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Enterococcus cecorum lesion strains cause more embryonic mortality after inoculation into the albumen of embryonated eggs compared to cloaca strains. We hypothesized that these strain differences are a result of differences in sensitivity to the antimicrobial effects of the albumen. In this study, the sensitivity of 14 lesion strains and 14 cloaca strains to albumen from 12-day incubated and non-incubated eggs was assessed. A major antimicrobial protein of the albumen is lysozyme and, therefore, the lysozyme resistance of all strains was determined. Albumen from 12-day incubated and non-incubated eggs was inoculated with four cloaca strains and four lesion strains (104 CFU/tube). Based on the results, in a subsequent experiment, all 28 strains were inoculated only into albumen from non-incubated eggs. For all strains, the minimal inhibitory concentrations (MIC) of lysozyme were determined using an agar dilution method and the growth rates in broth with (500 and 2000 µg/ml) or without lysozyme were assessed. Compared to cloaca strains, lesion strains had 320 times higher odds of being reisolated from albumen (95% confidence interval: 55–3962) and had 8.5 times higher reisolation ratios (reisolation dose/inoculation dose) (95% confidence interval: 4.8–15.3). Thirteen cloaca strains had a MIC ranging from 1000 to 8000 µg/ml, while one cloaca strain and all lesion strains were resistant to the lysozyme concentrations tested. Growth rates of cloaca strains were decreased more by lysozyme compared to lesion strains. In conclusion, lesion strains had higher survival rates in egg albumen and were more resistant to lysozyme compared to cloaca strains. Egg albumen inhibits Enterococcus cecorum cloaca strains more than lesion strains. Enterococcus cecorum lesion strains are resistant to high concentrations of lysozyme. Lysozyme resistance could enhance survival in albumen and body fluids. [ABSTRACT FROM AUTHOR]

The Major Histocompatibility Complex (MHC) genes play a key role in a number of biological processes, most notably in immunological responses. The MHCI and MHCII genes incorporate a complex set of highly polymorphic and polygenic series of genes, which, due to the technical limitations of previously available technologies, have only been partially characterized in non-model but economically important species such as the horse. The advent of high-throughput sequencing platforms has provided new opportunities to develop methods to generate high-resolution sequencing data on a large scale and apply them to the analysis of complex gene sets such as the MHC. In this study, we developed and applied a MiSeq-based approach for the combined analysis of the expressed MHCI and MHCII repertoires in cohorts of Thoroughbred, Icelandic, and Norwegian Fjord Horses. The approach enabled us to generate comprehensive MHCI/II data for all of the individuals (n = 168) included in the study, identifying 152 and 117 novel MHCI and MHCII sequences, respectively. There was limited overlap in MHCI and MHCII haplotypes between the Thoroughbred and the Icelandic/Norwegian Fjord horses, showcasing the variation in MHC repertoire between genetically divergent breeds, and it can be inferred that there is much more MHC diversity in the global horse population. This study provided novel insights into the structure of the expressed equine MHC repertoire and highlighted unique features of the MHC in horses. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (S. aureus), a major mastitis pathogen in dairy goats, is classified as a contagious pathogen. Although previous research has shown that extramammary body sites can be colonized with S. aureus, it is unknown whether these sites are reservoirs for intramammary infections. The aim of this research was to determine whether extramammary sites can be colonized with mastitis-associated S. aureus strains in dairy goats. Milk samples were collected from 207 primiparous goats and from 120 of these goats, extramammary site samples (hock, groin, nares, vulva and udder) were collected from a large commercial dairy goat herd in the Netherlands during four sampling visits. Extramammary site swabs and milk samples were (selectively) cultured and S. aureus isolates were spa genotyped. The prevalence of colonization of the extramammary sites at goat level was 51.7% and the prevalence of S. aureus intramammary infections was 7.2%. The nares were colonized most frequently (45%), while the groin area was colonized the least (2.5%). Six spa genotypes were identified in this herd and there was no significant difference in the distribution of spa genotypes between the milk or the extramammary sites (p = 0.141). Both in the extramammary sites and in the milk, spa genotypes t544 (82.3% and 53.3%) and t1236 (22.6% and 33.3%) were the dominant genotypes. These results show that in goats, extramammary sites, particularly the nares, are frequently colonized with mastitis-associated S. aureus strains. Extramammary sites may, thus, be a source of S. aureus intramammary infections that are not targeted by the intervention measures aimed at preventing transmission from infected udder glands. [ABSTRACT FROM AUTHOR]

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 as Mycobacterium 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 bacteria in vitro. Furthermore, MAIT cells in milk increased in frequency in cows with mastitis. Following challenge with virulent Mycobacterium 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. [ABSTRACT FROM AUTHOR]

