Scand. J. Immunol. 36, Suppl. 11, 103-106, 1992
Anaplasmosis in Uganda. I. Use of Dried Blood on Filter Paper and Serum Samples for Serodiagnosis of Anaplasmosis—a Comparative Study G. S. Z. S S E N Y O N G A , S. M O N T E N E G R O - J A M E S , I. K A K O M A & R. H A N S E N Makerere University, Faculty of Veterinary Medicine, Kampala, Uganda, and University of Illinois, College of Veterinary Medicine, Urbana, IL, USA
Ssenyonga GSZ, Montenegro-James S, Kakoma I, Hansen R. Anaplasmosis in Uganda. I. Use of Dried Blood on Filter Paper and Serum Samples for Serodiagnosis of Anaplasmosis—a Comparative Study. Seand J Immunol 1992;36(Suppl.ll):103-6 The suitability of blood eollected on filter papers in comparison with corresponding conventional serum samples in the diagnosis of bovine anaplasmosis was studied using the Complement Fixation Test (CFT), DOT-ELISA, Western immunoblot and Rapid Card Agglutination Test (RCAT). Dried blood on Whatman filter paper no. I was eluted in 1.8 ml of PBS 0.05% Tween 20 given an initial dilution of 1:100. The reactivity in both DOT-ELISA and Western immunoblotting was similar to that obtained with the sera diluted 1:100. Filter paper samples gave lower reactivity in all the tests as compared with corresponding serum samples. There was no significant difference in the reactivity between the eluates from filter papers stored at room temperature and those stored at 4°C. Storage at room temperature did not significantly affect reactivity for up to 6 months. Eluates from filter papers stored for 6 months at room temperature continued to give similar reactivity to those from freshly prepared filter papers in both DOT-ELISA and Western blot, and in the Rapid Card Agglutination Test. It is concluded that collecting blood on filter papers is a suitable technique for large-scale screening and for seroepidemiological studies on anaplasmosis, and offers many advantages especially in developing countries where transport and cold chain facilities are a major constraint. G. S. Z. Ssenyonga, Makerere University. Faculty of Veterinary Medicine, P.O. Box 7062, Kampala, Uganda
Bovine anaplasmosis caused by Anaplasma marginale is a worldwide tick-borne disease affecting cattle, goats and sheep and some wild ruminants such as deer and buffalo. The disease is of great economic importance in cattle in tropical and subtropical regions due to losses through morbidity and mortality, especially in the newly introduced highly susceptible exotic dairy cattle where it results in reduced milk production and poor weight gains. Preventive measures through chemotherapy, vector control and vaccination, where they are practised, are expensive and affect the economics of livestock production. Different diagnostic methods currently available include parasitological examination of blood smears and serological methods such as Capillary Agglutination Test (CAT) [1], Rapid Card Agglutination Test (RCAT) [2], Complement Fixation Test
(CFT), Indirect Fluorescent Antibody Test (IFAT), conventional enzyme-linked immunoassay (ELISA) and, more recently, DOT-ELISA [3]. One of the biggest problems in developing countries, and one which must be solved in order to achieve successful diagnosis of infectious diseases, is the collection and preservation of biological materials. In many of these countries, lack of cold chain facilities, quick means of transportation and of diagnostic laboratories within the country combined with high ambient temperatures constitute major constraints in that many biological materials collected for disease diagnosis deteriorate while in transit to the central diagnostic laboratory. Under these conditions there is an urgent need to devise a simple and inexpensive sample collection technique to pre103
