RESUMEN
Maternal colostrum (MC) is an important source of nutrients and immune factors for newborn calves. However, when colostrum is unavailable or of poor quality, a colostrum replacer (CR) may be a suitable alternative to MC. As stock-raising farmers must make informed decisions about colostrum feeding management, this study was conducted to determine the effect of feeding MC versus CR on the promotion of immunological status, growth, and health in pre-weaned Japanese black (JB) calves. Sixteen newborn JB calves were fed MC after birth, and 16 JB calves were fed CR. For the MC group, the numbers of γδ T cells, CD4+ cells, CD8+ cells, CD4+CD8+ cells, B cells, and MHC class II+ cells were significantly higher compared with the CR group. Furthermore, the expression levels of interleukin (IL)-1ß-, IL-2-, and interferon-γ (IFN-γ)-encoding mRNAs were significantly higher in the MC group compared with the CR group. A lower incidence of disease in 1-month-old calves and higher carcass weight in the MC group were observed compared with the CR group. These results suggest that CR activates the immune system delayed in calves compared with MC. MC increases populations of various immunocompetent cells, which can reduce infection rates and improve body weight gain.
RESUMEN
Shiga toxin-producing Escherichia coli (STEC) is a major intestinal pathogen and causes serious gastrointestinal illness, which includes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. The major virulence factors of STEC are Shiga toxins (Stx1 and Stx2), which belong to the AB-type toxin family. Among several subtypes of Stx1 and Stx2, the production of Stx2a is thought to be a risk factor for severe STEC infections, but Stx2a production levels vary markedly between STEC strains, even strains with the same serotype. Therefore, quantitative analyses of Stx2 production by STEC strains are important to understand the virulence potential of specific lineages or sublineages. In this study, we developed a novel Stx2 quantification method by utilizing homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technology. To determine suitable "sandwich" assay conditions, we tested 6 combinations of fluorescence-labeled monoclonal antibodies (mAbs) specific to Stx2 and compared the HTRF signal intensities obtained at various incubation times. Through this analysis, we selected the most suitable mAb pair, one recognizing the A subunit and the other recognizing the B subunit, thus together detecting Stx holotoxins. The optimal incubation time was also determined (18 h). Then, we optimized the concentrations of the two mAbs based on the range for linearity. The established HTRF assay detected 0.5 ng/ml of the highly purified recombinant Stx2a and Stx2e proteins and the working range was 1-64 ng/ml for both Stx2a and Stx2e. Through the quantification analysis of Stx proteins in STEC cell lysates, we confirmed that other Stx2 subtypes (Stx2b, Stx2c, Stx2d and Stx2g) can also be quantified at a certain level of accuracy, while this assay system does not detect Stx2f, which is highly divergent in sequence from other Stx2 subtypes, and Stx1. As the HTRF protocol we established is simple, this assay system should prove useful for the quantitative analysis of Stx2 production levels of a large number of STEC strains.