Bovine Tuberculosis (bTB) caused by Mycobacterium bovis is a livestock disease of global economic and public health importance. There are currently no effective vaccines available for livestock and so control relies on animal level surveillance and pasteurization of dairy products. A new alternative control approach is to exploit the genetic variability of the host; recent studies have demonstrated that breeding European taurine cattle, such as Holsteins for increased resistance to bTB is feasible. The utility of such an approach is still unknown for African cattle populations. This study aims to assess genetic variation in bTB resistance and the underlying genomic architecture of cattle in Cameroon. We conducted a cross-sectional study of 2,346 slaughter cattle in Cameroon. Retropharyngeal lymph node samples were collected and cultured on Lowenstein Jensen media and the BACTEC MGIT 960 system, and M. bovis was identified using the Hain® Genotype kits. A total of 153 cattle were positive for M. bovis and were archived along with a random selection of negative samples. In this study, we genotyped archived samples from 212 cattle. Their genomic diversity was characterized using PCA, hierarchical clustering and admixture analysis. We assessed genetic variation in bTB resistance using heritability analysis and compared quantitative trait loci. Previous research on this study population have shown that Fulani cattle are more susceptible to bTB than mixed breeds. However, here we show that these apparent phenotypic differences in breeds are not reflected by clear genomic differences. At the genetic level, both the Fulani and mixed cattle show similar patterns of admixture with evidence of both taurine and indicine ancestry. There was little European taurine introgression within the studied population. Hierarchical clustering showed clusters of cattle that differed in their susceptibility to bTB. Our findings allude to bTB resistance being polygenic in nature. This study highlights the potential for genetic control of bTB in Africa and the need for further research into the genetics of bTB resistance within African cattle populations. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis (bTB), caused by Mycobacterium bovis , is a chronic disease of cattle with a detrimental impact on food quality and production. Research on bTB vaccines has predominantly been focused on proteinaceous antigens. However, mycobacteria have a thick and intricate lipid outer layer and lipids as well as lipopeptides are important for immune-evasion and virulence. In humans, lipid extracts of M. tuberculosis have been shown to elicit immune responses effective against M. tuberculosis in vitro. Chloroform-methanol extraction (CME) was applied to M. bovis BCG to obtain a hydrophobic antigen extract (CMEbcg) containing lipids and lipopeptides. CMEbcg stimulated IFN-γ+IL-2+ and IL-17A+IL-22+ polyfunctional T cells and elicited T cell responses with a Th1 and Th17 cytokine release profile in both M. bovis BCG vaccinated and M. bovis challenged calves. Lipopeptides were shown to be the immunodominant antigens in CMEbcg, stimulating CD4 T cells via MHC class II. CMEbcg expanded T cells killed CMEbcg loaded monocytes and the CMEbcg-specific CD3 T cell proliferative response following M. bovis BCG vaccination was the best predictor for reduced pathology following challenge with M. bovis. Although the high predictive value of CMEbcg-specific immune responses does not confirm a causal relationship with protection against M. bovis challenge, when taking into account the in vitro antimycobacterial phenotype of CMEbcg-specific T cells (e.g. Th1/Th17 cytokine profile), it is indicative that CMEbcg-specific immune responses could play a functional role in immunity against M. bovis. Based on these findings we conclude that lipopeptides of M. bovis are potential novel subunit vaccine candidates and that further studies into the functional characterization of lipopeptide-specific immune responses together with their role in protection against bovine tuberculosis are warranted. [ABSTRACT FROM AUTHOR]

Introduction: Bovine neonatal pancytopenia (BNP) is a hemorrhagic disease that emerged in calves across Europe in 2007. Its occurrence is attributed to immunization of the calf’s mother with a vaccine produced using an allogeneic cell line. Vaccine-induced alloantibodies specific for major-histocompatibility class I antigens are transferred from the mother to the calf via colostrum, leading to profound depletion of peripheral blood and bone marrow cells that is often fatal. Areas covered: Pubmed and Web of Science were used to search for literature relevant to BNP and the use of allogeneic vaccine cell lines. Following a review of the pathology and pathogenesis of this novel condition, we discuss potential risks associated with the use of allogeneic vaccine cell lines. Expert commentary: Although BNP is associated with a specific vaccine, it highlights safety concerns common to all vaccines produced using allogeneic cell lines. Measures to prevent similar vaccine-induced alloimmune-mediated adverse events in the future are discussed. [ABSTRACT FROM PUBLISHER]

Although maternal vaccination is generally considered to be safe, the occurrence of Bovine Neonatal Pancytopenia (BNP) in cattle shows that maternal vaccination may pose a risk to the offspring. Pregsure © BVD-induced maternal alloantibodies cause BNP in newborn calves. The occurrence of BNP years after last Pregsure © BVD vaccination indicates that alloantibody levels may remain high in dams. Since pregnancy induces alloantibodies we hypothesized that pregnancy boosts the vaccine-induced alloantibody response. Alloantibody levels in Pregsure © BVD-vaccinated dams increased from conception towards the end of gestation and declined after parturition. In parallel, BVDV-antibody levels remained constant, indicating that there is specific boosting of alloantibodies. Since the rise in alloantibodies coincides with pregnancy and other alloantigen sources were excluded, we concluded that fetal alloantigens expressed during pregnancy boost the alloimmune response in the dam. These results help explain why BNP cases occur even years after Pregsure © BVD has been taken off the market. [ABSTRACT FROM AUTHOR]

