Detection of antibodies against Anaplasma marginale in milk using a recombinant MSP5 indirect ELISA

• Published in: Veterinary microbiology Vol. 106; no. 3; pp. 287 - 292
• Main Authors: de Echaide, Susana Torioni, Bono, María Florencia, Lugaresi, Cecilia, Aguirre, Nerina, Mangold, Atilio, Moretta, Rosalía, Farber, Marisa, Mondillo, Carolina
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 10-04-2005; Elsevier Science
• Subjects: acute course; Agglutination Tests - veterinary; Anaplasma marginale; Anaplasma marginale - immunology; Anaplasmosis - diagnosis; Anaplasmosis - microbiology; Animals; Antibodies, Bacterial - isolation & purification; bacterial antigens; Bacterial Outer Membrane Proteins - immunology; Bacteriology; Biological and medical sciences; bovine anaplasmosis; carrier state; Cattle; Cattle Diseases - diagnosis; Cattle Diseases - microbiology; Dairy cattle; dairy cows; Diagnosis; disease detection; disease diagnosis; disease outbreaks; disease surveillance; Electrophoresis, Agar Gel - veterinary; ELISA; enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; Enzyme-Linked Immunosorbent Assay - veterinary; Female; Fundamental and applied biological sciences. Psychology; immunoglobulin G; Microbiology; Milk; Milk - immunology; Milk - microbiology; Miscellaneous; monoclonal antibodies; peroxidase; Polymerase Chain Reaction - veterinary; recombinant antigens; ROC Curve; Sensitivity and Specificity; test sensitivity; test specificity; validity; Argentina; Anaplasma marginale; Diagnosis; Dairy cattle; ELISA; Milk; Rickettsiales; Microbiology; Antibody; Anaplasmataceae; Bacteria; Detection; Veterinary; ELISA assay
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/j.vetmic.2004.12.026

An indirect enzyme linked immunosorbent assay (iELISA) for diagnosis of anaplasmosis using undiluted individual milk samples from dairy cows was developed. The recombinant 19
kDa major surface protein 5 (rMSP5) of
Anaplasma marginale was used as antigen. A monoclonal antibody against bovine IgG1 conjugated with peroxidase and the chromogen 3,5,3′,5′-tetramethylbenzidine were used in the test. Strong and weak, positive and negative milk samples were set up as reference controls. Results were expressed as percentage of positivity (PP) contrasting with the strongest positive control. The test was evaluated in two groups (G1 and G2) of lactating dairy cows from herds located in
A. marginale non-endemic areas of Argentina. The infection status of both groups, G1 (
n
=
128) sampled after anaplasmosis outbreak, and G2 (
n
=
216) free of anaplasmosis was established by polymerase chain reaction (PCR). Serum samples of cows from G1 and G2 were analyzed by card agglutination test (CAT) and competitive ELISA (cELISA), while the novel iELISA was evaluated in their corresponding milk samples.
At a cutoff of 42 PP, the ELISA has 98% sensitivity and 95% specificity. A significant difference (
P
<
0.0001) was found between the mean PP value of negative samples from G1 (17.4
±
14.9), and G2 (8.6
±
7.1). The agreement and kappa (
κ) value between iELISA and PCR was 96%,
κ
=
0.919; between iELISA and CAT was 97%,
κ
=
0.880; and between iELISA and cELISA was 97%,
κ
=
0.899. These results strongly support the usefulness of iELISA to detect
A. marginale antibodies in milk. Additional studies are necessary to define the ability of the milk iELISA to detect not only acutely infected, but also carrier cattle.