No abstract

Using a chicken Class II MHC clone in Northern blot analysis, tissue-specific expression of turkey Class II MHC genes was observed in the embryonic bursa of Fabricius as well as in the adult spleen. In contrast, there was no detectable expression in the embryonic liver, brain, or spleen. Southern blot analysis of BamHI-digested turkey DNA revealed two restriction fragment length polymorphism (RFLP) patterns that did not deviate significantly from single-gene Mendelian inheritance. Further analysis of PvuII-digested DNA from 325 turkeys showed four distinct RFLP patterns that segregated within the turkey lines studied. Because the chicken Class II MHC clone hybridized specifically to mRNA in immune-associated tissues, and because it identified polymorphisms among turkeys, the chicken clone is suggested to identify four turkey Class II MHC genotypes. The current study provides good evidence that RFLP analysis of DNA can be used as a means for molecular genotyping at the MHC in turkeys.

Aims:  To detect and quantify Lactobacillus buchneri in plant samples with the aid of polymerase chain reaction (PCR) methods. Methods and Results:  DNA from silage samples spiked with different amounts of L. buchneri cells was isolated using a lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform extraction method. The DNA served as a template for PCR amplification with primers specific for the bacterium. The primers were developed by comparison of 16S rDNA sequences from different lactic acid bacteria (LAB) and testing for specificity with 11 different strains of LAB. As few as 100 L. buchneri colony‐forming units per gram of silage could be detected. Additionally, the technique was successfully applied to quantify the population of L. buchneri in two cultivars of corn with or without inoculation. Conclusions:  The PCR assay provided a specific and rapid tool for identifying and enumerating L. buchneri in silage samples. Significance and Impact of the Study:  The use of microbial inoculants for silage production is a safe and environment friendly practice, but the full potential of such additives can only be achieved with a better understanding of the fate and activity of the microbes involved. The current study describes a methodology to detect and enumerate L. buchneri, a micro‐organism used as an inoculant.

A turkey subline at the Ohio Agricultural Research and Development Center was developed by DNA typing of the MHC using a chicken MHC Class II probe, and it segregated for specific MHC genotypes. Histocompatibility was examined between turkeys of known MHC genotype using skin graft procedures, mixed lymphocyte reactions (MLR), and graft-versus-host reactions (GVHR). Skin grafts were exchanged among 3-wk-old turkeys and it was found that when birds shared DNA patterns (genotypes), the skin grafts were usually accepted. In contrast, skin grafts were always rejected when birds did not share the identical DNA pattern. Similarly, MLR only occurred when the lymphocytes were derived from birds that did not share the same DNA pattern. Lastly, GVHR were examined in embryos injected with either sire or dam blood. The GVHR in embryos was dependent on the parental MHC genotype. Four MHC haplotypes were identified in the turkey subline. The turkey MHC has been designated MhcMega-B, and each of the haplotypes, Mega-B(1) through Mega-B(4).

To investigate the origin of disease-associated IgM autoantibodies (AAb), we compared the genetic and structural characteristics of IgM AAb from autoimmune prone motheaten (mev) mice with natural autoantibodies (NAAb) from normal background C57/BL6 strain. Six hybridoma-derived IgM molecules each were obtained both from mev mice, at the terminal stage of systemic autoimmune disease, and from mitogen-stimulated C57/BL6 mice. These were randomly selected for VH J558 gene expression (aberrantly expressed in mev mice). The variable regions of the IgM molecules, both from autoimmune and normal mice, were encoded by unmutated germline VH genes. Disease-associated AAb from mev mice were predominantly encoded by the J558 subfamily 186.2, whereas five J558 subfamilies were utilized in NAAb originating from normal mice. Junctional diversity as a result of N or P nucleotide insertions and D-D fusions was noted among IgMs originating from both mev (mostly B-1 lymphocytes) and C57BL/6 (mostly B-2 lymphocytes) mice. Interestingly, all six J558+ IgMs from mev mice showed a restricted CDR3 length of 10 amino acids, with similar hydrophobicity indices. Four unique V-D-J rearrangements were observed among these IgMs. None of the IgMs were polyreactive and three of the six were subsequently observed to express monospecific autoreactivity with synthetic peptides (residues 81-92 and 37-53) representing segments of the T cell CD4-accessory molecule. Three IgM antibodies had hydrophilic arginine residues in their CDR3 heavy chain region. By contrast, all six J558+ IgMs from C57/BL6 mice had variable CDR3 length, distinct VDJ rearrangements and a local negative charge in the CDR3 region. Four of these IgMs demonstrated polyreactivity with multiple conserved autoantigens and, hence, were classified as NAAb. These findings provide evidence for either positive or impaired negative selection of B-1 lymphocytes secreting disease-associated IgM AAb in mev mice. This likely results from a reduced threshold of responsiveness to autoantigens due to PTP1C deficiency, which is targeted at the CDR3 length of the variable region of the heavy chain. In addition, characteristic differences in the size and hydrophobicity pattern of the CDR3 of the heavy chain allow structural distinction between monospecific disease-associated IgM AAb and the polyreactive IgM NAAb.