Virulence related sequences; insights provided by comparative genomics of Streptococcus uberis of differing virulence.

BACKGROUND: Streptococcus uberis, a Gram-positive, catalase-negative member of the family Streptococcaceae is an important environmental pathogen responsible for a significant proportion of subclinical and clinical bovine intramammary infections. Currently, the genome of only a single reference strain (0140J) has been described. Here we present a comparative analysis of complete draft genome sequences of an additional twelve S. uberis strains. RESULTS: Pan and core genome analysis revealed the core genome common to all strains to be 1,550 genes in 1,509 orthologous clusters, complemented by 115-246 accessory genes present in one or more S. uberis strains but absent in the reference strain 0140J. Most of the previously predicted virulent genes were present in the core genome of all 13 strains but gene gain/loss was observed between the isolates in CDS associated with clustered regularly interspaced short palindromic repeats (CRISPRs), prophage and bacteriocin production. Experimental challenge experiments confirmed strain EF20 as non-virulent; only able to infect in a transient manner that did not result in clinical mastitis. Comparison of the genome sequence of EF20 with the validated virulent strain 0140J identified genes associated with virulence, however these did not relate clearly with clinical/non-clinical status of infection. CONCLUSION: The gain/loss of mobile genetic elements such as CRISPRs and prophage are a potential driving force for evolutionary change. This first "whole-genome" comparison of strains isolated from clinical vs non-clinical intramammary infections including the type virulent vs non-virulent strains did not identify simple gene gain/loss rules that readily explain, or be confidently associated with, differences in virulence. This suggests that a more complex dynamic determines infection potential and clinical outcome not simply gene content.

A core genome multi-locus sequence typing scheme for Streptococcus uberis: an evolution in typing a genetically diverse pathogen.

Streptococcus uberis is a globally endemic and poorly controlled cause of bovine mastitis impacting the sustainability of the modern dairy industry. A core genome was derived from 579 newly sequenced S. uberis isolates, along with 305 publicly available genome sequences of S. uberis isolated from 11 countries around the world and used to develop a core genome multi-locus sequence typing (cgMLST) scheme. The S. uberis core genome comprised 1475 genes, and these were used to identify 1447 curated loci that were indexed into the cgMLST scheme. This was able to type 1012 of 1037 (>97  %) isolates used and differentiated the associated sequences into 932 discrete core genome sequence types (cgSTs). Analysis of the phylogenetic relationships of cgSTs revealed no clear clustering of isolates based on metadata such as disease status or year of isolation. Geographical clustering of cgSTs was limited to identification of a UK-centric clade, but cgSTs from UK isolates were also dispersed with those originating from other geographical regions across the entire phylogenetic topology. The cgMLST scheme offers a new tool for the detailed analysis of this globally important pathogen of dairy cattle. Initial analysis has re-emphasized and exemplified the genetically diverse nature of the global population of this opportunistic pathogen.

Unveiling the gut bacteriome diversity and distribution in the national fish hilsa (Tenualosa ilisha) of Bangladesh.

The field of fish microbiome research has rapidly been advancing, primarily focusing on farmed or laboratory fish species rather than natural or marine fish populations. This study sought to reveal the distinctive gut bacteriome composition and diversity within the anadromous fish species Tenualosa ilisha (hilsa), which holds the status of being the national fish of Bangladesh. We conducted an analysis on 15 gut samples obtained from 15 individual hilsa fishes collected from three primary habitats (e.g., freshwater = 5, brackish water = 5 and marine water = 5) in Bangladesh. The analysis utilized metagenomics based on 16S rRNA gene sequencing targeting the V3-V4 regions. Our comprehensive identification revealed a total of 258 operational taxonomic units (OTUs). The observed OTUs were represented by six phyla, nine classes, 19 orders, 26 families and 40 genera of bacteria. Our analysis unveiled considerable taxonomic differences among the habitats (freshwater, brackish water, and marine water) of hilsa fishes, as denoted by a higher level of shared microbiota (p = 0.007, Kruskal-Wallis test). Among the identified genera in the gut of hilsa fishes, including Vagococcus, Morganella, Enterobacter, Plesiomonas, Shigella, Clostridium, Klebsiella, Serratia, Aeromonas, Macrococcus, Staphylococcus, Proteus, and Hafnia, several are recognized as fish probiotics. Importantly, some bacterial genera such as Sinobaca, Synechococcus, Gemmata, Serinicoccus, Saccharopolyspora, and Paulinella identified in the gut of hilsa identified in this study have not been reported in any aquatic or marine fish species. Significantly, we observed that 67.50% (27/40) of bacterial genera were found to be common among hilsa fishes across all three habitats. Our findings offer compelling evidence for the presence of both exclusive and communal bacteriomes within the gut of hilsa fishes, exhibiting potential probiotic properties. These observations could be crucial for guiding future microbiome investigations in this economically significant fish species.

