JOURNAL
OF
Vol. 30, No. 7
CLINICAL MICROBIOLOGY, JUIY 1992, p. 1870-1873
0095-1137/92/071870-04$02.00/0 Copyright C) 1992, American Society for Microbiology
Distribution of Heartwater in the Caribbean Determined on the Basis of Detection of Antibodies to the Conserved 32-Kilodalton Protein of Cowdria ruminantium ANNEKE MULLER KOBOLD,' DOMINIQUE MARTINEZ,2 EMMANUEL CAMUS,2 AND FRANS JONGEJANl* Department of Parasitology and Tropical Veterinary Medicine, Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, P. O. Box 80.165, 3508 TD Utrecht, The Netherlands, and Institut d'Elevage et de Medecine Veterinaire des Pays Tropicaux, Mission Antilles-Guyane, Institut National de la Recherche Agronomique, 97184 Pointe-a-Pitre Cedex,
Guadeloupe, French West Indies2 Received 9 December 1991/Accepted 7 April 1992
A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect immunoglobulin G antibodies to the major 32-kDa protein of Cowdria ruminantium. A total of 1,804 serum samples collected from cattle on 19 islands in the eastern Caribbean Basin were tested by this cELISA. A total of 133 serum samples from 10 islands (Antigua, Dominica, Grenada, Guadeloupe, Martinique, Montserrat, St. Kitts, St. Lucia, St. Martin, and St. Vincent) were found to be positive. The presence of antibodies to C. ruminantium in cattle on these islands was confirmed by immunofluorescence and Western blotting (immunoblotting). In earlier studies, C. ruminantium has been demonstrated only on Guadeloupe, Antigua, and Marie Galante. This study shows that the causative agent of heartwater is now firmly established in the Caribbean.
Heartwater is an infectious disease of domestic and wild ruminants. It is caused by the rickettsia Cowdria ruminantium, an organism that is transmitted transstadially by Amblyomma ticks. Typical symptoms are high fever, respiratory distress, nervous symptoms, and hydropericardium (17). The disease is endemic in sub-Saharan Africa, including the islands of Madagascar, Mauritius, La Reunion, Sao Tome (17), and Zanzibar (8) as well as the Comoros Islands (7). Heartwater was also observed on the Caribbean island of Guadeloupe for the first time in 1980 (16). It was probably introduced 160 years earlier by the tick Amblyomma variegatum on cattle from West Africa. Outside Guadeloupe the disease has now, with certainty, been diagnosed on two other Caribbean islands, Marie Galante (19) and Antigua (3). From the late 1960s onward, there appeared to be an increasing dissemination of the tick A. variegatum throughout the Caribbean region (5). The countries and islands in the region have been put into the following risk classes on the basis of the estimated risk of infestation and present infestation byA. variegatum (21): risk class I, the island is free of the tick and is at a low risk of infestation; risk class II, the island is presently uninfested or ticks have been reported on the islands, but the island is not considered to have established tick populations and/or to be at a higher risk of infestation; risk class III, the tick is established on the island, but with limited distribution; and risk class IV, the tick is widespread on the island. The distribution of A. variegatum in the Caribbean is given in Fig. 1. A possible explanation for the spread of A. variegatum is the increased migration of the cattle egret (Bubulcus ibis), which is frequently infested with larvae and, to a lesser extent, nymphs of A. variegatum (1, 18). This means of spread of ticks infected with C. ruminantium makes heartwater a serious threat to livestock on other Caribbean *
