Have a personal or library account? Click to login
Protective efficacy of the recombinantly expressed C-terminal domain of Mannheimia haemolytica leukotoxin in mice and goats Cover

Protective efficacy of the recombinantly expressed C-terminal domain of Mannheimia haemolytica leukotoxin in mice and goats

Journal of Veterinary Research
AHEAD OF PRINT
By: Thu-Dung Doan,  Teerajet Laohasatian,  Hsing-Chieh Wu and  Chun-Yen Chu  
Open Access
|Oct 2025

Figures & Tables

Fig. 1.

Constructions of pET32a/N- and C-terminal Mannheimia haemolytica leukotoxin A (nLktA and cLktA) plasmids. (A) Truncation of LktA. (B) Locations of the BamHI and EcoRI restriction sites for the N-terminal region of leukotoxin A (LktA) and of EcoRI and XhoI for the C-terminal region. LktB – leukotoxin B; LktC – leukotoxin C; aa – amino acids; bp – base pairs
Constructions of pET32a/N- and C-terminal Mannheimia haemolytica leukotoxin A (nLktA and cLktA) plasmids. (A) Truncation of LktA. (B) Locations of the BamHI and EcoRI restriction sites for the N-terminal region of leukotoxin A (LktA) and of EcoRI and XhoI for the C-terminal region. LktB – leukotoxin B; LktC – leukotoxin C; aa – amino acids; bp – base pairs

Fig. 2.

Antigens, immunisation schedule and immune evaluation of a truncated C-terminal leukotoxin A (cLktA) of Mannheimia haemolytica in mice and goat models. (A) Vaccination groups. (B) Immunisation schedule: primary vaccination administered in week (W) 0 and followed by a booster dose in W2. (C) Immune response evaluation through ELISA assay and challenge test. Mh 13948 – Mannheimia haemolytica strain 13948; CFU – colony-forming units; ISA201 VG – Seppic water-in-oil-in-water adjuvant 201; PBS – phosphate-buffered saline; IgG – immunoglobulin G
Antigens, immunisation schedule and immune evaluation of a truncated C-terminal leukotoxin A (cLktA) of Mannheimia haemolytica in mice and goat models. (A) Vaccination groups. (B) Immunisation schedule: primary vaccination administered in week (W) 0 and followed by a booster dose in W2. (C) Immune response evaluation through ELISA assay and challenge test. Mh 13948 – Mannheimia haemolytica strain 13948; CFU – colony-forming units; ISA201 VG – Seppic water-in-oil-in-water adjuvant 201; PBS – phosphate-buffered saline; IgG – immunoglobulin G

Fig. 3.

(A) Epitope prediction for the full-length LktA leukotoxin A protein of Mannheimia haemolytica with a threshold value of 0.5. Positive peaks above the threshold line indicate amino acids likely to be part of an epitope. (B) Eleven potential epitopes ranked based on their mean prediction scores, listed from highest to lowest (1–11)
(A) Epitope prediction for the full-length LktA leukotoxin A protein of Mannheimia haemolytica with a threshold value of 0.5. Positive peaks above the threshold line indicate amino acids likely to be part of an epitope. (B) Eleven potential epitopes ranked based on their mean prediction scores, listed from highest to lowest (1–11)

Fig. 4.

PCR amplification of C-terminal Mannheimia haemolytica leukotoxin A (cLktA) and confirmation of cloning cLktA into pET32a. (A) Product of 1,035 bp after amplification, indicating cLktA. (B) Restriction enzyme digestion with EcoRI and XhoI confirming the pET32a/cLktA construct
PCR amplification of C-terminal Mannheimia haemolytica leukotoxin A (cLktA) and confirmation of cloning cLktA into pET32a. (A) Product of 1,035 bp after amplification, indicating cLktA. (B) Restriction enzyme digestion with EcoRI and XhoI confirming the pET32a/cLktA construct

Fig. 5.

Expression of N- and C-terminal Mannheimia haemolytica leukotoxin A (nLktA and cLktA) in E. coli. (A) Isopropyl ß-D-1-thiogalactopyranoside-induced expression of nLktA, cLktA and control pET32a at 0 h and 4 h. (B) Western blot confirmation of recombinant cLktA protein using anti-M. haemolytica B2 strain antibody. (C) Further Western blot confirmation using anti–secreted leukotoxin (sLkt) serum
Expression of N- and C-terminal Mannheimia haemolytica leukotoxin A (nLktA and cLktA) in E. coli. (A) Isopropyl ß-D-1-thiogalactopyranoside-induced expression of nLktA, cLktA and control pET32a at 0 h and 4 h. (B) Western blot confirmation of recombinant cLktA protein using anti-M. haemolytica B2 strain antibody. (C) Further Western blot confirmation using anti–secreted leukotoxin (sLkt) serum

Fig. 6.

