Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Bacterial Cultures and Growth Conditions
2.2. Serum Samples and Groups of Dogs
2.3. Detection of Leptospira in Sera Using a Genus-Specific Nested PCR Assay
2.4. A Preliminary Study Using the Microscopic Agglutination Test (MAT)
2.5. Leptospiral Protein Preparations for Modified ELISAs
2.5.1. Whole-Cell Protein Using Sonicated Leptospiral Preparations
2.5.2. Total Membrane Protein Fraction Obtained Using Lysis Buffer
2.5.3. Outer Membrane Protein Isolation Using Triton X-114
2.6. Confirmation of Protein Components via SDS-PAGE and Western Blotting
2.7. Indirect Immunoglobulin G (IgG) ELISAs
2.7.1. Optimisation of Indirect ELISAs and Cut-Off OD Values
2.7.2. IgG Antibody Detection
2.8. Statistical Analysis
3. Results
3.1. Detection of Leptospira in Serum via Nested PCR
3.2. Antibody Titres in the Microscopic Agglutination Test
3.3. The Protein Components Confirmed via SDS-PAGE and Western Blot Analysis Using Serum from a Dog with Leptospirosis
3.4. Determination of the Optimal OD Cut-Off Values for Indirect ELISAs
3.5. Detection of Antibody Levels via the Modified ELISAs
3.6. Diagnostic Performance of All Modified ELISAs
3.7. Receiver–Operator Curve (ROC) Analysis, Correlation Analysis, and Agreement Analysis
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Serovars/Isolates | MAT | ELISA |
---|---|---|
24 representative reference isolates (belonging to 24 serovars) | ||
Australis | Used | - |
Aumtumnalis | Used | - |
Ballum | Used | - |
Bataviae | Used | - |
Canicola | Used | Used |
Cellidoni | Used | - |
Cynopteri | Used | - |
Djasiman | Used | - |
Grippotyphosa | Used | Used |
Hebdonadis | Used | - |
Icterohaemorrhagiae | Used | Used |
Javanica | Used | - |
Louisaina | Used | - |
Manhao | Used | - |
Mini | Used | - |
Panama | Used | - |
Pomona | Used | Used |
Pyrogenes | Used | - |
Ranarum | Used | - |
Sarmin | Used | - |
Sejroe | Used | - |
Shermani | Used | - |
Tarasovi | Used | - |
Semaranga | Used | - |
5 local Thai isolates (belonging to 4 serovars) | ||
Paidjan strain CUDO5 | Used | Used |
Dadas strain CUDO8 | Used | Used |
Bataviae strain D64 | Used | Used |
Mini strain CUDO6 | Used | Used |
Mini strain CUD13 | Used | Used |
Groups | Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Total | |
---|---|---|---|---|---|---|---|
Infected Dogs from Nan Province That Were Confirmed as Infected via Positive PCR and Isolation | Unvaccinated Dogs from Nan Province | Vaccinated Dogs from Non-Endemic Areas | Unvaccinated Dogs from Non-Endemic Areas | Unvaccinated Puppies from Non-Endemic Areas | |||
Number of samples | 6 | 21 | 23 * | 89 ** | 108 | 13 | 260 |
112 | |||||||
Area | Endemic area: Nan Province | Endemic area: Nan Province | Non-endemic area: Bangkok, Samut Prakan, and Chonburi provinces | Non-endemic area: Bangkok, Samut Prakan, and Chonburi provinces | Non-endemic area: Bangkok, Samut Prakan, and Chonburi provinces | - | |
Age | >1 year | >1 year | >1 year | >1 year | Two months | - | |