islands and, eventually, on the North and South American mainlands as well (2). Recently, we developed a competitive enzyme-linked immunosorbent assay (cELISA) based on the C. ruminantium antigen derived from endothelial cell cultures and mediated by monoclonal antibodies (MAbs) against an immunodominant and antigenically conserved Cowdria protein of 32 kDa (Cr32) (12, 13). In this report, we evaluated the cELISA in a survey for the presence of Cowdria antibodies in cattle in the Caribbean. Samples were obtained from a serum collection maintained at the Institut d'Elevage et de Medecine Veterinaire des Pays Tropicaux, Pointe-a-Pitre, Guadeloupe, containing cattle sera collected in the Caribbean Basin between 1982 and 1991 (Table 1). Antisera against C. ruminantium (Senegal isolate) were raised in experimentally infected Frisian calves in The Netherlands. Negative control sera were obtained from Dutch calves which had never been in contact with C. ruminantium. Two stocks of C. ruminantium were used: the Senegal isolate (14) and an isolate from Guadeloupe, Gardel (20). Cowdria antigens for cELISA, immunofluorescence, and Western blotting (immunoblotting) were prepared from rickettsiae that were cultivated in bovine umbilical endothelial cells (BUE) as described previously (10). Briefly, endothelial cells were grown until confluency in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered RPMI 1640 medium supplemented with antibiotics and 10% newborn calf serum. Monolayers were inoculated with BUE cell culture supernatant infected with elementary bodies of C. ruminantium. Cultures were maintained in Glasgow minimal essential medium supplemented with penicillin (100 IU/ml), streptomycin (100 ,ug/ml), amphotericin B (1.25 ,ug/ml), HEPES buffer (20 mM; pH 7.0 to 7.2), L-glutamine (2 mM), and 10% newborn calf serum. Incubation was carried out at 37°C on a slowly rocking platform. Samples of BUE cells were scraped from the bottom of the culture flask, smeared
Corresponding author. 1870
VOL.
30, 1992
NOTES
VIRGIN ISLANDS Jost van Dyke Tortoil
>Z~tse
c
Anegada
0Virgin Gorda
Anguilla
eV St. Marin0
Porto Rico 1974
_
Saba
St. Croix 1967
a
4,0 > Barbuda
St. Eustatius 0 0 St. Kilts
1978%
Nevis 1977
Antigua
ige
Century I~
( Montserrat 1983 t 1830 t)
0D
(0
d Deirade
I (0
cn
Cowdria ruminantium isolated Tick widespread
_
a
00
C ELISA positive
Dominica 1983
T= ick limited in dstribution Tick reported, not established
(0
%4 Martinique 19480
= TTick not reported
(0)
4 St.
1988 = Year tick first reported
y St. Vincent 1988 (0 GRENADINES
@1
b0
°Carnacou aGrenada (?
&
Tobago
TRINIDAD VENEZUELA
FIG. 1. Distribution of A. variegatum ticks in the Western Hemisphere (eastern Caribbean Basin) and the presence of antibodies (indicated by a plus sign) to C. ruminantium in cattle on various Caribbean islands as determined by cELISA.
onto a glass slide, and examined for Cowdria inclusions after the slide was stained with Diff-Quik (Merz & Dada AG, Dudingen, Switzerland). Cultures with virtually all BUE cells infected with reticulate bodies of C. ruminantium and containing large numbers of extracellular elementary bodies in the supernatant were centrifuged at 4°C for 15 min at 15,000 x g. Pellets were resuspended in phosphate-buffered saline (PBS) and sonicated on ice four times for 15 s each time with a 1-min interval between sonications (Vibracell disintegrator; Sonics & Materials Inc.). The protein concentration was determined and sonic extracts were stored at -200C. In the cELISA an immunoglobulin G3 MAb (4F10B4 [13]) was used; the MAb reacted with the Cr32 Cowdria surface protein (12). The cELISA was performed as described previously (13), with modifications. Polystyrene 96-well flatbottom plates (Nunc) were coated overnight at 370C with 6 TABLE 1. Origin of cattle sera tested in this study Caribbean island
Guadeloupe St. Lucia
Martinique Remaining islandsb
Yr of serum collection
1991 1990 1989 1982-1984
" IEMVT, Institut d'Elevage et caux; UF, University of Florida. h See Table 2.
