Johne's disease of cattle is widespread and causes significant economic loss to producers. Control has been hindered by limited understanding of the immune response to the causative agent,Mycobacterium aviumsubsp.paratuberculosis, and lack of an effective vaccine and sensitive specific diagnostic assays. The present study was conducted to gain insight into factors affecting the immune response toM. aviumsubsp.paratuberculosis. A persistent proliferative response toM. aviumsubsp.paratuberculosispurified protein derivative and solubleM. aviumsubsp.paratuberculosisantigens was detected in orally infected neonatal calves 6 months postinfection (p.i.) by flow cytometry (FC). CD4⁺T cells with a memory phenotype (CD45R0⁺) expressing CD25 and CD26 were the predominant cell type responding to antigens. Few CD8⁺T cells proliferated in response to antigens until 18 months p.i. γδ T cells did not appear to respond to antigen until 18 months p.i. The majority of WC1⁺CD2⁻and a few WC1⁻CD2⁺γδ T cells expressed CD25 at time zero. By 18 months, however, subsets of γδ T cells from both control and infected animals showed an increase in expression of CD25, ACT2, and CD26 in the presence of the antigens. Two populations of CD3⁻non-T non-B null cells, CD2⁺and CD2⁻, proliferated in cell cultures from some control and infected animals during the study, with and without antigen. The studies clearly show multicolor FC offers a consistent reliable way to monitor the evolution and changes in the immune response toM. aviumsubsp.paratuberculosisthat occur during disease progression.

Background: Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. BLV infection may remain clinically silent at the aleukemic (AL) stage, cause persistent lymphocytosis (PL), or, more rarely, B cell lymphoma. BLV has been identified in B cells, CD2⁺T cells, CD3⁺T cells, CD4⁺T cells, CD8⁺T cells, γ/δ T cells, monocytes, and granulocytes in infected cattle that do not have tumors, although the most consistently infected cell is the CD5⁺B cell. The mechanism by which BLV causes uncontrolled CD5⁺B cell proliferation is unknown. Recently, we developed a new quantitative real-time polymerase chain reaction (PCR) method, BLV-CoCoMo-qPCR, which enabled us to demonstrate that the proviral load correlates not only with BLV infection, as assessed by syncytium formation, but also with BLV disease progression. The present study reports the distribution of BLV provirus in peripheral blood mononuclear cell subpopulations isolated from BLV-infected cows at the subclinical stage of EBL as examined by cell sorting and BLV-CoCoMo-qPCR.Results: Phenotypic characterization of five BLV-infected but clinically normal cattle with a proviral load of > 100 copies per 1 × 10⁵cells identified a high percentage of CD5⁺IgM⁺cells (but not CD5^-IgM⁺B cells, CD4⁺T cells, or CD8⁺T cells). These lymphocyte subpopulations were purified from three out of five cattle by cell sorting or using magnetic beads, and the BLV proviral load was estimated using BLV-CoCoMo-qPCR. The CD5⁺IgM⁺B cell population in all animals harbored a higher BLV proviral load than the other cell populations. The copy number of proviruses infecting CD5^-IgM⁺B cells, CD4⁺cells, and CD8⁺T cells (per 1 ml of blood) was 1/34 to 1/4, 1/22 to 1/3, and 1/31 to 1/3, respectively, compared with that in CD5⁺IgM⁺B cells. Moreover, the BLV provirus remained integrated into the genomic DNA of CD5⁺IgM⁺B cells, CD5^-IgM⁺B cells, CD4⁺T cells, and CD8⁺T cells, even in BLV-infected cattle with a proviral load of <100 copies per 10⁵cells.Conclusions: The results of the recent study showed that, although CD5⁺IgM⁺B cells were the main cell type targeted in BLV-infected but clinically normal cattle, CD5^-IgM⁺B cells, CD4⁺cells, and CD8⁺T cells were infected to a greater extent than previously thought.

Mycobacterium avium subsp. paratuberculosis is the causative pathogen of Johne's disease, a chronic inflammatory wasting disease in ruminants. This disease has been difficult to control because of the lack of an effective vaccine. To address this need, we adapted a specialized transduction system originally developed for M. tuberculosis and modified it to improve the efficiency of allelic exchange in order to generate site-directed mutations in preselected M. avium subsp. paratuberculosis genes. With our novel optimized method, the allelic exchange frequency was 78 to 100% and the transduction frequency was 1.1 × 10 ⁻⁷ to 2.9 × 10 ⁻⁷ . Three genes were selected for mutagenesis: pknG and relA , which are genes that are known to be important virulence factors in M. tuberculosis and M. bovis , and lsr2 , a gene regulating lipid biosynthesis and antibiotic resistance. Mutants were successfully generated with a virulent strain of M. avium subsp. paratuberculosis ( M. avium subsp. paratuberculosis K10) and with a recombinant K10 strain expressing the green fluorescent protein gene, gfp . The improved efficiency of disruption of selected genes in M. avium subsp. paratuberculosis should accelerate development of additional mutants for vaccine testing and functional studies.