Identification of single nucleotide polymorphisms in the bovine Toll-like receptor 1 gene and association with health traits in cattle.

Bovine mastitis remains the most common and costly disease of dairy cattle worldwide. A complementary control measure to herd hygiene and vaccine development would be to selectively breed cattle with greater resistance to mammary infection. Toll-like receptor 1 (TLR1) has an integral role for the initiation and regulation of the immune response to microbial pathogens, and has been linked to numerous inflammatory diseases. The objective of this study was to investigate whether single nucleotide polymorphisms (SNPs) within the bovine TLR1 gene (boTLR1) are associated with clinical mastitis (CM).Selected boTLR1 SNPs were analysed within a Holstein Friesian herd. Significant associations were found for the tagging SNP -79 T > G and the 3'UTR SNP +2463 C > T. We observed favourable linkage of reduced CM with increased milk fat and protein, indicating selection for these markers would not be detrimental to milk quality. Furthermore, we present evidence that some of these boTLR1 SNPs underpin functional variation in bovine TLR1. Animals with the GG genotype (from the tag SNP -79 T > G) had significantly lower boTLR1 expression in milk somatic cells when compared with TT or TG animals. In addition, stimulation of leucocytes from GG animals with the TLR1-ligand Pam3csk4 resulted in significantly lower levels of CXCL8 mRNA and protein.SNPs in boTLR1 were significantly associated with CM. In addition we have identified a bovine population with impaired boTLR1 expression and function. This may have additional implications for animal health and warrants further investigation to determine the suitability of identified SNPs as markers for disease susceptibility.

Therapeutic targeting of the innate immune system in domestic animals.

Since first being described in the fruit fly Drosophila melanogaster, the knowledge regarding Toll-like receptors (TLRs) has transformed our understanding of immunology. TLRs are a family of conserved pattern recognition receptors (PRR) that recognise specific microbial-associated molecular patterns and allow the cell to distinguish between self and non-self materials. The very property of the TLRs, to link innate and adaptive immunity, offers a novel opportunity to develop vaccines that engage TLR signalling. The presence of TLR ligands as adjuvants in conjunction with a vaccine is shown to increase the efficacy and response to the immunisation with a particular antigen. Here, we focus on the findings pertaining to TLR ligands as adjuvants and discuss the importance of these studies in the development of an optimal vaccine in farm and companion animals.

The bovine chemokine receptors and their mRNA abundance in mononuclear phagocytes.

BACKGROUND: The chemokine and chemokine receptor families play critical roles in both the healthy and diseased organism mediating the migration of cells. The chemokine system is complex in that multiple chemokines can bind to one chemokine receptor and vice versa. Although chemokine receptors have been well characterised in humans, the chemokine receptor repertoire of cattle is not well characterised and many sequences are yet to be experimentally validated. RESULTS: We have identified and sequenced bovine homologs to all identified functional human chemokine receptors. The bovine chemokine receptors show high levels of similarity to their human counterparts and similar genome arrangements. We have also characterised an additional bovine chemokine receptor, not present in the available genome sequence of humans or the more closely related pigs or horses. This receptor shows the highest level of similarity to CCR1 but shows significant differences in regions of the protein that are likely to be involved in ligand binding and signalling. We have also examined the mRNA abundance levels of all identified bovine chemokine receptors in mononuclear phagocytic cells. Considerable differences were observed in the mRNA abundance levels of the receptors, and interestingly the identified novel chemokine receptor showed differing levels of mRNA abundance to its closest homolog CCR1. The chemokine receptor repertoire was shown to differ between monocytes, macrophages and dendritic cells. This may reflect the differing roles of these cells in the immune response and may have functional consequences for the trafficking of these cells in vivo. CONCLUSIONS: In summary, we have provided the first characterisation of the complete bovine chemokine receptor gene repertoire including a gene that is potentially unique to cattle. Further study of this receptor and its ligands may reveal a specific role of this receptor in cattle. The availability of the bovine chemokine receptor sequences will allow further characterisation of the function of these genes and will confer wide-reaching benefits to the study of this important aspect of the bovine immune response.