No. of serum samples
83 66
Collection' IEMVT
IEMVT 283 IEMVT UF 1,372 de MWdecine VWterinaire des Pays Tropi-
1871
,ug of sonicated Cowdnia antigen per ml in 0.05 M carbonatebicarbonate buffer (pH 9.6). Plates were washed three times with tap water. Test serum (1:50 dilution) was applied simultaneously with MAb 4F13B4 (1:400), and both were diluted in phosphate-buffered saline (PBS; pH 7.2) containing 3% skim milk and 0.05% Tween 20 and incubated at 37°C for 1 h. After washing, peroxidase-labeled rabbit anti-mouse immunoglobulin (Dakopatts, Glostrup, Denmark) was added at a 1:750 dilution, and the solution was incubated at 37°C for 1 h. After washing, 100 p.l of 0.1 M citrate buffer (pH 5.5) containing o-phenylenediamine and hydrogen peroxide was added to each well. After a 30-min incubation at room temperature in the dark, reactions were stopped by adding 50 p.l of 1 M H2S04 per well, and the optical density (OD) was read at 405 nm on a Bioteck ELISA plate reader. A maximum of 34 test samples were tested in duplicate on each test plate. Two Cowdria-positive serum samples with antibody titers of 5,210 determined by immunofluorescence and 10 negative control serum samples were included on each plate, in duplicate. A mean and standard deviation of the OD for the control serum samples were calculated following each test. A serum sample was considered positive for anti-Cr32 antibodies if it inhibited binding of MAb 4F10B4 such that the mean duplicate OD for that sample was at least 2 standard deviations below the mean OD of the negative serum samples run simultaneously. The cELISA was done twice on the same serum samples. To detect antibodies by immunofluorescence, BUE cell cultures that were heavily infected with C. ruminantium were centrifuged at 15,000 x g at 4°C for 15 min. Pellets were resuspended in PBS, spotted onto microscope slides (10 pL. per well; Bio-Merieux), dried, and fixed in acetone for 10 min (15). Sera were titrated on the slides in twofold dilutions in PBS starting at 1:80 and going up to 1:1,280. Fluorescein isothiocyanate-labeled rabbit anti-bovine immunoglobulins were used as secondary antibodies. Specific fluorescence was observed with a Leitz Laborlux 11 microscope at a magnification of x500. Endothelial cell culture sonic extracts similar to the sonic extracts used for the cELISA were subjected to sodium dodecyl sulfate-gel electrophoresis on a 12.5% polyacrylamide gel. Western blotting was carried out essentially as described earlier (13), but electrophoretic transfer was carried out for 1 h at 100 V or overnight at 20 V. Blots were quenched for 1 h in PBS-5% skim milk and incubated for 1 h with test serum or with positive or negative control serum diluted 1:150 in PBS containing 0.02% Tween 20 and 5% milk. Bound antibodies were visualized by incubation with rabbit anti-bovine immunoglobulins conjugated with alkaline phosphatase (Sigma) at a dilution of 1:2,000. Binding of conjugate was visualized by the addition, after washing, of 100 mM Tris-HCl buffer (pH 9.5) containing 100 mM NaCl and 5 mM MgCl2 buffer with nitroblue tetrazolium and 5-bromo-4-dichloro-3-indolyl phosphate. The reaction was stopped with a 20 mM Tris-HCl buffer (pH 8.0) containing 5 mM EDTA. Anti-Cr32 antibodies were detected by cELISA in 133 of 1,804 cattle serum samples from 10 (Antigua, Dominica, Guadeloupe, Grenada, Martinique, Montserrat, St. Kitts, St. Lucia, St. Martin, and St. Vincent) of the 19 islands (Table 2; Fig. 1). On Guadeloupe and Antigua, where A. variegatum is widespread (risk class IV), 36.6 and 4.9% of serum samples, respectively, were cELISA positive. Since C. ruminantium has previously been isolated from both islands (3, 16), no further confirmation was needed. The low incidence found on Antigua may be related to low infection
J. CLIN. MICROBIOL.
NOTES
1872
TABLE 2. Results of a survey for C. rmminantium antibodies in cattle in the Caribbean by using a cELISA Risk 1S class
Islands
III IV II III II II IV III III II II IV III III II
Anguilla Antigua Barbuda Dominica Grenada Carriacou (Grenadines) Guadeloupe Martinique Montserrat Saba St. Eustatius St. Kitts St. Lucia St. Martin St. Vincent Virgin Islands Anegada Jost Van Dyke Tortola Virgin Gorda
II II II II
No. positive by cELISA/total no. tested (%)
1/25a (4.0) 3/61 (4.9)
1/28a (3.6) 32/136 (24.0) 9/166 (5.4) 0/175 (0) 32/83 (38.6) 19/283 (6.7) 4/155 (2.6) 0/28 (0) 0/64 (0) 7/66 (10.6) 3/66 (4.5) 3/93 (3.2) 18/181 (9.9) 1/19" (5.2) 0/12 (0) 0/140 (0)
0/23 (0)
133/1,804
Total
aNot confirmed by immunofluorescence or Western blotting.