104
G. S. Z. Ssenyonga et al.
serve the samples for a considerable time pending analysis. Whole blood specimens dried on filter papers have been used in human medicine for serological and other screening tests for phenylketonuria [4], haemoglobinopathies [5,6], antibodies to measles virus [7], hepatitis B surface antigens [8] and more recently for Acquired Immunodeficiency Syndrome (AIDS) [9]. In veterinary medicine, filter paper blood samples have been used in screening antibodies against Newcastle disease [10], in studies on Eastern Equine Encephalomyelitis [11] and in various haemoprotozoan infections [12, 13]. Collecting blood specimens on filter paper is suitable for large-scale screening and for seroepidemiological studies. The dried samples are light, easy and economical to store and mail, cannot be broken or spilled, and take up little space. Another major advantage of this technique is the ease with which one can obtain blood samples from the ear vein of animals. This study was undertaken in order to evaluate the suitability of blood collected on filter papers in comparison with corresponding conventional serum samples in the serodiagnosis of bovine anaplasmosis using DOT-ELISA, RCAT, and Western immunoblotting. In addition, the same samples were submitted to the USDA Diagnostic Laboratory for independent confirmation for seropositivity to anaplasmosis by the Official Complement Fixation Test. MATERIALS AND METHODS Collection of conventional serum samples and dried blood on filter paper. Ten millilitres of blood was collected from the jugular vein of each of 288 heads of cattle in clean universal bottles. The bottles containing blood were left at room temperature for 3-6 h in order to allow separation of serum from the clot. The separated serum was transferred into plastic vials and frozen at — 70°C until required. About 0.2 ml ofthe corresponding blood sample was applied in duplicate on Whatman's filter paper no. 1 immediately after collection from the jugular vein. The filter papers were allowed to dry in the air. They were placed individually in self-sealing plastic bags. One group was stored at room temperature and the other at 4''C. Elution of serum from dried blood on filter paper. The sample areas on the filter paper were cut out using a sharp scalpel blade and each placed in a wide-mouth plastic centrifuge bottle containing 1.8 ml of PBS 0.05% Tween 20, pH 7.2 to give a dilution of l;100. The samples were left to elute overnight at room temperature.
Serum samples. (1) Known positive and negative control sera. Sixteen samples of known positive control sera were obtained from cattle experimentally infected with A. marginale Florida strain and from naturally infected animals, and were parasitologically positive to the infection. Sixteen samples of negative sera were obtained from cattle known never to have been exposed to the infection and which had proved negative to anaplasma by both parasitological and serological tests. Filter paper samples were prepared from reference positive and negative sera as described above. (2) Anaplasma marginale antigens (Florida isolate). The antigens used in both DOT-ELISA and Western immunoblot were prepared in exactly the same way as described in Ref. 3. (3) Serological tests performed. All samples were tested in serial dilution starting at 1:25 through 1:200. The following serological tests were performed on both the filter paper eluates and the conventional serum samples: (i) DOT-ELISA. The test was performed as described in Ref. 3. Briefly, approximately 1 lA oi Anaplasma marginale anXx^tnai a d\\uy\on of 1:20 was dotted onto 6-mm diameter nitrocellulose discs using a Pasteur pipette. The discs were dried at room temperature for 10 min, blocked for 15 min with PBS 0.3% Tween 20, pH 7.2, and washed for 5 min in PBS pH 7.2 without Tween 20. The appropriate serum/ eluate dilutions in PBS 0.05% Tween 20 were added and incubated for 1 h at room temperature into 24-well flatbottomed microtitre plates followed by three washings of 10 min each with PBS 0.1 % Tween 20, pH 7.2. About 0.5 ml of alkaline phosphase Protein A conjugate diluted 1:500 in PBS 0.05% Tween 20 was added and the solution was incubated for 30 min at room temperature followed by three washes as above except for the last wash which was with PBS alone. Commercially prepared enzyme substrate [Nitroblue tetrazolium (NBT)/5 bromo-4-chloroindoxyl phosphate (BICP) in O.I M Tris buffer] pH 9.5 was added and the colour reaction allowed to develop for 20-30 min during which time purple-coloured dots of variable intensity appeared on positive nitrocellulose discs and no colour developed on negative discs, (ii) Western immunoblotting. The procedure was performed as described in Ref. 14. Alkaline phosphatase anti-bovine IgG conjugate was used at a dilution of 1:1000 in PBS 0.05% Tween 20 pH 7.2 and the same substrate as for the DOT-ELISA was used, (iii) Rapid Card Agglutination Test (RCAT). The test was performed using the USDA antigen and following the procedures previously described by Amerault & Roby [15] and Amerault et al. [16]. Whole sera/ eluates were used without further dilution, (iv) Complement Fixation Test (CFT). CFT was kindly performed by the USDA Animal Diseases Diagnostic Laboratory at Centralia, Illinois, according to the USPHS protocol [17].