PCR-Based Direct Detection of Streptococcus uberis from Subclinical and Clinical Dairy Cattle Milk Samples.

Streptococcus uberis is one of the leading causes worldwide of mastitis in the dairy industry, with the most likely sources of infection attributed to environmental reservoirs such as contaminated bedding materials. Early detection of those cases most likely to progress to clinical disease would lead to improved animal welfare, a critical component of overall health and productivity. A multiplex PCR-based diagnostic test was developed for detection of S. uberis directly from milk and targeting two genes previously identified as important for intramammary colonisation and persistence in dairy cattle. Results indicated the threshold for detection directly from milk was 20,000 CFU/ml and this was achieved without the need for preenrichment. In addition, S. uberis could be identified from milk samples collected during intramammary challenge studies, prior to clinical signs of infection and at much lower detection limits. The PCR test developed for confirmation of the presence of S. uberis directly from infected milk has potential value as a diagnostic test to identify early infection and/or to confirm that antibiotic therapy has been successful.

A Paradox in Bacterial Pathogenesis: Activation of the Local Macrophage Inflammasome Is Required for Virulence of Streptococcus uberis.

Streptococcus uberis is a common cause of intramammary infection and mastitis in dairy cattle. Unlike other mammary pathogens, S. uberis evades detection by mammary epithelial cells, and the host-pathogen interactions during early colonisation are poorly understood. Intramammary challenge of dairy cows with S. uberis (strain 0140 J) or isogenic mutants lacking the surface-anchored serine protease, SUB1154, demonstrated that virulence was dependent on the presence and correct location of this protein. Unlike the wild-type strain, the mutant lacking SUB1154 failed to elicit IL-1ß from ex vivo CD14+ cells obtained from milk (bovine mammary macrophages, BMM), but this response was reinstated by complementation with recombinant SUB1154; the protein in isolation elicited no response. Production of IL-1ß was ablated in the presence of various inhibitors, indicating dependency on internalisation and activation of NLRP3 and caspase-1, consistent with inflammasome activation. Similar transcriptomic changes were detected in ex vivo BMM in response to the wild-type or the SUB1154 deletion mutant, consistent with S. uberis priming BMM, enabling the SUB1154 protein to activate inflammasome maturation in a transcriptionally independent manner. These data can be reconciled in a novel model of pathogenesis in which, paradoxically, early colonisation is dependent on the innate response to the initial infection.

The calf model of immunity for development of a vaccine against tuberculosis.

Tuberculosis (TB) remains a major threat to public health. The identification of safe TB vaccine candidates beyond Mycobacterium bovis BCG, is an exciting prospect for control of human TB and necessary in the context of the human immunodeficiency virus (HIV) pandemic. Selection of vaccine candidates for human trials which are ultimately targeted for use in children less than 5 years of age or in newborns will require an animal model that closely approximates immune function and disease. We propose that the bovine neonate and adolescent is a robust animal model for preclinical safety and efficacy evaluation of TB candidate vaccines targeting this special human population. Parallel studies conducted in bovine neonates and non-human primates with a leading auxotrophic mutant with demonstrated efficacy/safety in a rodent TB model of TB demonstrated similar findings with respect to gross pathology scoring relative to BCG. The findings indicated more numerous and severe lesions in the lung in addition to higher levels of IFN-gamma producing cells. BCG vaccinates demonstrated higher levels of FoxP3 transcripts and lower levels of IL-4 mRNA.

Identification and functional characterization of a bovine orthologue to DC-SIGN.

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) C-type lectin is almost exclusively expressed at the cell surface of DC. In addition to its normal function facilitating contact of DC with T cells, DC-SIGN has been shown to bind a variety of pathogens, including Mycobacterium bovis, and HIV-1 envelope protein gp120. In this study, we identified the bovine ortholog of the human DC-SIGN gene within the bovine genome, which exists as a single copy. PCR amplified a product, showing a 100% match with the predicted sequences as well as a sequence predicted to be similar to that of SIGNR7. Furthermore, a protein with the same molecular weight as human DC-SIGN was detected by Western blot in cell lysate derived from bovine DC. To characterize this molecule functionally, the uptake of FITC-labeled OVA and FITC-labeled gp120 (FITC-gp120) by bovine and human DC was assessed. FITC-gp120 was shown to bind to bovine DC in a time- and temperature-dependent manner. Binding was blocked by a polyclonal anti-DC-SIGN antibody but not by a control antibody. Furthermore, blocking of this molecule also reduced the binding of M. bovis bacillus Calmette-Guerin expressing GFP. Confocal microscopy showed that DC-SIGN was expressed on the surface of bovine DC. Subsequent pulse-chase studies revealed that FITC-gp120 was internalized by bovine monocyte-derived DC as early as 10 min. Thus, there is evidence of a DC-SIGN-like molecule expressed specifically by bovine DC. This molecule may play an important role in the infection of bovine (DC) cells with M. bovis.

