Laboratory, Ifakara, Tanzania), Dr T. Arap Siongok (Ministry of Health, Kenya) and all officials for their support during this project. Sincere thanks to Dr C. Wood (AMREF), Dr R. Gray (ILRAD) and Dr M. J. Howell (Australian National University) for comments on the manuscript and to A. Jones, E. Lundu and Z. Sigira for technical assistance. Thanks also to the International Committee of the Red Cross for their support. Financial support was provided by the Australian Counterpart Wheat Fund, made available to AMREF by the Kenyan government. REFERENCES CRAIG P. S., MACPHERSON C. N., WATSON-JONES D. L. & NELSONG. S. 1988. Immunodetection ofEchinococcuseggs from naturally infected dogs and from environmental
Research Note contamination sites in settlements in Turkana, Kenya. Transactions of the Royal Society of Tropical Medicine and Hygiene 82: 268-214. DEP&ZESP., GOT~STEIN B., STINCELIN Y. & ECKERTJ. 1990. Detection of Taenia hydatigena copro-antigens by ELISA in dogs. Veterinary Parasitology 36: 91-139. GASSERR. B., LIGHTOWLER~ M. W., OBENDORF D. L., JENKINS D. J. & RICKARDM. D. 1988. Evaluation of a serological test system for the diagnosis of natural Echinococcus grantdosus infection in dogs using E. granulosus protoscolex and oncosphere antigens. Australian Veterinary Journal 65: 369-373. GASSERR. B., LIGHTOWLERS M. W. & RICKARD M. D. 1990. A recombinant antigen with potential for serodiagnosis of Echinococcus granulosus infection in dogs. International Journal for Parasitology 20: 943-950. GOTTSTEIN B., ECKERTJ. & FEY H. 1983. Serological differentiation between Echinococcus granulosus and E. multilocukaris infections in man. Zeitschrtft fur Parasitenkunde 69: 347-356. HEATHD. D., LAWRENCE S. B. & OUDEMANS G. 1988. A
253
blind test of the serological response of dogs to infection with Taenia ovis. New Zealand Veterinary Journal 36: 143145. JENKINSD. J. & RICKARDM. D. 1985. Specific antibody responses to Taenia hydatigena, Taenia pistformis and Echinococcus granulosus infection in dogs. Australian Veterinary Journal 62: 72-78. JENKINSD. J., GA~SER R. B., ZEYHLE E., ROMIG T. & MACPHERSON C. N. L. 1990. Assessment of a serological test for diagnosis of Echinococcus granulosus infection in dogs in Kenya. Acta Tropica 47: 245-248. RICKARDM. D. 1983. Immunity. In: Biology of the Eucestoda, Vol. 2 (Edited by PAPPASP. & ARMESC.), pp. 539-579. Academic Press, London. THORNERR. M. & REMEINQ. R. 1967. Principles and procedures in the evaluation of screening for disease. Public Health Service Publication No. 846, pp. l-24. U.S. Department of Health, Education and Welfare, Virginia. VERSTER A. 1969. A taxonomic revision of the genus Taenia Linnaeus, 1758 s. str. Onderstepoort Journal of Veterinary Research 36: 3-58.