RESULTS The results ofthe four serological tests performed on the sera and the corresponding filter paper eluates are given in Table I. These results show 100% agreement between the sera and the corre-
Anaplasmosis in Uganda I TABLE I. Per cent agreement between sera and corresponding filter paper eluates in various serological tests Serological test DOT-ELISA Western Blot CFT RCAT
% agreement 100
100 93.8 83.3
Percentage agreement was calculated on the basis of pairs of serum samples and their corresponding filter paper eluates which gave similar reaction in any given serological test.
sponding filter paper eluates in both DOTELISA and Western immunoblotting, and 93.8% and 83.3% agreement in CFT and RCAT, respectively. The filter paper eluates generally gave lower reactivity in all the tests compared with their corresponding serum samples. The reactivity in both DOT-ELISA and Western immunoblotting was similar to those obtained with sera diluted 1:100. There was 100% agreement in the reactivity between the eluates from filter papers stored at room temperature and those stored at 4°C. Storage at room temperature did not significantly affect reactivity. Eluates from filter papers stored for 6 months at room temperature continued to give similar reactivity to those from freshly prepared filter papers in all the tests. Similar polypeptide profiles were demonstrated using sera and filter paper eluates and there was no selective loss ofthe major profiles. The immunodominant AMF 36-kDa and AMF 105-kDa proteins of Anaplasma marginale (Florida isolate) were consistently demonstrated by both the sera and the corresponding filter paper eluates in Western immunoblotting (results not shown).
DISCUSSION The data presented here support the suitability of collecting blood samples on filter paper for seroepidemiological studies of bovine anaplasmosis in Uganda. There was 100% agreement between the serological results from filter paper samples and those from serum samples in DOTELISA and Western blot, and 93.8% and 83.3% agreement in CFT and RCAT, respectively. The Western blot results are consistent with previously published data [18] with respect to the AMF 36-kDa and AMF 105-kDa polypeptides of
105
A. marginale (Florida isolate) which have been shown to be highly conserved and immunodominant. This would indicate that the Uganda isolate of A. marginale is antigenieally closely related to other East African isolates. Storage at 4°C and at room temperature for over 6 months did not affect the reactivity. The use of dried blood on filter papers for AIDS diagnosis has been evaluated along with corresponding serum samples using HLISA and Western blot tests. The concordance was good, with consistent confirmation of ELISA results by Western blots [19]. Dried blood samples on filter paper have also been used successfully in studies of Eastern Equine Encephalomyelitis [11], Newcastle disease [10] and various haemoprotozoan infections [12, 13]. The technique has also been highly recommended and found suitable for ELISA test in the diagnosis of a number of poultry diseases [20]. This technique of specimen collection should be of great value in seroepidemiological investigation of haemotropic diseases in developing countries as it offers a number of advantages: it requires a minimum amount of equipment, no special training is needed and specimens are less bulky. It eliminates many problems such as bacterial spoilage, chemical denaturation, breakage of glass vials or spillage and other public health hazards associated with handling and transportation of conventional serum samples. The specimens can be mailed to any laboratory in sealed envelopes with minimum risk of spreading infection and it eliminates the costs of handling, storage, and packaging of the specimens. The samples may be stored for a considerable period of time without deterioration even at room temperature, which eliminates special storage facilities. This also allows laboratory personnel considerable flexibility in scheduling the tests. The ease with which filter papers can be saturated with whole blood makes the technique more favourable for sampling very small animals where serum would otherwise be difficult to obtain.
ACKNOWLEDGMENTS The authors wish to thank the staff of USDA at Centralia, Illinois, for the CFT analysis, the Fulbright program for supporting the senior author, and the staff of the Word Processing Centre, UIUC for preparing this manuscript.