Granulocyte chemotactic properties of M. tuberculosis versus M. bovis-infected bovine alveolar macrophages.

The incidence of bovine tuberculosis (TB) continues to rise, and causes significant economic losses worldwide. The causative agent of bovine TB, Mycobacterium bovis, is closely related to the human pathogen M. tuberculosis, and yet these two organisms differ profoundly in their ability to cause disease in cattle. The innate immune system is primarily responsible for controlling disease, with the alveolar macrophage (AlvMvarphi) acting as one of the first points of contact between host and respiratory pathogens. In this study we have examined some of the differences in this component of the host immune response to M. bovis and M. tuberculosis, with the aim of improving our understanding of why M. bovis is able to cause disease in cattle whereas M. tuberculosis is efficiently controlled. Initial studies using microarray technology revealed that chemokines represented some of the most differentially expressed genes between M. tuberculosis and M. bovis-infected bovine AlvMvarphi. M. tuberculosis-infected bovine AlvMvarphi expressed significantly higher levels of the chemokines CCL3, CCL4, CCL5 and CXCL8, whereas M. bovis-infected AlvMvarphi were shown to express higher levels of CCL23. We further demonstrated the role of chemokines in bovine TB by showing that supernatants from AlvMvarphi infected with M. tuberculosis were significantly more effective than those from M. bovis-infected cells at attracting bovine granulocytes in an in vitro chemotaxis assay. These results have significant implications in vivo as they suggest that the M. bovis-infected macrophage is able to circumvent activation of the host chemotactic response and thereby evade killing by the host immune system.

Bovine Neonatal Monocytes Display Phenotypic Differences Compared With Adults After Challenge With the Infectious Abortifacient Agent Neospora caninum.

The neonatal period represents a window of susceptibility for ruminants given the abundance of infectious challenges in their environment. Maternal transfer of immunity does not occur in utero but post-parturition, however this does not compensate for potential deficits in the cellular compartment. Here we present a cellular and transcriptomic study to investigate if there is an age-related difference in the monocyte response in cattle during intra-cellular protozoan infection. We utilized Neospora caninum, an obligate intracellular protozoan parasite that causes abortion and negative economic impacts in cattle worldwide, to study these responses. We found neonatal animals had a significant greater percentage of CD14+ monocytes with higher CD80 cell surface expression. Adult monocytes harbored more parasites compared to neonatal monocytes; additionally greater secretion of IL-1ß was observed in neonates. Microarray analysis revealed neonates have 535 genes significantly upregulated compared to adult with 23 upregulated genes. Biological pathways involved in immune response were evaluated and both age groups showed changes in the upregulation of tyrosine phosphorylation of STAT protein and JAK-STAT cascade pathways. However, the extent to which these pathways were upregulated in neonates was much greater. Our findings suggest that neonates are more resistant to cellular invasion with protozoan parasites and that the magnitude of the responses is related to significant changes in the JAK-STAT network.

The Applied Development of a Tiered Multilocus Sequence Typing (MLST) Scheme for Dichelobacter nodosus.

Dichelobacter nodosus (D. nodosus) is the causative pathogen of ovine footrot, a disease that has a significant welfare and financial impact on the global sheep industry. Previous studies into the phylogenetics of D. nodosus have focused on Australia and Scandinavia, meaning the current diversity in the United Kingdom (U.K.) population and its relationship globally, is poorly understood. Numerous epidemiological methods are available for bacterial typing; however, few account for whole genome diversity or provide the opportunity for future application of new computational techniques. Multilocus sequence typing (MLST) measures nucleotide variations within several loci with slow accumulation of variation to enable the designation of allele numbers to determine a sequence type. The usage of whole genome sequence data enables the application of MLST, but also core and whole genome MLST for higher levels of strain discrimination with a negligible increase in experimental cost. An MLST database was developed alongside a seven loci scheme using publically available whole genome data from the sequence read archive. Sequence type designation and strain discrimination was compared to previously published data to ensure reproducibility. Multiple D. nodosus isolates from U.K. farms were directly compared to populations from other countries. The U.K. isolates define new clades within the global population of D. nodosus and predominantly consist of serogroups A, B and H, however serogroups C, D, E, and I were also found. The scheme is publically available at https://pubmlst.org/dnodosus/.