rates in ticks, since in a previous study 500 ticks were required to demonstrate the presence of heartwater on this island (4). On Dominica (risk class III), 32 of 136 samples were positive by cELISA (24%). Thirty of these cELISA-positive serum samples were confirmed by immunofluorescence, with titers ranging from 1:320 to 1:1,280, whereas 29 of 30 immunofluorescence-positive serum samples recognized the Cr32 protein on Western blots (Table 3; Fig. 2, lanes 9 and 10). Although a previous survey by immunofluorescence with Cowdria-infected mouse macrophage antigen remained inconclusive about the presence of C. ruminantium on Dominica (4), the data obtained from all three assays in the present study show that C. ruminantium is present on the island. Furthermore, on Martinique, Montserrat, St. Martin, and St. Kitts, where A. variegatum is firmly established (risk TABLE 3. Comparison of cELISA, immunofluorescence, and Western blotting for detection of antibodies to C. ruminantium in cattle sera collected in 10 Caribbean islands No. of cattle sera positive/total no. tested by:
Island
Antigua Dominica Grenada Guadeloupe Martinique Montserrat St. Kitts St. Lucia St. Martin St. Vincent
cELISA
Immunofluorescencea
Western blot
3/61 32/136 9/166 32/83 19/283 4/155 7/66 3/66 3/93 18/181
ND* 30/136 8/166 ND 16/283 4/155 ND ND ND ND
ND 29/136 6/166 ND 12/283 4/155 3/66 ND 3/93 11/181
"Titers ranged between 320 and 1,280.
"ND, not determined.
kDa 946743-
30-
*
OF
Vol. 30, No. 7
CLINICAL MICROBIOLOGY, JUIY 1992, p. 1870-1873
0095-1137/92/071870-04$02.00/0 Copyright C) 1992, American Society for Microbiology
Distribution of Heartwater in the Caribbean Determined on the Basis of Detection of Antibodies to the Conserved 32-Kilodalton Protein of Cowdria ruminantium ANNEKE MULLER KOBOLD,' DOMINIQUE MARTINEZ,2 EMMANUEL CAMUS,2 AND FRANS JONGEJANl* Department of Parasitology and Tropical Veterinary Medicine, Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, P. O. Box 80.165, 3508 TD Utrecht, The Netherlands, and Institut d'Elevage et de Medecine Veterinaire des Pays Tropicaux, Mission Antilles-Guyane, Institut National de la Recherche Agronomique, 97184 Pointe-a-Pitre Cedex,
Guadeloupe, French West Indies2 Received 9 December 1991/Accepted 7 April 1992
A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect immunoglobulin G antibodies to the major 32-kDa protein of Cowdria ruminantium. A total of 1,804 serum samples collected from cattle on 19 islands in the eastern Caribbean Basin were tested by this cELISA. A total of 133 serum samples from 10 islands (Antigua, Dominica, Grenada, Guadeloupe, Martinique, Montserrat, St. Kitts, St. Lucia, St. Martin, and St. Vincent) were found to be positive. The presence of antibodies to C. ruminantium in cattle on these islands was confirmed by immunofluorescence and Western blotting (immunoblotting). In earlier studies, C. ruminantium has been demonstrated only on Guadeloupe, Antigua, and Marie Galante. This study shows that the causative agent of heartwater is now firmly established in the Caribbean.