106
G. S. Z. Ssenyonga et al.
REFERENCES 1 Ristic M. A capillary tube-agglutination test for anaplasmosis. A preliminary report. J Am Vet Med Assoc 1962;l41:588-94. 2 Amerault TE, Roby TO. Rapid Card Agglutination Test. Department of Agriculture, Animal Parasitology Institute, Beltsville, MD, 1976. 3 Montenegro-James S, Guillen AT, Ma SJ, Ristic M. Use of the DOT-ELISA with isolated A. marginale initial bodies for the serodiagnosis of bovine anaplasmosis. Am J Vet Res 1988. 4 Guthrie R, Susi A. A simple phenylalanine method for detecting phenylketonuria in a large population of newborn infants. Pediatrics 1963;32:338'43. 5 Thielman K, Moreira Aquino A. Whole blood samples dried and dotted on filter papers as a substrate for the electrophoretic separation of hemoglobin S from hemoglobin A. A screening procedure. Clin Chim Acta 1971;35:237-8. 6 Garrick MD, Dembyre P, Guthrie R. Sickle-cell anaemia and other hemoglobinopathies. Procedures and strategy for screening employing spots of blood on filter paper as specimens. N Engl J Med 1973;288:1265-8. 7 Wassilak SGF, Bernier RH, Herrmann KL, Orenstein WA, Bart KJ, Amler R. Measles seroconfirmation using dried blood specimens in filter paper. Pediatr Infect Dis 1984;3:117-21. 8 Farzadegan H, Noori KH, Ala F. Detection of hepatitis-B surface antigen in blood and blood products on filter paper. Lancet 1978; 1:362-3. 9 Farzadegan H, Quinn T, Polk BF. Detecting antibodies to human immunodeficiency virus in dried blood on filter papers. J Infect Dis 1987; 155:1073-4. 10 Beard CW, Brugh M Jr. Use of the Nabuto blood sampling paper strip for Newcastle disease serology. Avian Dis 1977;21:630-6. U Karslad L, Spalatin J, Hanson RP. Application of the paper disc technique to the collection of whole
12
13
14
15
16
17
18
19 20
blood and serum samples in studies on Eastern Equine Encephalomyelitis. J Infect Dis 1957; 101:295-9. Kimber CD, Burridge MJ. The indirect fluorescent antibody test for experimental East Coast Fever {Theileria parva infection of cattle). Evaluation of dried blood samples as a source of antibody. Res Vet Sci 1972; 13:133-5. Young ER, Purnell RE. Evaluation of dried blood samples as a source of antibody in the micro-ELlSA test for Babesia divergerts. Vet Rec I98O;19:6O-1. Kwak Young R. (1988) Diagnosis of bovine anaplasmosis using Anaplasma antigenic markers identified by Western blotting. PhD Thesis, University oflllinois, 1988:27-8. Amerault TE, Roby TO. A. rapid card agglutination test for bovine anaplasmosis. J Am Vet Med Assoc 1968;153:I828-31. Amerault TE, Rose JE, Roby TO. Modified card agglutination test for bovine anaplasmosis: Evaluation with serum and plasma from experimental and natural cases of anaplasmosis. Proc 76th Ann Meet US Anim Hlth Assoc, 1972:736-44. United States Department of Health, Education, and Welfare, Public Health Service (USDHEW/ P H S ) . A guide to the performance of the standardized complement fixation method and adaptation to the microtest, 1st edn. USDHEW/PHS/HS/MHA, Atlanta, Georgia, USA. Palmer GH, Barbet AF, Musoke AJ, Katende JM, Rurangirwa F, Shkap N, Papano E, Davis WC, McGuire TC. Recognition of conserved surface protein epitopes on Anaplasma centrale and A. marginale isolates from Israel, Kenya or USA. Int J Parasit 1988; 18:33-8. Arya SC. Testing for AIDS on samples of dried blood prepared on filter papers. Vaccine 1988;6:210. Synder DB. Latest developments in the EnzymeLinked lmmunosorbent Assay (ELISA). Avian Dis 1985;30:19-23.
