MHC peptide binding and presentation is the most selective event defining the landscape of T cell epitopes. Consequently, understanding the diversity of MHC alleles in a given population and the parameters that define the set of ligands that can be bound and presented by each of these alleles (the immunopeptidome) has an enormous impact on our capacity to predict and manipulate the potential of protein Ags to elicit functional T cell responses. Liquid chromatography–mass spectrometry analysis of MHC-eluted ligand data has proven to be a powerful technique for identifying such peptidomes, and methods integrating such data for prediction of Ag presentation have reached a high level of accuracy for both MHC class I and class II. In this study, we demonstrate how these techniques and prediction methods can be readily extended to the bovine leukocyte Ag class II DR locus (BoLA-DR). BoLA-DR binding motifs were characterized by eluted ligand data derived from bovine cell lines expressing a range of DRB3 alleles prevalent in Holstein–Friesian populations. The model generated (NetBoLAIIpan, available as a Web server at www.cbs.dtu.dk/services/NetBoLAIIpan) was shown to have unprecedented predictive power to identify known BoLA-DR–restricted CD4 epitopes. In summary, the results demonstrate the power of an integrated approach combining advanced mass spectrometry peptidomics with immunoinformatics for characterization of the BoLA-DR Ag presentation system and provide a prediction tool that can be used to assist in rational evaluation and selection of bovine CD4 T cell epitopes.

Staphylococcus aureus, a major cause of bovine mastitis, produces a wide range of immune-evasion molecules. The bi-component leukocidin LukMF’ is a potent killer of bovine neutrophils in vitro. Since the role of LukMF’ in development of bovine mastitis has not been studied in natural infections, we aimed to clarify whether presence of the lukM-lukF’ genes and production levels of LukMF’ are associated with clinical severity of the disease. Staphylococcus aureus was isolated from mastitis milk samples (38 clinical and 17 subclinical cases) from 33 different farms. The lukM-lukF’ genes were present in 96% of the isolates. Remarkably, 22% of the lukM-lukF’-positive S. aureus isolates displayed a 10-fold higher in vitro LukMF’ production than the average of the lower-producing ones. These high producing isolates were cultured significantly more frequently from clinical than subclinical mastitis cases. Also, the detection of LukM protein in milk samples was significantly associated with clinical mastitis and high production in vitro. The high producing LukMF’ strains all belonged to the same genetic lineage, spa-type t543. Analysis of their global toxin gene regulators revealed a point mutation in the Repressor of toxins (rot) gene which results in a non-functional start codon, preventing translation of rot. This mutation was only identified in high LukMF’ producing isolates and not in low LukMF’ producing isolates. Since rot suppresses the expression of various toxins including leukocidins, this mutation is a possible explanation for increased LukMF’ production. Identification of high LukMF’ producing strains is of clinical relevance and can potentially be used as a prognostic marker for severity of mastitis.

Staphylococcus aureus is an important mastitis pathogen, causing both clinical mastitis (CM) and subclinical mastitis (SCM) in small ruminants. In general, CM has a low incidence in sheep and goats but can be very severe and costly. In contrast, subclinical mastitis (SCM) is common but is associated with less cost. For both sheep and goats, S. aureus is the main cause of CM and is associated with SCM cases with a high SCC. Recently, specific lineages of S. aureus have been identified that are associated with CM rather than SCM in dairy cows. It is unknown whether specific S. aureus lineages are associated with CM in goats and sheep. The aim of this study was to compare the clonal complex (CC), staphylococcal protein A (spa) type, leukocidin lukM-lukF' presence, and potential to produce LukMF' in vitro between CM and SCM S. aureus mastitis isolates obtained from sheep and goats. Differences between isolates from different host species were also compared. Ovine (CM, n = 12; SCM, n = 29) and caprine (CM, n = 14; SCM, n = 30) isolates were obtained from 8 sheep flocks and 8 goat herds in the Netherlands. Overall, the isolates belonged to CC133 (85%), CC398 (7%), CC425 (5%), and CC45 (2%). Seventeen spa types were found, including 6 novel types; the predominant types were t2678 (34%), t544 (18%), and t3583 (18%). Although CC133 was dominant among both sheep and goat isolates, spa type CC133/t2678 was associated with ovine isolates, whereas CC133/t544 and CC133/t3583 were found mostly in goats. The presence of lukM-lukF' among the S. aureus isolates was high (87%), especially in CC133 (96%) and CC425 (100%), but the genes were absent in CC45 and CC398. In vitro-cultured lukM-lukF'-positive isolates produced LukM (71 out of 74 positive isolates tested) in the range of 0.4 to 5.0 µg/mL. Interestingly, the goat-associated lineages CC133/t544 and CC133/t3583 produced more LukM in vitro than the sheep-associated CC133/t2678. We found no difference in LukMF' production potential between CM and SCM isolates. In sheep as well as in goats, no association was found between genotype and CM or SCM, demonstrating that the same lineages of S. aureus are responsible for both CM and SCM. These results suggest that subclinically infected animals in a herd or flock likely act as the reservoir of S. aureus causing CM. This highlights the importance of early identification and control of SCM and suggests that controlling SCM within a herd is an effective intervention to prevent CM in small ruminants.

Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells that utilize a semi-invariant T cell receptor (TCR) α chain and are restricted by the highly conserved antigen presenting molecule MR1. MR1 presents microbial riboflavin biosynthesis derived metabolites produced by bacteria and fungi. Consistent with their ability to sense ligands derived from bacterial sources, MAIT cells have been associated with the immune response to a variety of bacterial infections, such asMycobacterium spp., Salmonella spp. and Escherichia coli. To date, MAIT cells have been studied in humans, non-human primates and mice. However, they have only been putatively identified in cattle by PCR based methods; no phenotypic or functional analyses have been performed. Here, we identified a MAIT cell population in cattle utilizing MR1 tetramers and high-throughput TCR sequencing. Phenotypic analysis of cattle MAIT cells revealed features highly analogous to those of MAIT cells in humans and mice, including expression of an orthologous TRAV1-TRAJ33 TCR α chain, an effector memory phenotype irrespective of tissue localization, and expression of the transcription factors PLZF and EOMES. We determined the frequency of MAIT cells in peripheral blood and multiple tissues, finding that cattle MAIT cells are enriched in mucosal tissues as well as in the mesenteric lymph node. Cattle MAIT cells were responsive to stimulation by 5-OP-RU and riboflavin biosynthesis competent bacteriain vitro. Furthermore, MAIT cells in milk increased in frequency in cows with mastitis. Following challenge with virulentMycobacterium bovis, a causative agent of bovine tuberculosis and a zoonosis, peripheral blood MAIT cells expressed higher levels of perforin. Thus, MAIT cells are implicated in the immune response to two major bacterial infections in cattle. These data suggest that MAIT cells are functionally highly conserved and that cattle are an excellent large animal model to study the role of MAIT cells in important zoonotic infections.

Vaccines against S. aureus bovine mastitis are scarce and show limited protection only. All currently available vaccines are applied via the parenteral (usually intramuscular) route. It is unknown, however, whether this route is the most suitable to specifically increase intramammary immunity to combat S. aureus at the site of infection. Hence, in the present study, immunization via mucosal (intranasal; IN), intramuscular (triangle of the neck; IM), intramammary (IMM) and subcutaneous (suspensory ligament; SC) routes were analyzed for their effects on the quantity of the antibody responses in serum and milk as well as the neutralizing capacity of the antibodies within serum. The experimental vaccine comprised the recombinant S. aureus immune evasion proteins extracellular fibrinogen-binding protein (Efb) and the leukotoxin subunit LukM in an oil-in-water adjuvant combined with a hydrogel and alginate. The highest titer increases for both Efb and LukM specific IgG1 and IgG2 antibody levels in serum and milk were observed following SC/SC immunizations. Furthermore, the harmful effects of Efb and leukotoxin LukMF’ on host-defense were neutralized by serum antibodies in a route-dependent manner. SC/SC immunization resulted in a significant increase in the neutralizing capacity of serum antibodies towards Efb and LukMF’, shown by increased phagocytosis of S. aureus and increased viability of bovine leukocytes. Therefore, a SC immunization route should be considered when aiming to optimize humoral immunity against S. aureus mastitis in cattle.