Development of a new recombinant p24 ELISA system for diagnosis of bovine leukemia virus in serum and milk
Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leucosis. Here, we designed a p24 enzyme-linked immunosorbent assay (ELISA) to detect antibodies specific for BLV capsid protein p24 (encoded by the gag gene) in bovine serum samples. The p24 gene was inserted into an Escherichia coli expression system, and recombinant proteins (GST-p24, p24, and His-p24) were purified. His-p24 was the most suitable antigen for using in the ELISA. The cut-off point was calculated from a receiver operating characteristic curve derived from a set of 582 field samples that previously tested positive or negative by BLV-CoCoMo-qPCR-2, which detects BLV provirus. The new p24 ELISA showed almost the same specificity and sensitivity as a commercial gp51 ELISA kit when used to test field serum samples, and allowed monitoring of p24 antibodies in raw milk and whey. Comparing the results for the p24 ELISA and gp51 ELISA revealed that p24 antibodies were detected earlier than gp51 antibodies in three out of eight calves experimentally infected with BLV, indicating improved detection without diminishing BLV serodiagnosis. Thus, the p24 ELISA is a robust and reliable assay for detecting BLV antibodies in serum or milk, making it is a useful tool for large-scale BLV screening.
We thank Dr. Junko Kohara, Mr. Sunao Hirai, Mr. Susumu Ogawa, Mr. Kenji Mizushiri, Mr. Naoya Takahashi, and Mr. Yoshihiro Okada of the Animal Research Center, Hokkaido Research Organization, for help with experimental infection and drawing of blood. We are also grateful to all members of the Viral Infectious Diseases Field, Nano Medical Engineering Laboratory, RIKEN Cluster for Pioneering Research, for technical assistance, help, and helpful suggestions. We also thank the Support Unit for Biomaterial Analysis, RIKEN BSI Research Resources Center, for help with sequence analysis.
This work was supported by Grants-in-Aid for Scientific Research [A (16H02590) and C (16K02590)] from the Japan Society for the Promotion of Science (JSPS), by a grant from the Project of the NARO Bio-oriented Technology Research Advancement Institution (the special scheme project on regional developing strategy) (Grant No. 16817983), by a grant from the Project of the NARO Bio-oriented Technology Research Advancement Institution (the special scheme project on vitalizing management entities of agriculture, forestry and fisheries) (Grant No. 16930548), and by the Japan Society for the JSPS Postdoctoral Fellowship (Grant No. 16F16404).
Compliance with ethical standards
Conflicts of interest
The authors declare no conflicts of interest.
Animal handling and research ethics
All animals were handled by veterinarians from the Animal Research Center in Hokkaido, and RIKEN, Japan in strict accordance with good animal practice following the guidelines of Hokkaido Research Organization and RIKEN. The study was approved by the Animal Ethical Committee and the Animal Care and Use Committee of the Animal Research Center, Hokkaido Research Organization (approval number 1302) and RIKEN Animal Experiments Committee (approval number H29-2-104).
- 4.APHIS (2008) Info sheet: bovine leukosis virus (BLV) on US dairy operations, 2007. US Department of Agriculture Animal and Plant Health Services, Veterinary Services, Center for Epidemiology and Animal Health. http://www.aphis.usda.gov/animal_health/nahms/dairy/downloads/dairy07/Dairy07_is_BLV.pdf. Accessed 11 Nov 2015
- 14.Gillet N, Florins A, Boxus M, Burteau C, Nigro A, Vandermeers F, Balon H, Bouzar AB, Defoiche J, Burny A, Reichert M, Kettmann R, Willems L (2007) Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in human. Retrovirology 4:18CrossRefGoogle Scholar
- 29.Miyasaka T, Takeshima SN, Jimba M, Matsumoto Y, Kobayashi N, Matsuhashi T, Sentsui H, Aida Y (2013) Identification of Bovine leukocyte antigen class II haplotypes associated with variations in Bovine leukemia virus proviral load in Japanese Black cattle. Tissue Antigens 81:72–82CrossRefGoogle Scholar
- 37.OIE (2018) World animal health information database-version enzootic Bovine leucosis. In: Manual of diagnostic tests and vaccines for terrestrial animals 2018. Chapter 2.4.10. 1–12. World organisation for animal Health, Paris. http://www.oie.int/en/international-standard-setting/terrestrial-manual/access-online/
- 39.Panei CJ, Takeshima SN, Omori T, Nunoya T, Davis WC, Ishizaki H, Matoba K, Aida Y (2013) Estimation of Bovine leukemia virus (BLV) proviral load harbored by lymphocyte subpopulations in BLV-infected cattle at the subclinical stage of enzootic bovine leucosis using BLV-CoCoMo-qPCR. BMC Vet Res 9:95CrossRefGoogle Scholar
- 42.Polat M, Takeshima SN, Hosomichi K, Kim J, Miyasaka T, Yamada K, Arainga M, Murakami T, Matsumoto Y, de la Barra Diaz V, Panei CJ, Gonzalez ET, Kanemaki M, Onuma M, Giovambattista G, Aida Y (2016) A new genotype of Bovine leukemia virus in South America identified by NGS-based whole genome sequencing and molecular evolutionary genetic analysis. Retrovirology 13:4CrossRefGoogle Scholar
- 56.Yuan Y, Kitamura-Muramatsu Y, Saito S, Ishizaki H, Nakano M, Haga S, Matoba K, Ohno A, Murakami H, Takeshima SN, Aida Y (2015) Detection of the BLV provirus from nasal secretion and saliva samples using BLV-CoCoMo-qPCR-2: comparison with blood samples from the same cattle. Virus Res 210:248–254CrossRefGoogle Scholar