Anaplasmosis in Uganda. I. Use of Dried Blood on Filter Paper and Serum Samples for Serodiagnosis of Anaplasmosis—a Comparative Study G. S. Z. S S E N Y O N G A , S. M O N T E N E G R O - J A M E S , I. K A K O M A & R. H A N S E N Makerere University, Faculty of Veterinary Medicine, Kampala, Uganda, and University of Illinois, College of Veterinary Medicine, Urbana, IL, USA
Ssenyonga GSZ, Montenegro-James S, Kakoma I, Hansen R. Anaplasmosis in Uganda. I. Use of Dried Blood on Filter Paper and Serum Samples for Serodiagnosis of Anaplasmosis—a Comparative Study. Seand J Immunol 1992;36(Suppl.ll):103-6 The suitability of blood eollected on filter papers in comparison with corresponding conventional serum samples in the diagnosis of bovine anaplasmosis was studied using the Complement Fixation Test (CFT), DOT-ELISA, Western immunoblot and Rapid Card Agglutination Test (RCAT). Dried blood on Whatman filter paper no. I was eluted in 1.8 ml of PBS 0.05% Tween 20 given an initial dilution of 1:100. The reactivity in both DOT-ELISA and Western immunoblotting was similar to that obtained with the sera diluted 1:100. Filter paper samples gave lower reactivity in all the tests as compared with corresponding serum samples. There was no significant difference in the reactivity between the eluates from filter papers stored at room temperature and those stored at 4°C. Storage at room temperature did not significantly affect reactivity for up to 6 months. Eluates from filter papers stored for 6 months at room temperature continued to give similar reactivity to those from freshly prepared filter papers in both DOT-ELISA and Western blot, and in the Rapid Card Agglutination Test. It is concluded that collecting blood on filter papers is a suitable technique for large-scale screening and for seroepidemiological studies on anaplasmosis, and offers many advantages especially in developing countries where transport and cold chain facilities are a major constraint. G. S. Z. Ssenyonga, Makerere University. Faculty of Veterinary Medicine, P.O. Box 7062, Kampala, Uganda
Bovine anaplasmosis caused by Anaplasma marginale is a worldwide tick-borne disease affecting cattle, goats and sheep and some wild ruminants such as deer and buffalo. The disease is of great economic importance in cattle in tropical and subtropical regions due to losses through morbidity and mortality, especially in the newly introduced highly susceptible exotic dairy cattle where it results in reduced milk production and poor weight gains. Preventive measures through chemotherapy, vector control and vaccination, where they are practised, are expensive and affect the economics of livestock production. Different diagnostic methods currently available include parasitological examination of blood smears and serological methods such as Capillary Agglutination Test (CAT) [1], Rapid Card Agglutination Test (RCAT) [2], Complement Fixation Test
(CFT), Indirect Fluorescent Antibody Test (IFAT), conventional enzyme-linked immunoassay (ELISA) and, more recently, DOT-ELISA [3]. One of the biggest problems in developing countries, and one which must be solved in order to achieve successful diagnosis of infectious diseases, is the collection and preservation of biological materials. In many of these countries, lack of cold chain facilities, quick means of transportation and of diagnostic laboratories within the country combined with high ambient temperatures constitute major constraints in that many biological materials collected for disease diagnosis deteriorate while in transit to the central diagnostic laboratory. Under these conditions there is an urgent need to devise a simple and inexpensive sample collection technique to pre103