Vaccination status | No vaccination | No vaccination | Complete vaccination | No vaccination | No vaccination | - | |
Methods (Nested PCR, Isolation, and MAT assays) | |||||||
Nested PCR from urine | 6 (6/6; 100%) | 0 (0/21; 0%) | N/A | N/A | N/A | 6 (6/27; 22%) | |
Isolation from urine | 4 (4/6; 67%) | 0 (0/21; 0%) | N/A | N/A | N/A | 4 (4/27; 15%) | |
Nested PCR from sera | 6 (6/6; 100%) | 0 (0/21; 0%) | 0 (0/112; 0%) | 0 (0/108; 0%) | 0 (0/13; 0%) | 6 (6/260; 2%) | |
MAT from sera | 0 (0/6; 0%) | 0 (0/21; 0%) | 0 (0/23; 0%) | N/A | N/A | N/A | 14 (14/50; 28%) |
Methods (modified ELISAs), protein preparation, and local isolates of serovars used in the ELISAs | |||||||
WCP-Dadas/IgG-ELISA | 6 (6/6; 100%) | 14 (14/21; 67%) | 12 (12/23; 52%) | 25 (25/89; 28%) | 0 (0/108; 0%) | 0 (0/13; 0%) | 57 (57/260; 22%) |
37 (37/112; 33%) | |||||||
TMP-Dadas/IgG-ELISA | 6 (6/6; 100%) | 13 (13/21; 62%) | 6 (6/23; 26%) | 12 (12/89; 13%) | 0 (0/108; 0%) | 0 (0/13; 0%) | 37 (37/260; 14%) |
18 (18/112; 16%) | |||||||
OMP-Dadas/IgG-ELISA | 6 (6/6; 100%) | 15 (10/21; 48%) | 0 (0/23; 0%) | 0 (0/89; 0%) | 0 (0/108; 0%) | 0 (0/13; 0%) | 21 (21/260; 8%) |
0 (0/112; 0%) |
Groups | Number | MAT | Serovars and Serological Titres in MAT | ||||
---|---|---|---|---|---|---|---|
Hebdonadis | Sejroe | Shermani | Paidjan (CUDO5) | ||||
Isolation and PCR-confirmed infected dogs from Nan Province (Group 1) | 6 | 0 (0/6; 0%) | - | - | - | - | |
Unvaccinated dogs from Nan Province (Group 2) | 21 | 11 (11/21; 52%) | 1 * | 1 | 9 * | 1 | |
1:20 | 1:80 | 4 | 5 | ||||
1:20 | 1:40 | ||||||
Vaccinated dogs from Bangkok (Group 3) | 23 | 3 (3/23; 13%) | - | - | 3 | - | |
1:20 | |||||||
Total | 50 | 14 (28%) | 1 | 1 | 12 | 1 |
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Boonciew, P.; Saisongkorh, W.; Brameld, S.; Thongpin, M.; Kurilung, A.; Krangvichian, P.; Niyomtham, W.; Patarakul, K.; Phichitraslip, T.; Hampson, D.J.; et al. Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs. Animals 2024, 14, 893. https://doi.org/10.3390/ani14060893
Boonciew P, Saisongkorh W, Brameld S, Thongpin M, Kurilung A, Krangvichian P, Niyomtham W, Patarakul K, Phichitraslip T, Hampson DJ, et al. Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs. Animals. 2024; 14(6):893. https://doi.org/10.3390/ani14060893
Chicago/Turabian StyleBoonciew, Pannawich, Watcharee Saisongkorh, Suppalak Brameld, Matsaya Thongpin, Alongkorn Kurilung, Pratomporn Krangvichian, Waree Niyomtham, Kanitha Patarakul, Thanmaporn Phichitraslip, David J. Hampson, and et al. 2024. "Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs" Animals 14, no. 6: 893. https://doi.org/10.3390/ani14060893
APA StyleBoonciew, P., Saisongkorh, W., Brameld, S., Thongpin, M., Kurilung, A., Krangvichian, P., Niyomtham, W., Patarakul, K., Phichitraslip, T., Hampson, D. J., & Prapasarakul, N. (2024). Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs. Animals, 14(6), 893. https://doi.org/10.3390/ani14060893