Purification of recombinant cLktA protein. (A) Solubility screening of cLktA demonstrating that it was a soluble protein. (B) Purification of cLktA using affinity chromatography
Purification of recombinant cLktA protein. (A) Solubility screening of cLktA demonstrating that it was a soluble protein. (B) Purification of cLktA using affinity chromatography

Fig. 7.

Results of indirect ELISA to determine the levels of antigen-specific total immunoglobulin G (IgG) in the sera of mice immunised with inactivated Mannheimia haemolytica 13948 (Mh 13948) or recombinant C-terminal protein of M. haemolytica leukotoxin A (cLktA) or injected with phosphate-buffered saline (control). Different superscript letters present statistically significant differences (P-value < 0.05)
Results of indirect ELISA to determine the levels of antigen-specific total immunoglobulin G (IgG) in the sera of mice immunised with inactivated Mannheimia haemolytica 13948 (Mh 13948) or recombinant C-terminal protein of M. haemolytica leukotoxin A (cLktA) or injected with phosphate-buffered saline (control). Different superscript letters present statistically significant differences (P-value < 0.05)

Fig. 8.

Results of indirect ELISA to determine the levels of antigen-specific total immunoglobulin G in the sera of goats immunised with inactivated Mannheimia haemolytica 13948 (Mh 13948) or recombinant C-terminal protein of M. haemolytica leukotoxin A (cLktA) or injected with phosphate-buffered saline (control). Different superscript letters present statistically significant differences (P-value < 0.05)
Results of indirect ELISA to determine the levels of antigen-specific total immunoglobulin G in the sera of goats immunised with inactivated Mannheimia haemolytica 13948 (Mh 13948) or recombinant C-terminal protein of M. haemolytica leukotoxin A (cLktA) or injected with phosphate-buffered saline (control). Different superscript letters present statistically significant differences (P-value < 0.05)

Fig. 9.

Survival rates of mice immunised with inactivated Mannheimia haemolytica 13948 (Mh 13948) or recombinant C-terminal protein of M. haemolytica leukotoxin A (cLktA) or injected with phosphate-buffered saline (control) when challenged with (A) Mannheimia haemolytica 13948 strain or (B) M. haemolytica B2 field isolate strain
Survival rates of mice immunised with inactivated Mannheimia haemolytica 13948 (Mh 13948) or recombinant C-terminal protein of M. haemolytica leukotoxin A (cLktA) or injected with phosphate-buffered saline (control) when challenged with (A) Mannheimia haemolytica 13948 strain or (B) M. haemolytica B2 field isolate strain

Primers for cloning nLktA and cLktA

Target geneSenseSequence (5ʹ–3ʹ)Restriction enzyme siteLength (base pairs)
nLktAForwardCGgaattcTCCCTTATGGGAACTAGACTTBamHI1,131
ReverseGCCctcgagTTATGCAGCAGCAGACACACCEcoRI
cLktAForwardAAAggatccAAACTTGGTGAAGGTGATEcoRI1,035
ReverseAAActcgagTTAAGCTGCTCTAGCAAAXhoI
DOI: https://doi.org/10.2478/jvetres-2025-0056 | Journal eISSN: 2450-8608
Journal RSS Feed
Language: English
Submitted on: Apr 1, 2025
Accepted on: Oct 9, 2025
Published on: Oct 18, 2025
Published by: National Veterinary Research Institute in Pulawy
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year
Keywords:
bovine respiratory disease (BRD),
cLktA,
cross-strain efficacy,
field-strain protection,
murine challenge model
Related subjects:
Life sciences,
Molecular biology,
Microbiology and virology,
Life sciences, other,
Medicine,
Veterinary medicine

© 2025 Thu-Dung Doan, Teerajet Laohasatian, Hsing-Chieh Wu, Chun-Yen Chu, published by National Veterinary Research Institute in Pulawy
This work is licensed under the Creative Commons Attribution 4.0 License.

AHEAD OF PRINT