104
G. S. Z. Ssenyonga et al.
serve the samples for a considerable time pending analysis. Whole blood specimens dried on filter papers have been used in human medicine for serological and other screening tests for phenylketonuria [4], haemoglobinopathies [5,6], antibodies to measles virus [7], hepatitis B surface antigens [8] and more recently for Acquired Immunodeficiency Syndrome (AIDS) [9]. In veterinary medicine, filter paper blood samples have been used in screening antibodies against Newcastle disease [10], in studies on Eastern Equine Encephalomyelitis [11] and in various haemoprotozoan infections [12, 13]. Collecting blood specimens on filter paper is suitable for large-scale screening and for seroepidemiological studies. The dried samples are light, easy and economical to store and mail, cannot be broken or spilled, and take up little space. Another major advantage of this technique is the ease with which one can obtain blood samples from the ear vein of animals. This study was undertaken in order to evaluate the suitability of blood collected on filter papers in comparison with corresponding conventional serum samples in the serodiagnosis of bovine anaplasmosis using DOT-ELISA, RCAT, and Western immunoblotting. In addition, the same samples were submitted to the USDA Diagnostic Laboratory for independent confirmation for seropositivity to anaplasmosis by the Official Complement Fixation Test. MATERIALS AND METHODS Collection of conventional serum samples and dried blood on filter paper. Ten millilitres of blood was collected from the jugular vein of each of 288 heads of cattle in clean universal bottles. The bottles containing blood were left at room temperature for 3-6 h in order to allow separation of serum from the clot. The separated serum was transferred into plastic vials and frozen at — 70°C until required. About 0.2 ml ofthe corresponding blood sample was applied in duplicate on Whatman's filter paper no. 1 immediately after collection from the jugular vein. The filter papers were allowed to dry in the air. They were placed individually in self-sealing plastic bags. One group was stored at room temperature and the other at 4''C. Elution of serum from dried blood on filter paper. The sample areas on the filter paper were cut out using a sharp scalpel blade and each placed in a wide-mouth plastic centrifuge bottle containing 1.8 ml of PBS 0.05% Tween 20, pH 7.2 to give a dilution of l;100. The samples were left to elute overnight at room temperature.
Serum samples. (1) Known positive and negative control sera. Sixteen samples of known positive control sera were obtained from cattle experimentally infected with A. marginale Florida strain and from naturally infected animals, and were parasitologically positive to the infection. Sixteen samples of negative sera were obtained from cattle known never to have been exposed to the infection and which had proved negative to anaplasma by both parasitological and serological tests. Filter paper samples were prepared from reference positive and negative sera as described above. (2) Anaplasma marginale antigens (Florida isolate). The antigens used in both DOT-ELISA and Western immunoblot were prepared in exactly the same way as described in Ref. 3. (3) Serological tests performed. All samples were tested in serial dilution starting at 1:25 through 1:200. The following serological tests were performed on both the filter paper eluates and the conventional serum samples: (i) DOT-ELISA. The test was performed as described in Ref. 3. Briefly, approximately 1 lA oi Anaplasma marginale anXx^tnai a d\\uy\on of 1:20 was dotted onto 6-mm diameter nitrocellulose discs using a Pasteur pipette. The discs were dried at room temperature for 10 min, blocked for 15 min with PBS 0.3% Tween 20, pH 7.2, and washed for 5 min in PBS pH 7.2 without Tween 20. The appropriate serum/ eluate dilutions in PBS 0.05% Tween 20 were added and incubated for 1 h at room temperature into 24-well flatbottomed microtitre plates followed by three washings of 10 min each with PBS 0.1 % Tween 20, pH 7.2. About 0.5 ml of alkaline phosphase Protein A conjugate diluted 1:500 in PBS 0.05% Tween 20 was added and the solution was incubated for 30 min at room temperature followed by three washes as above except for the last wash which was with PBS alone. Commercially prepared enzyme substrate [Nitroblue tetrazolium (NBT)/5 bromo-4-chloroindoxyl phosphate (BICP) in O.I M Tris buffer] pH 9.5 was added and the colour reaction allowed to develop for 20-30 min during which time purple-coloured dots of variable intensity appeared on positive nitrocellulose discs and no colour developed on negative discs, (ii) Western immunoblotting. The procedure was performed as described in Ref. 14. Alkaline phosphatase anti-bovine IgG conjugate was used at a dilution of 1:1000 in PBS 0.05% Tween 20 pH 7.2 and the same substrate as for the DOT-ELISA was used, (iii) Rapid Card Agglutination Test (RCAT). The test was performed using the USDA antigen and following the procedures previously described by Amerault & Roby [15] and Amerault et al. [16]. Whole sera/ eluates were used without further dilution, (iv) Complement Fixation Test (CFT). CFT was kindly performed by the USDA Animal Diseases Diagnostic Laboratory at Centralia, Illinois, according to the USPHS protocol [17].
