Comparison of latex agglutination, indirect immunofluorescent antibody, and enzyme immunoassay methods for serodiagnosis of Rocky Mountain spotted fever in dogs

Craig E. Greene From the Department of Small Animal Medicine, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602 (Greene, Marks, Lappin, Wolski); Department of Companion and Special Species Medicine, School of Veterinary Medicine, The North Carolina State University, Raleigh, NC 27606 (Breitschwerdt); and The National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, MT 59840 (Burgdorfer).

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M. Amanda Marks From the Department of Small Animal Medicine, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602 (Greene, Marks, Lappin, Wolski); Department of Companion and Special Species Medicine, School of Veterinary Medicine, The North Carolina State University, Raleigh, NC 27606 (Breitschwerdt); and The National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, MT 59840 (Burgdorfer).

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Michael R. Lappin From the Department of Small Animal Medicine, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602 (Greene, Marks, Lappin, Wolski); Department of Companion and Special Species Medicine, School of Veterinary Medicine, The North Carolina State University, Raleigh, NC 27606 (Breitschwerdt); and The National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, MT 59840 (Burgdorfer).

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Edward B. Breitschwerdt From the Department of Small Animal Medicine, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602 (Greene, Marks, Lappin, Wolski); Department of Companion and Special Species Medicine, School of Veterinary Medicine, The North Carolina State University, Raleigh, NC 27606 (Breitschwerdt); and The National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, MT 59840 (Burgdorfer).

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Nancy A. Wolski From the Department of Small Animal Medicine, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602 (Greene, Marks, Lappin, Wolski); Department of Companion and Special Species Medicine, School of Veterinary Medicine, The North Carolina State University, Raleigh, NC 27606 (Breitschwerdt); and The National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, MT 59840 (Burgdorfer).

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Willy Burgdorfer From the Department of Small Animal Medicine, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602 (Greene, Marks, Lappin, Wolski); Department of Companion and Special Species Medicine, School of Veterinary Medicine, The North Carolina State University, Raleigh, NC 27606 (Breitschwerdt); and The National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, MT 59840 (Burgdorfer).

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SUMMARY

Indirect immunofluorescent antibody (ifa), latex agglutination (la), and enzyme immunoassay (eia) methods were compared for evaluation of the serum antibody responses of dogs experimentally and naturally exposed to spotted fever-group rickettsiae. Selected sera (obtained on days 1, 42, 53, 124, 145, 236, 255, 264, and 292) were examined from three 8-month-old female Beagles inoculated with Rickettsia rickettsii on days 34 and 250 of the study. A second group of dogs comprised three 8-month-old female Beagles inoculated with R montana on days 34 and 102. Subsequently, these dogs were inoculated with R rickettsii on day 250. Serum samples were obtained from the second group of dogs on days 1, 96, 103, 132, 180, 215, 292, and 494. A third group consisted of 21 naturally exposed dogs, from which sequentially obtained serum samples were available, and which had clinical signs compatible with Rocky Mountain spotted fever. Clinical signs of disease in dogs of the third group resolved after treatment with tetracycline (22 mg/kg of body weight, po, q 8 h) was instituted. At least 2 sequentially obtained serum samples from each dog were tested. In general, the first sample was obtained just prior to treatment and the convalescent serum samples were obtained at weekly or greater intervals thereafter.

For correlation and reactivity data, an ifa test for IgG/IgM (using heavy and light chains-specific conjugate) was used as the reference standard for comparison of results with those of the other tests.

