Development and Evaluation of a Recombinant Antigen,Monoclonal Antibody-Based Competitive ELISA for Heartwater Serodiagnosis

Abstract

Cowdria ruminantium is the etiologic agent of heartwater, a tick-transmitted foreign animal disease with considerable potential for entrance into the USA. A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect serologic responses to C. ruminantium infection. The cELISA utilized a recombinant form of the C. ruminantium major antigenic protein (MAP-1) as the antigen and an anti-MAP-1 monoclonal antibody as the competing indicator reagent. Experimental antisera to C. ruminantium and a wide variety of related ehrlichial organisms were used to evaluate cELISA reactivity. Only sera against C. ruminantium, Ehrlichia canis, E. chaffeensis, and a recently discovered cervine ehrlichia-like organism reacted positively in the cELISA. Specificity of the cELISA was 299.5% in a survey of 1,774 southeastern US and Puerto Rican slaughter cattle sera but was only 85% in a group of 79 hunter-killed white-tailed deer (Odocoileus virginianus) from the southeastern USA. Reference true-positive and cELISA false-positive sera were further analyzed by end point titrations using the cELISA and by indirect fluorescent antibody (IFA) tests for reactivity with C. ruminantium, E. canis, and E. chaffeensis antigens. True heartwater-positive sera were significantly more reactive using the cELISA and C. ruminantium IFA procedures (P < 0.05), whereas false-positive sera were significantly more reactive with the antigens used in the E. chaffeensis IFA procedure (P < 0.05). A group of sera from 210 field-origin ruminants residing on known or potentially heartwater-endemic Caribbean islands revealed a substantial (12.4%) prevalence of cELISA-positive specimens. The cELISA is a relatively specific serodiagnostic test for heartwater in cattle and could be used to monitor for possible introduction of the disease into the USA. The cELISA may also be an excellent tool for monitoring the success of an ongoing Caribbean Amblyomma tick eradication program designed to eliminate the biological vector responsible for the perpetuation and spread of this dangerous foreign animal disease.

References

Camus

Barre

Martinez

Uilenberg

: 1996, Heartwater (cowdriosis)-a review, 2nd ed. Office International des Epizooties, Paris, France

Dawson

Childs

Biggie

: 1994, White-tailed deer as a potential reservoir of Ehrlichia spp J Wild Dis 30:162–168

Dawson

Warner

Baker

: 1996, Ehrlichia-like 16S rDNA sequence from wild white tailed deer (Odocoileus virginianus) J Parasitol 82:52–58

Dilbeck

Evermann

Crawford

: 1990, Isolation of a previously undescribed rickettsia from an aborted bovine fetus J Clin Microbiol 28:814–816

Du Pleiss

Bezuidenhout

Brett

: 1993, The serodiagnosis of heartwater: A comparison of five tests Rev élév Méd Vét Pays Trop 46:123–129

Gooding

: 1987, Production of monoclonal antibodies In: Monoclonal antibodies: Principles and practices, 2nd ed., pp. 59–103. Academic Press, San Diego, CA

Jongejan

De Vries

Nieuwenhuijs

: 1993, The immunodominant protein of Cowdria ruminantium is conserved within the genus Ehrlichia Rév élév Méd Vét Pays Trop 46:145–152

Jongejan

Thielmans

MJC

De Groot

: 1991, Competitive enzyme-linked immunosorbent assay for heartwater using monoclonal antibodies to a Cowdria ruminantium-specific 32-kilodalton protein Vet Microbiol 28:199–211

Katz

Barbet

Mahan

: 1996, A recombinant antigen from the heartwater agent (Cowdria ruminantium) reactive with antibodies in some southeastern United States white-tailed deer (Odocoileus virginianus), but not cattle sera J Wildl Dis 32:424–430

10.

Katz

Green

Barbet

: 1995, Sheep and cattle inoculated with Ehrlichia canis, Ehrlichia chaffeensis, and blood from Ehrlichia-seropositive and PCR-positive southeastern U.S. white-tailed deer remain seronegative to a recombinant heartwater antigen Proc Annu Meet Conf Res Workers Anim Dis 76: 173. [Abstr.]

11.

Kobold

Martinez

Camus

: 1992, Distribution of heartwater in the Caribbean determined on the basis of detection of antibodies to the conserved 32-kilodalton protein of Cowdria ruminantium J Clin Microbiol 30:1870–1873

12.

Logan

Holland

Mebus

: 1986, Serological relationship between Cowdria ruminantium and certain Ehrlichia Vet Rec 119:458–459

13.

Mahan

Tebels

Mukwedeya

: 1993, An immunoblotting diagnostic assay for heartwater based on the immunodominant 32-kilodalton protein of Cowdria ruminantium detects false-positives in field sera J Clin Microbiol 31:2729–2737

14.

Perreau

Morel

Barre

Durand

: 1980, Existence de la cowdriose a Cowdria ruminantium, chez les petits ruminants des Antilles Francaises (la Guadeloupe) et des Mascareignes (la Reunion et Ile Maurice) Rév élév Méd Vét Pays Trop 33:21–22

15.

Rikihisa

: 1991, The tribe Ehrlichieae and ehrlichial diseases Clin Microbiol Rev 4:286–308

16.

Rossouw

Neitz

AWH

De Waal

: 1990, Identification of the antigenic proteins of Cowdria ruminantium Onderstepoort J Vet Res 57:215–221

17.

Snedecor

Cochran

: 1967, Statistical methods, 6th ed., pp. 20–62. Iowa State University Press, Ames, IA

18.

Van Vliet

Jongejan

Van der Zeijst

: 1992, Phylogenetic position of Cowdria ruminantium (Rickettsiales) determined by analysis of amplified 16S ribosomal DNA sequences Int J Syst Bacteriol 42:494–498

19.

Van Vliet

Van der Zeist

Camus

: 1995, Use of a specific immunogenic region on the Cowdria ruminantium MAP 1 protein in a serologic assay J Clin Microbiol 33:2405–2410

20.

Walker

: 1987, The tick vectors of Cowdria ruminantium (Ixodoidea, Ixodidae, genus Amblyomma) and their distribution Onderstepoort J Vet Res 54:353–379