RESULTS The results ofthe four serological tests performed on the sera and the corresponding filter paper eluates are given in Table I. These results show 100% agreement between the sera and the corre-
Anaplasmosis in Uganda I TABLE I. Per cent agreement between sera and corresponding filter paper eluates in various serological tests Serological test DOT-ELISA Western Blot CFT RCAT
% agreement 100
100 93.8 83.3
Percentage agreement was calculated on the basis of pairs of serum samples and their corresponding filter paper eluates which gave similar reaction in any given serological test.
sponding filter paper eluates in both DOTELISA and Western immunoblotting, and 93.8% and 83.3% agreement in CFT and RCAT, respectively. The filter paper eluates generally gave lower reactivity in all the tests compared with their corresponding serum samples. The reactivity in both DOT-ELISA and Western immunoblotting was similar to those obtained with sera diluted 1:100. There was 100% agreement in the reactivity between the eluates from filter papers stored at room temperature and those stored at 4°C. Storage at room temperature did not significantly affect reactivity. Eluates from filter papers stored for 6 months at room temperature continued to give similar reactivity to those from freshly prepared filter papers in all the tests. Similar polypeptide profiles were demonstrated using sera and filter paper eluates and there was no selective loss ofthe major profiles. The immunodominant AMF 36-kDa and AMF 105-kDa proteins of Anaplasma marginale (Florida isolate) were consistently demonstrated by both the sera and the corresponding filter paper eluates in Western immunoblotting (results not shown).
DISCUSSION The data presented here support the suitability of collecting blood samples on filter paper for seroepidemiological studies of bovine anaplasmosis in Uganda. There was 100% agreement between the serological results from filter paper samples and those from serum samples in DOTELISA and Western blot, and 93.8% and 83.3% agreement in CFT and RCAT, respectively. The Western blot results are consistent with previously published data [18] with respect to the AMF 36-kDa and AMF 105-kDa polypeptides of
105
A. marginale (Florida isolate) which have been shown to be highly conserved and immunodominant. This would indicate that the Uganda isolate of A. marginale is antigenieally closely related to other East African isolates. Storage at 4°C and at room temperature for over 6 months did not affect the reactivity. The use of dried blood on filter papers for AIDS diagnosis has been evaluated along with corresponding serum samples using HLISA and Western blot tests. The concordance was good, with consistent confirmation of ELISA results by Western blots [19]. Dried blood samples on filter paper have also been used successfully in studies of Eastern Equine Encephalomyelitis [11], Newcastle disease [10] and various haemoprotozoan infections [12, 13]. The technique has also been highly recommended and found suitable for ELISA test in the diagnosis of a number of poultry diseases [20]. This technique of specimen collection should be of great value in seroepidemiological investigation of haemotropic diseases in developing countries as it offers a number of advantages: it requires a minimum amount of equipment, no special training is needed and specimens are less bulky. It eliminates many problems such as bacterial spoilage, chemical denaturation, breakage of glass vials or spillage and other public health hazards associated with handling and transportation of conventional serum samples. The specimens can be mailed to any laboratory in sealed envelopes with minimum risk of spreading infection and it eliminates the costs of handling, storage, and packaging of the specimens. The samples may be stored for a considerable period of time without deterioration even at room temperature, which eliminates special storage facilities. This also allows laboratory personnel considerable flexibility in scheduling the tests. The ease with which filter papers can be saturated with whole blood makes the technique more favourable for sampling very small animals where serum would otherwise be difficult to obtain.
ACKNOWLEDGMENTS The authors wish to thank the staff of USDA at Centralia, Illinois, for the CFT analysis, the Fulbright program for supporting the senior author, and the staff of the Word Processing Centre, UIUC for preparing this manuscript.