Heartwater is an infectious disease of domestic and wild ruminants. It is caused by the rickettsia Cowdria ruminantium, an organism that is transmitted transstadially by Amblyomma ticks. Typical symptoms are high fever, respiratory distress, nervous symptoms, and hydropericardium (17). The disease is endemic in sub-Saharan Africa, including the islands of Madagascar, Mauritius, La Reunion, Sao Tome (17), and Zanzibar (8) as well as the Comoros Islands (7). Heartwater was also observed on the Caribbean island of Guadeloupe for the first time in 1980 (16). It was probably introduced 160 years earlier by the tick Amblyomma variegatum on cattle from West Africa. Outside Guadeloupe the disease has now, with certainty, been diagnosed on two other Caribbean islands, Marie Galante (19) and Antigua (3). From the late 1960s onward, there appeared to be an increasing dissemination of the tick A. variegatum throughout the Caribbean region (5). The countries and islands in the region have been put into the following risk classes on the basis of the estimated risk of infestation and present infestation byA. variegatum (21): risk class I, the island is free of the tick and is at a low risk of infestation; risk class II, the island is presently uninfested or ticks have been reported on the islands, but the island is not considered to have established tick populations and/or to be at a higher risk of infestation; risk class III, the tick is established on the island, but with limited distribution; and risk class IV, the tick is widespread on the island. The distribution of A. variegatum in the Caribbean is given in Fig. 1. A possible explanation for the spread of A. variegatum is the increased migration of the cattle egret (Bubulcus ibis), which is frequently infested with larvae and, to a lesser extent, nymphs of A. variegatum (1, 18). This means of spread of ticks infected with C. ruminantium makes heartwater a serious threat to livestock on other Caribbean *
islands and, eventually, on the North and South American mainlands as well (2). Recently, we developed a competitive enzyme-linked immunosorbent assay (cELISA) based on the C. ruminantium antigen derived from endothelial cell cultures and mediated by monoclonal antibodies (MAbs) against an immunodominant and antigenically conserved Cowdria protein of 32 kDa (Cr32) (12, 13). In this report, we evaluated the cELISA in a survey for the presence of Cowdria antibodies in cattle in the Caribbean. Samples were obtained from a serum collection maintained at the Institut d'Elevage et de Medecine Veterinaire des Pays Tropicaux, Pointe-a-Pitre, Guadeloupe, containing cattle sera collected in the Caribbean Basin between 1982 and 1991 (Table 1). Antisera against C. ruminantium (Senegal isolate) were raised in experimentally infected Frisian calves in The Netherlands. Negative control sera were obtained from Dutch calves which had never been in contact with C. ruminantium. Two stocks of C. ruminantium were used: the Senegal isolate (14) and an isolate from Guadeloupe, Gardel (20). Cowdria antigens for cELISA, immunofluorescence, and Western blotting (immunoblotting) were prepared from rickettsiae that were cultivated in bovine umbilical endothelial cells (BUE) as described previously (10). Briefly, endothelial cells were grown until confluency in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered RPMI 1640 medium supplemented with antibiotics and 10% newborn calf serum. Monolayers were inoculated with BUE cell culture supernatant infected with elementary bodies of C. ruminantium. Cultures were maintained in Glasgow minimal essential medium supplemented with penicillin (100 IU/ml), streptomycin (100 ,ug/ml), amphotericin B (1.25 ,ug/ml), HEPES buffer (20 mM; pH 7.0 to 7.2), L-glutamine (2 mM), and 10% newborn calf serum. Incubation was carried out at 37°C on a slowly rocking platform. Samples of BUE cells were scraped from the bottom of the culture flask, smeared
Corresponding author. 1870
VOL.
30, 1992
NOTES
VIRGIN ISLANDS Jost van Dyke Tortoil
>Z~tse
c
Anegada
0Virgin Gorda
Anguilla
eV St. Marin0
Porto Rico 1974
_
Saba
St. Croix 1967
a
4,0 > Barbuda
St. Eustatius 0 0 St. Kilts
1978%
Nevis 1977
Antigua
ige
Century I~
( Montserrat 1983 t 1830 t)
0D
(0
d Deirade
I (0
cn
Cowdria ruminantium isolated Tick widespread
_
a
00
C ELISA positive
Dominica 1983
T= ick limited in dstribution Tick reported, not established
(0
%4 Martinique 19480
= TTick not reported
(0)
4 St.
1988 = Year tick first reported
y St. Vincent 1988 (0 GRENADINES
@1
b0
°Carnacou aGrenada (?
