Bovine CD4 (BoCD4)

Cluster of differentiation 4 (CD4) is the accessory protein non-covalently bounded to T cell receptor (TCR) that recognize an invariant region of MHC class II on antigen presenting cell (APC). Its cytoplasmic tail, physically associated with a protein tyrosine kinase, is important in the activation of helper/inducer T (T_H) lymphocytes. The anti-bovine CD4 monoclonal antibody (mAb) CC8 is a clone usefully used in flow cytometry assay in the last 20 years and no anomalous labelling has been described in previous reports. The aim of this study was to investigate the lack and reduced binding of clone CC8 to T_H lymphocytes in three different breeds of dairy cattle: Italian Friesian (FRI), Pezzata Rossa Friulana (PRI) and their crossbreed (MET). Two unexpected flow cytometric phenotypes were found: CC8⁺ (reduced labelling) in 25%, 35% and 15% of the animals in the three breeds respectively, and CC8⁻ (complete lack of labelling) in 15% of PRI cows. The complete sequencing of CD4 coding region (CDS) has allowed to identify two new missense mutations in the exon 5: Q306H and K310 N associated with the two phenotypes. A further new synonymous mutation (g.103634725 G > A) was found in exon 6. Further studies are needed to understand the effects of the news mutations, but these results demonstrate that flow cytometry is a powerful tool to discover polymorphic determinants of antigens involved in the immune responses.

Precalving feeding level affects dry matter intake, postcalving energy balance, the risk of hepatic lipidosis and metabolic disease, and gene expression in liver and adipose tissue. These coincide with a higher risk of disease postpartum and, very likely, a failure to reach optimum production as well as reproductive targets. Current interpretation of the available evidence suggest that metabolic stressors affect the immune system of transition dairy cows and lead to reduced immunocompetence. The objective of the current study was to investigate the effect of precalving body condition score (BCS) and level of feeding on immunocompetence during the peripartum period. Twenty-three weeks before calving, 78 cows were allocated randomly to 1 of 6 treatment groups (n = 13) in a 2 × 3 factorial arrangement: 2 precalving BCS categories (4.0 and 5.0, based on a 10-point scale) and 3 levels of energy intake during the 3 wk preceding calving (75, 100, and 125% of estimated requirements). Blood was sampled precalving and at 1, 2 and 4 wk after calving. Cells were analyzed by flow cytometry and quantitative real-time PCR. The numbers of T helper lymphocytes (CD4+), cytotoxic T lymphocytes (CD8+), natural killer cells (CD335+), and γδ T lymphocytes (WC1+) as well as their activation status [IL-2 receptor (CD25)+ cells] were highly variable between animals, but there was no evident effect of BCS, feeding level, or time. All groups presented with an increase in expression of cytokines in unstimulated blood cells in the week after calving, although this was significant only for IFNG in the BCS 4.0 group. Analysis of in vitro-stimulated cells allowed 2 general observations: (1) cows with high energy intake precalving (125%) had increased cytokine expression precalving, and (2) all cows had increased cytokine expression in the week after calving. The present study provides evidence that prepartum feed management can affect immunocompetence during the transition period. Considering the current results, optimally conditioned animals might benefit from a restricted precalving diet, whereas underconditioned cows can be fed to requirements.

Peripartum, and especially during the transition period, dairy cows undergo dramatic physiological changes. These coincide with an increased risk of disease during the first 2 wk after calving and have been linked to dairy cows failing to achieve production as well as reproductive targets. Previous evidence suggests that these physiological changes affect the immune system and that transition dairy cows experience some form of reduced immunocompetence. However, almost all of these studies were undertaken in high-production, housed dairy cows. Grazing cows have much lower levels of production and this study aimed to provide clarity whether or not the dysfunctional attributes of the peripartum immune system reported in high production housed cows are evident in these animals. Therefore, cell culture techniques, flow cytometry, and quantitative PCR were applied to analyze the cellular composition of peripheral blood mononuclear cells from transition dairy cows as well as the performance of these cells in an in vitro assay. First, a combination of in vitro stimulation and quantitative PCR for cytokines was validated as a quantifiable immunocompetence assay in 29 cattle and a correlation of quantitative PCR and ELISA demonstrated. Second, the relative number of T helper cells, cytotoxic T cells, B cells, γδ T cells, natural killer cells, and monocytes in peripheral blood was measured, of which B cells and natural killer cells increased in number postcalving (n = 29) compared with precalving. Third, following in vitro stimulation cytokine profiles indicated decreased expression of IFNγ, tumor necrosis factor, and IL-17 and increased expression of IL-10 wk 1 after calving, which later all returned to precalving values (n = 39). Additionally, treatment of transition cows with a nonsteroidal anti-inflammatory drug (i.e., carprofen) administered on d 1, 3, and 5 postcalving (n = 19; untreated control n = 20) did not affect the cytokine expression at any time point. In conclusion, an immunocompetence assay has been developed that highlights a characteristic expression pattern for IFNγ, tumor necrosis factor, IL-17, and IL-10 that reflects a state of reduced immunocompetence in moderate-yielding pasture-based transition cows after calving, which is similar to that described for higher-yielding housed cows.

The immuno-staining patterns of skin leukocytes were investigated in three breeds of cattle: Holstein–Friesian, Brahman and Santa Gertrudis of similar age before and after tick infestation. The antibodies specific for CD45 and CD45RO reacted with cells in the skin of all Holstein–Friesian cattle but did not react with cells in the skin of any Brahman cattle. The same antibodies reacted with cells from the skin of four (CD45) and seven (CD45RO) of twelve Santa Gertrudis cattle. The antibodies specific for T cells and γδ subset of T cells recognized cells from all three breeds of cattle. The antibody specific for MHC class II molecules labelled cells of mostly irregular shape, presumably dermal dendritic cells and/or macrophages and Langerhans cells. The antibody specific for granulocytes (mAb CH138) reacted with cells only in sections cut from skin with lesions. The antibody specific for CD25⁺ cells labelled regularly shaped cells that showed a wide range of intensities of staining.

Evaluation of the changes induced by immunological interventions requires a baseline against which to compare those changes. The age-related changes in the CD8⁺ T-cell population of cattle were studied. The results indicate that CD8⁺ T-cells could be divided into γ/δ TCR1⁺ and γ/δ TCR1⁻ according to their expression of the γ/δ T-cell receptor. As a proportion, the CD8⁺ γ/δ TCR1⁺ population appears to increase with age. Within the CD8⁺γ/δ TCR1⁻ a population of cells expressing a profile of surface molecules previously associated with effector memory T cells (CD45RO⁺, CD62L⁻, CD27⁻, CD45RA⁻ and CD28⁻) increases with age. Furthermore, a parallel increase with age in the proportion of CD8⁺CD45RO⁺ T cells that express the cytotoxic granule protein perforin was observed. In peripheral tissues, namely lungs, it was found that the majority of CD8⁺ T cells present expressed a phenotype indicative of previously primed T cells (high expression of CD45RO and perforin). In contrast, only a small population of memory CD8⁺ T cells was present in lymphoid tissue where most of the CD8⁺ T cells expressed a naïve phenotype. In conclusion, in cattle, like in human, CD8⁺ T cells that express a phenotype associated with antigen experience accumulate with age that may play a role in immunocompetence as the individual ages.

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3⁺, CD4⁺, CD8⁺ and γδ T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for γδ T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.