106
G. S. Z. Ssenyonga et al.
REFERENCES 1 Ristic M. A capillary tube-agglutination test for anaplasmosis. A preliminary report. J Am Vet Med Assoc 1962;l41:588-94. 2 Amerault TE, Roby TO. Rapid Card Agglutination Test. Department of Agriculture, Animal Parasitology Institute, Beltsville, MD, 1976. 3 Montenegro-James S, Guillen AT, Ma SJ, Ristic M. Use of the DOT-ELISA with isolated A. marginale initial bodies for the serodiagnosis of bovine anaplasmosis. Am J Vet Res 1988. 4 Guthrie R, Susi A. A simple phenylalanine method for detecting phenylketonuria in a large population of newborn infants. Pediatrics 1963;32:338'43. 5 Thielman K, Moreira Aquino A. Whole blood samples dried and dotted on filter papers as a substrate for the electrophoretic separation of hemoglobin S from hemoglobin A. A screening procedure. Clin Chim Acta 1971;35:237-8. 6 Garrick MD, Dembyre P, Guthrie R. Sickle-cell anaemia and other hemoglobinopathies. Procedures and strategy for screening employing spots of blood on filter paper as specimens. N Engl J Med 1973;288:1265-8. 7 Wassilak SGF, Bernier RH, Herrmann KL, Orenstein WA, Bart KJ, Amler R. Measles seroconfirmation using dried blood specimens in filter paper. Pediatr Infect Dis 1984;3:117-21. 8 Farzadegan H, Noori KH, Ala F. Detection of hepatitis-B surface antigen in blood and blood products on filter paper. Lancet 1978; 1:362-3. 9 Farzadegan H, Quinn T, Polk BF. Detecting antibodies to human immunodeficiency virus in dried blood on filter papers. J Infect Dis 1987; 155:1073-4. 10 Beard CW, Brugh M Jr. Use of the Nabuto blood sampling paper strip for Newcastle disease serology. Avian Dis 1977;21:630-6. U Karslad L, Spalatin J, Hanson RP. Application of the paper disc technique to the collection of whole
12
13
14
15
16
17
18
19 20
blood and serum samples in studies on Eastern Equine Encephalomyelitis. J Infect Dis 1957; 101:295-9. Kimber CD, Burridge MJ. The indirect fluorescent antibody test for experimental East Coast Fever {Theileria parva infection of cattle). Evaluation of dried blood samples as a source of antibody. Res Vet Sci 1972; 13:133-5. Young ER, Purnell RE. Evaluation of dried blood samples as a source of antibody in the micro-ELlSA test for Babesia divergerts. Vet Rec I98O;19:6O-1. Kwak Young R. (1988) Diagnosis of bovine anaplasmosis using Anaplasma antigenic markers identified by Western blotting. PhD Thesis, University oflllinois, 1988:27-8. Amerault TE, Roby TO. A. rapid card agglutination test for bovine anaplasmosis. J Am Vet Med Assoc 1968;153:I828-31. Amerault TE, Rose JE, Roby TO. Modified card agglutination test for bovine anaplasmosis: Evaluation with serum and plasma from experimental and natural cases of anaplasmosis. Proc 76th Ann Meet US Anim Hlth Assoc, 1972:736-44. United States Department of Health, Education, and Welfare, Public Health Service (USDHEW/ P H S ) . A guide to the performance of the standardized complement fixation method and adaptation to the microtest, 1st edn. USDHEW/PHS/HS/MHA, Atlanta, Georgia, USA. Palmer GH, Barbet AF, Musoke AJ, Katende JM, Rurangirwa F, Shkap N, Papano E, Davis WC, McGuire TC. Recognition of conserved surface protein epitopes on Anaplasma centrale and A. marginale isolates from Israel, Kenya or USA. Int J Parasit 1988; 18:33-8. Arya SC. Testing for AIDS on samples of dried blood prepared on filter papers. Vaccine 1988;6:210. Synder DB. Latest developments in the EnzymeLinked lmmunosorbent Assay (ELISA). Avian Dis 1985;30:19-23.