For analyzing sensitivity and specificity data in naturally exposed dogs, the assumption was made that fourfold change in the ifa IgG/IgM titer for R rickettsii or titer ≥ 1,024 identified infected dogs. Threshold titer indicative of specific rickettsial antibody was determined for each test: ifa IgG/IgM, 64; ifa IgM, 8; la, 16; EIA IgG, 64; and EIA IgM, 64. Marked cross-reactivity of sera with R rickettsii, R rhipicephali, and R montana was apparent for the ifa method using these antigens; lesser cross-reactivity was observed for R bellii. Although the ifa test for IgM, using R rickettsii as antigen, had high specificity (88.2%), it had low sensitivity (21.7%), limiting its use as a sole diagnostic test. The la test had a higher sensitivity (58.5%) than did any of the IgM ifa tests. The la test results correlated best with IgM ifa test results, and the closest agreement was obtained when R bellii was used as antigen. Because of its relatively high sensitivity (58.5%) and specificity (83.6%), the la procedure may be a valuable screening test for diagnosis of Rocky Mountain spotted fever in dogs. The IgG and IgM EIA methods had high sensitivity- 90.6 and 83.1%, respectively. The IgM EIA performed by us had low specificity; it detected infection with R montana in inoculated dogs. Comparing results of all test methods in naturally exposed dogs, the IgM EIA results were positive for the greatest number of samples with corresponding negative results by the reference IgG/IgM ifa test. This low specificity may preclude its routine use as a diagnostic test. No single serologic method correctly identified all affected dogs. On the basis of comparison with results obtained for the other serologic procedures and antigen-detection methods in 1 dog, the IgG/IgM ifa test used as a reference test may not have detected some infected dogs. Combination of a test that detects predominantly IgG, with either the la or IgM ifa test, would help to identify most infected dogs.

SUMMARY

Indirect immunofluorescent antibody (ifa), latex agglutination (la), and enzyme immunoassay (eia) methods were compared for evaluation of the serum antibody responses of dogs experimentally and naturally exposed to spotted fever-group rickettsiae. Selected sera (obtained on days 1, 42, 53, 124, 145, 236, 255, 264, and 292) were examined from three 8-month-old female Beagles inoculated with Rickettsia rickettsii on days 34 and 250 of the study. A second group of dogs comprised three 8-month-old female Beagles inoculated with R montana on days 34 and 102. Subsequently, these dogs were inoculated with R rickettsii on day 250. Serum samples were obtained from the second group of dogs on days 1, 96, 103, 132, 180, 215, 292, and 494. A third group consisted of 21 naturally exposed dogs, from which sequentially obtained serum samples were available, and which had clinical signs compatible with Rocky Mountain spotted fever. Clinical signs of disease in dogs of the third group resolved after treatment with tetracycline (22 mg/kg of body weight, po, q 8 h) was instituted. At least 2 sequentially obtained serum samples from each dog were tested. In general, the first sample was obtained just prior to treatment and the convalescent serum samples were obtained at weekly or greater intervals thereafter.

For correlation and reactivity data, an ifa test for IgG/IgM (using heavy and light chains-specific conjugate) was used as the reference standard for comparison of results with those of the other tests.

For analyzing sensitivity and specificity data in naturally exposed dogs, the assumption was made that fourfold change in the ifa IgG/IgM titer for R rickettsii or titer ≥ 1,024 identified infected dogs. Threshold titer indicative of specific rickettsial antibody was determined for each test: ifa IgG/IgM, 64; ifa IgM, 8; la, 16; EIA IgG, 64; and EIA IgM, 64. Marked cross-reactivity of sera with R rickettsii, R rhipicephali, and R montana was apparent for the ifa method using these antigens; lesser cross-reactivity was observed for R bellii. Although the ifa test for IgM, using R rickettsii as antigen, had high specificity (88.2%), it had low sensitivity (21.7%), limiting its use as a sole diagnostic test. The la test had a higher sensitivity (58.5%) than did any of the IgM ifa tests. The la test results correlated best with IgM ifa test results, and the closest agreement was obtained when R bellii was used as antigen. Because of its relatively high sensitivity (58.5%) and specificity (83.6%), the la procedure may be a valuable screening test for diagnosis of Rocky Mountain spotted fever in dogs. The IgG and IgM EIA methods had high sensitivity- 90.6 and 83.1%, respectively. The IgM EIA performed by us had low specificity; it detected infection with R montana in inoculated dogs. Comparing results of all test methods in naturally exposed dogs, the IgM EIA results were positive for the greatest number of samples with corresponding negative results by the reference IgG/IgM ifa test. This low specificity may preclude its routine use as a diagnostic test. No single serologic method correctly identified all affected dogs. On the basis of comparison with results obtained for the other serologic procedures and antigen-detection methods in 1 dog, the IgG/IgM ifa test used as a reference test may not have detected some infected dogs. Combination of a test that detects predominantly IgG, with either the la or IgM ifa test, would help to identify most infected dogs.

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