Vaccines against S. aureus bovinemastitis 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. [ABSTRACT FROM AUTHOR]

Bovine mastitis is a costly disease to the dairy industry and intramammary infections (IMI) with Staphylococcus aureus are a major cause of mastitis. Staphylococcus aureus strains responsible for mastitis in cattle predominantly belong to ruminant-associated clonal complexes (CCs). Recognition of pathogens by bovine mammary epithelial cells (bMEC) plays a key role in activation of immune responsiveness during IMI. However, it is still largely unknown to what extent the bMEC response differs according to S. aureus CC. The aim of this study was to determine whether ruminant-associated S. aureus CCs differentially activate bMEC. For this purpose, the immortalized bMEC line PS was stimulated with S. aureus mastitis isolates belonging to four different clonal complexes (CCs; CC133, CC479, CC151 and CC425) and interleukin 8 (IL-8) release was measured as indicator of activation. To validate our bMEC model, we first stimulated PS cells with genetically modified S. aureus strains lacking (protein A, wall teichoic acid (WTA) synthesis) or expressing (capsular polysaccharide (CP) type 5 or type 8) factors expected to affect S. aureus recognition by bMEC. The absence of functional WTA synthesis increased IL-8 release by bMEC in response to bacterial stimulation compared to wildtype. In addition, bMEC released more IL-8 after stimulation with S. aureus expressing CP type 5 compared to CP type 8 or a strain lacking CP expression. Among the S. aureus lineages, isolates belonging to CC133 induced a significantly stronger IL-8 release from bMEC than isolates from the other CCs, and the IL-8 response to CC479 was higher compared to CC151 and CC425. Transcription levels of IL-8, tumor necrosis factor alpha (TNFα), serum amyloid A3 (SAA3), Toll-like receptor (TLR)-2 and nuclear factor κB (NF-κB) in bMEC after bacterial stimulation tended to follow a similar pattern as IL-8 release, but there were no significant differences between the CCs. This study demonstrates a differential activation of bMEC by ruminant-associated CCs of S. aureus, which may have implications for the severity of mastitis during IMI by S. aureus belonging to these lineages. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a major human and animal pathogen and a common cause of mastitis in cattle. S. aureus secretes several leukocidins that target bovine neutrophils, crucial effector cells in the defence against bacterial pathogens. In this study, we investigated the role of staphylococcal leukocidins in the pathogenesis of bovine S. aureus disease. We show that LukAB, in contrast to the γ-hemolysins, LukED, and LukMF′, was unable to kill bovine neutrophils, and identified CXCR2 as a bovine receptor for HlgAB and LukED. Furthermore, we assessed functional leukocidin secretion by bovine mastitis isolates and observed that, although leukocidin production was strain dependent, LukMF′ was most abundantly secreted and the major toxin killing bovine neutrophils. To determine the role of LukMF′ in bovine mastitis, cattle were challenged with high (S1444) or intermediate (S1449, S1463) LukMF′-producing isolates. Only animals infected with S1444 developed severe clinical symptoms. Importantly, LukM was produced in vivo during the course of infection and levels in milk were associated with the severity of mastitis. Altogether, these findings underline the importance of LukMF′ as a virulence factor and support the development of therapeutic approaches targeting LukMF′ to control S. aureus mastitis in cattle. [ABSTRACT FROM AUTHOR]

Bovine Neonatal Pancytopenia (BNP), a fatal bleeding syndrome of neonatal calves, is caused by maternal alloantibodies absorbed from colostrum and is characterized by lymphocytopenia, thrombocytopenia and bone marrow hypoplasia. An inactivated viral vaccine is the likely source of alloantigens inducing BNP-associated alloantibodies in the dam. In this study the specificity of BNP alloantibodies was assessed and was linked to the pathology of BNP. We demonstrated that Major Histocompatibility Complex class I (MHC I) and Very Late Antigen-3, an integrin α3/β1 heterodimer, were the major targets of BNP alloantibodies. However, alloantibody binding to various bovine cell types correlated with MHC I expression, rather than integrin β1 or α3 expression. Likewise, alloantibody-dependent complement-mediated cell lysis correlated strongly with MHC I expression. Examination of several tissues of third trimester bovine foetuses revealed that cells, shown to be affected in calves with BNP, were characterized by high MHC class I expression and high levels of alloantibody binding. We conclude that in spite of the heterogeneous specificity of BNP associated maternal alloantibodies, MHC I-specific antibodies mediate the pathogenicity of BNP in the calf and that cells with high MHC I expression were preferentially affected in BNP. [ABSTRACT FROM AUTHOR]