&
Tobago
TRINIDAD VENEZUELA
FIG. 1. Distribution of A. variegatum ticks in the Western Hemisphere (eastern Caribbean Basin) and the presence of antibodies (indicated by a plus sign) to C. ruminantium in cattle on various Caribbean islands as determined by cELISA.
onto a glass slide, and examined for Cowdria inclusions after the slide was stained with Diff-Quik (Merz & Dada AG, Dudingen, Switzerland). Cultures with virtually all BUE cells infected with reticulate bodies of C. ruminantium and containing large numbers of extracellular elementary bodies in the supernatant were centrifuged at 4°C for 15 min at 15,000 x g. Pellets were resuspended in phosphate-buffered saline (PBS) and sonicated on ice four times for 15 s each time with a 1-min interval between sonications (Vibracell disintegrator; Sonics & Materials Inc.). The protein concentration was determined and sonic extracts were stored at -200C. In the cELISA an immunoglobulin G3 MAb (4F10B4 [13]) was used; the MAb reacted with the Cr32 Cowdria surface protein (12). The cELISA was performed as described previously (13), with modifications. Polystyrene 96-well flatbottom plates (Nunc) were coated overnight at 370C with 6 TABLE 1. Origin of cattle sera tested in this study Caribbean island
Guadeloupe St. Lucia
Martinique Remaining islandsb
Yr of serum collection
1991 1990 1989 1982-1984
" IEMVT, Institut d'Elevage et caux; UF, University of Florida. h See Table 2.
No. of serum samples
83 66
Collection' IEMVT
IEMVT 283 IEMVT UF 1,372 de MWdecine VWterinaire des Pays Tropi-
1871
,ug of sonicated Cowdnia antigen per ml in 0.05 M carbonatebicarbonate buffer (pH 9.6). Plates were washed three times with tap water. Test serum (1:50 dilution) was applied simultaneously with MAb 4F13B4 (1:400), and both were diluted in phosphate-buffered saline (PBS; pH 7.2) containing 3% skim milk and 0.05% Tween 20 and incubated at 37°C for 1 h. After washing, peroxidase-labeled rabbit anti-mouse immunoglobulin (Dakopatts, Glostrup, Denmark) was added at a 1:750 dilution, and the solution was incubated at 37°C for 1 h. After washing, 100 p.l of 0.1 M citrate buffer (pH 5.5) containing o-phenylenediamine and hydrogen peroxide was added to each well. After a 30-min incubation at room temperature in the dark, reactions were stopped by adding 50 p.l of 1 M H2S04 per well, and the optical density (OD) was read at 405 nm on a Bioteck ELISA plate reader. A maximum of 34 test samples were tested in duplicate on each test plate. Two Cowdria-positive serum samples with antibody titers of 5,210 determined by immunofluorescence and 10 negative control serum samples were included on each plate, in duplicate. A mean and standard deviation of the OD for the control serum samples were calculated following each test. A serum sample was considered positive for anti-Cr32 antibodies if it inhibited binding of MAb 4F10B4 such that the mean duplicate OD for that sample was at least 2 standard deviations below the mean OD of the negative serum samples run simultaneously. The cELISA was done twice on the same serum samples. To detect antibodies by immunofluorescence, BUE cell cultures that were heavily infected with C. ruminantium were centrifuged at 15,000 x g at 4°C for 15 min. Pellets were resuspended in PBS, spotted onto microscope slides (10 pL. per well; Bio-Merieux), dried, and fixed in acetone for 10 min (15). Sera were titrated on the slides in twofold dilutions in PBS starting at 1:80 and going up to 1:1,280. Fluorescein isothiocyanate-labeled rabbit anti-bovine immunoglobulins were used as secondary antibodies. Specific fluorescence was observed with a Leitz Laborlux 11 microscope at a magnification of x500. Endothelial cell culture sonic extracts similar to