Poster
Comparison of whole-organism and leukotoxin ELISAs for detection of Mannheimia haemolytica antibodies in experimentally inoculated calves
| Date/Time: |
8/27/2026
|
| Author: |
Korakrit Poonsuk |
| Clinic: |
Washington State University |
| City, State, ZIP: |
Pullman, WA 99164 |
K. Poonsuk, DVM, MSc, PhD, DACVM
1
;
A. Lear, DVM, MS, PhD, DACVIM
2
;
A. Wilson, MS
2
;
M. Caldwell, DVM, PhD, DACVIM
2
;
1Washington Animal Disease Diagnostic Laboratory, Washington State University, Pullman, WA, 99163
2University of Tennessee College of Veterinary Medicine Large Animal Clinical Sciences, 37996
Introduction:
Mannheimia haemolytica is a major bacterial pathogen associated with bovine respiratory disease, and antibody detection is commonly used to evaluate exposure and immune response. A previously developed whole-organism (WO) ELISA detects broad antibody responses against outer membrane proteins but may not fully capture immune responses directed against exotoxins. Leukotoxin (LKT), a key exotoxin and component of commercial M. haemolytica vaccines, represents an important target for serologic evaluation. The objective of this study was to develop an LKT-based ELISA and evaluate its performance alongside the WO ELISA in calves.
Materials and methods:
Serum samples (n = 64) were collected from calves in an experimental inoculation study at multiple timepoints, including pre-inoculation (day post-inoculation [DPI] 0, 10, 18, 25, 32, and 39). Antibody responses were measured using three newly developed LKT ELISAs (LKT ELISA A, B, and C), each performed under different assay conditions, in comparison to a WO ELISA.
Results were expressed as sample-to-positive (S/P) ratios and interpreted as positive or negative based on predefined assay criteria. Longitudinal antibody responses were evaluated within animals across timepoints. Biological response status was defined based on time post-inoculation and antibody trends and was used as the reference standard for calculation of diagnostic sensitivity and specificity. Agreement between WO ELISA and LKT ELISAs was also assessed.
Results:
At DPI 0, most samples were negative or low across all assays. LKT ELISAs detected increasing antibody responses beginning at DPI 10–18, whereas WO ELISA primarily detected positive responses at later timepoints (DPI ≥25).
Seroconversion was first detected by LKT ELISAs at DPI 10–18 in a subset of calves, while WO ELISA detected most positive animals at DPI 25 and later. By DPI 25–39, all assays detected strong responders, with high agreement among assays for clearly positive and negative samples.
Using biological response as the reference standard, all assays demonstrated good diagnostic performance. Among the LKT ELISAs, LKT ELISA A showed balanced performance, with sensitivity of 83% and specificity of 86%. LKT ELISA B demonstrated the highest sensitivity (94%), indicating strong ability to detect responding animals, particularly at earlier timepoints. LKT ELISA C demonstrated the highest specificity (91%), indicating fewer false-positive results. In comparison, the WO ELISA showed lower sensitivity at earlier timepoints but high specificity for strong antibody responses.
Overall, LKT ELISAs demonstrated sensitivity ranging from 81% to 94% and specificity ranging from 82% to 91%. Agreement between WO and LKT ELISAs was highest for strongly positive and negative samples, with discordant results observed primarily at earlier timepoints.
Significance:
LKT ELISAs demonstrated strong analytical performance and reproducibility across multiple platforms using specimens from experimentally inoculated calves. Compared to the WO ELISA, all LKT ELISAs detected antibody responses earlier while maintaining strong diagnostic performance. These findings support the use of LKT-based assays as complementary tools to the WO ELISA for evaluating immune responses to Mannheimia haemolytica.
