Our current research on CVID in horses
Our laboratory studies a natural form of CVID in horses that is characterized by late-onset recurrent bacterial infections, hypo- or agammaglobulinemia, inadequate response to tetanus toxoid vaccination, and B cell lymphopenia or depletion. By the time of diagnosis, B cells are rare in peripheral blood and secondary lymphoid tissues, and essentially absent in the bone marrow.
Affected individuals are non-related adult horses (average age 10.7 ± 4.4 years) of both sexes, different breeds, living in distinct parts of the country. Clinical signs include most commonly recurrent fevers and pneumonia, but also meningitis and/or neurologic disorders, gingivitis, sinusitis, hepatitis, diarrhea, and skin abscesses. The majority of the affected horses were submitted to euthanasia due to severity of infections and poor prognosis. A few horses were managed for 1 to 5 years on continuous or intermittent antibiotic therapy.
Peripheral blood lymphocyte immunophenotyping reveals persistent, severe B cell lymphopenia in all patients. Affected horses frequently present with intermittent peripheral blood lymphopenia (<1,000 cells/µL). Serum IgM concentrations are undetectable or markedly reduced in all patients. Persistent IgG deficiency is common to all horses. Serum IgA concentrations are within normal values in early diagnosis in most patients despite severely low serum IgM and IgG concentrations, but levels reduce with progression of clinical disease and proximity to euthanasia.
The most outstanding finding comes from the immunohistochemical and histopathological analysis of lymphoid tissues of affected horses: there are no BSAP(PAX5), CD19 or IGM positive B cells found in the bone marrow and spleen; occasional B cells are present in the lymph node of many patients.
Our hypothesis is that B cell differentiation in the affected horses is impaired at the level of pro-B cells in the bone marrow due to abnormal gene regulation. We studied the expression of the major genes involved in the regulation of equine B cell differentiation in archive bone marrow samples from horses with CVID and healthy horses. Transcriptome analysis, standard and quantitative RT-PCR has revealed that most patients express significantly lower levels of E2A and PAX5 genes in their bone marrow. Importantly, expression of PAX5 target genes was also low, including CD19, CD79A, CD79B, and IGJ. We have not identified mutations in the PAX5 gene in the affected horses. PAX5, an early transcription factor that codes for B cell-specific activator protein (BSAP), is essential for B lineage commitment and development throughout B cell life up to plasma cells.
We are currently investigating epigenetic mechanisms that affect E2A and PAX5 expressions, and the expression of other EBF1 targets with PCR amplification and sequencing of bisulfite-treated genomic DNA and Methyl-Seq, which allows us comparison of the methylation status between affected and non-affected horses on a genome-wide scale.