the sonic extracts used for the cELISA were subjected to sodium dodecyl sulfate-gel electrophoresis on a 12.5% polyacrylamide gel. Western blotting was carried out essentially as described earlier (13), but electrophoretic transfer was carried out for 1 h at 100 V or overnight at 20 V. Blots were quenched for 1 h in PBS-5% skim milk and incubated for 1 h with test serum or with positive or negative control serum diluted 1:150 in PBS containing 0.02% Tween 20 and 5% milk. Bound antibodies were visualized by incubation with rabbit anti-bovine immunoglobulins conjugated with alkaline phosphatase (Sigma) at a dilution of 1:2,000. Binding of conjugate was visualized by the addition, after washing, of 100 mM Tris-HCl buffer (pH 9.5) containing 100 mM NaCl and 5 mM MgCl2 buffer with nitroblue tetrazolium and 5-bromo-4-dichloro-3-indolyl phosphate. The reaction was stopped with a 20 mM Tris-HCl buffer (pH 8.0) containing 5 mM EDTA. Anti-Cr32 antibodies were detected by cELISA in 133 of 1,804 cattle serum samples from 10 (Antigua, Dominica, Guadeloupe, Grenada, Martinique, Montserrat, St. Kitts, St. Lucia, St. Martin, and St. Vincent) of the 19 islands (Table 2; Fig. 1). On Guadeloupe and Antigua, where A. variegatum is widespread (risk class IV), 36.6 and 4.9% of serum samples, respectively, were cELISA positive. Since C. ruminantium has previously been isolated from both islands (3, 16), no further confirmation was needed. The low incidence found on Antigua may be related to low infection
J. CLIN. MICROBIOL.
NOTES
1872
TABLE 2. Results of a survey for C. rmminantium antibodies in cattle in the Caribbean by using a cELISA Risk 1S class
Islands
III IV II III II II IV III III II II IV III III II
Anguilla Antigua Barbuda Dominica Grenada Carriacou (Grenadines) Guadeloupe Martinique Montserrat Saba St. Eustatius St. Kitts St. Lucia St. Martin St. Vincent Virgin Islands Anegada Jost Van Dyke Tortola Virgin Gorda
II II II II
No. positive by cELISA/total no. tested (%)
1/25a (4.0) 3/61 (4.9)
1/28a (3.6) 32/136 (24.0) 9/166 (5.4) 0/175 (0) 32/83 (38.6) 19/283 (6.7) 4/155 (2.6) 0/28 (0) 0/64 (0) 7/66 (10.6) 3/66 (4.5) 3/93 (3.2) 18/181 (9.9) 1/19" (5.2) 0/12 (0) 0/140 (0)
0/23 (0)
133/1,804
Total
aNot confirmed by immunofluorescence or Western blotting.
rates in ticks, since in a previous study 500 ticks were required to demonstrate the presence of heartwater on this island (4). On Dominica (risk class III), 32 of 136 samples were positive by cELISA (24%). Thirty of these cELISA-positive serum samples were confirmed by immunofluorescence, with titers ranging from 1:320 to 1:1,280, whereas 29 of 30 immunofluorescence-positive serum samples recognized the Cr32 protein on Western blots (Table 3; Fig. 2, lanes 9 and 10). Although a previous survey by immunofluorescence with Cowdria-infected mouse macrophage antigen remained inconclusive about the presence of C. ruminantium on Dominica (4), the data obtained from all three assays in the present study show that C. ruminantium is present on the island. Furthermore, on Martinique, Montserrat, St. Martin, and St. Kitts, where A. variegatum is firmly established (risk TABLE 3. Comparison of cELISA, immunofluorescence, and Western blotting for detection of antibodies to C. ruminantium in cattle sera collected in 10 Caribbean islands No. of cattle sera positive/total no. tested by:
Island
Antigua Dominica Grenada Guadeloupe Martinique Montserrat St. Kitts St. Lucia St. Martin St. Vincent
cELISA
Immunofluorescencea
Western blot
3/61 32/136 9/166 32/83 19/283 4/155 7/66 3/66 3/93 18/181
ND* 30/136 8/166 ND 16/283 4/155 ND ND ND ND
ND 29/136 6/166 ND 12/283 4/155 3/66 ND 3/93 11/181
"Titers ranged between 320 and 1,280.
"ND, not determined.
kDa 946743-
30-
*