Differential responses of bovine macrophages to infection with bovine-specific and non-bovine specific mycobacteria.

Members of the Mycobacterium tuberculosis complex share a high level of genetic identity, however pathogenic ability appears to display host restriction. Interestingly M. tuberculosis, the primary cause of tuberculosis in humans, is non-pathogenic in cattle. Conversely Mycobacterium bovis, the cause of tuberculosis in cattle, is also responsible for a proportion of tuberculosis cases in humans. We hypothesise that differences in the abilities of M. bovis and M. tuberculosis to cause pathogenesis in cattle will be reflected in their interactions with bovine antigen presenting cells. To analyse the importance of host species in mycobacterial infection, bovine antigen presenting cells were infected with bovine or human mycobacterial strains. Levels of nitric oxide and tumour necrosis factor production, markers of antimicrobial activity, were found to be associated with a specific mycobacterial strain, and varied between cell subsets.

Multilocus-sequence typing analysis reveals similar populations of Streptococcus uberis are responsible for bovine intramammary infections of short and long duration.

Multilocus-sequence typing (MLST) was used to analyse Streptococcus uberis isolates from a single herd associated with long duration (50-260 days) and rapidly cleared (less than 1 month) bovine intramammary infections to determine whether the bacterial type had any impact on the duration of infection. Most chronic infections (24 of 33) were due to continuous infection of the mammary quarter with the same sequence type, and infections were found to persist for many months. The remaining quarters were re-infected with a different sequence type within a single lactation. No particular sequence type or clonal complex (lineage) was associated with persisting infections, indicating that the outcome of intramammary infections with S. uberis is more likely to be dependent on host factors than on inter-strain differences. Analysis of these strains alongside others obtained from the same herd at a later date revealed the shift in the predominant genotypes with time.

Characterisation of bovine inducible nitric oxide synthase.

Inducible nitric oxide (iNOS) is an enzyme that catalyzes the production of the reactive nitrogen intermediate nitric oxide (NO). NO is an important signalling molecule, released by numerous cells, that acts in many tissues to regulate a diverse range of physiological and biological processes, including neurotransmission, immune defence and the regulation of cell death (apoptosis). NO plays a major role in the killing of intracellular pathogens as part of the innate immune response. iNOS is known to be induced by a number of stimuli including cytokines as well as pathogens and their components. As yet, a full-length bovine iNOS sequence has only been predicted from the genome, although partial sequences from cDNA are available. Here, we have identified a 3471bp transcript for bovine iNOS, isolated from RNA from bovine alveolar macrophages stimulated with the intracellular pathogen Mycobacterium bovis. When translated this gives a protein of 1156 amino acids. Bovine iNOS shows a high degree of similarity to iNOS from other species, and also shares a common protein domain structure.

Pattern recognition receptors in companion and farm animals - the key to unlocking the door to animal disease?

The innate immune system is essential for host defence and is responsible for early detection of potentially pathogenic microorganisms. Upon recognition of microbes by innate immune cells, such as macrophages and dendritic cells, diverse signalling pathways are activated that combine to define inflammatory responses that direct sterilisation of the threat and/or orchestrate development of the adaptive immune response. Innate immune signalling must be carefully controlled and regulation comes in part from interactions between activating and inhibiting signalling receptors. In recent years, an increasing number of pattern recognition receptors (PRRs), including C-type lectin receptors and Toll-like receptors (TLRs), has been described that participate in innate recognition of microbes, especially through the so called pathogen-associated molecular patterns (PAMPs). Recent studies demonstrate strong interactions between signalling through these receptors. Whereas useful models to study these receptors in great detail in the murine and human system are now emerging, relatively little is known regarding these receptors in companion and farm animals. In this review, current knowledge regarding these receptors in species of veterinary relevance is summarised.

A rapid IL-17 response to Cryptosporidium parvum in the bovine intestine.

Cryptosporidium parvum causes diarrhoea, due to villi damage, in livestock and humans globally. Immunity develops after repeated infections but initial infections can be severe, highlighting the importance of early infection dynamics. We have modelled early C. parvum infection in bovine jejunum biopsies. IL-17A accumulated over time peaking at 9h post-infection, with no effect of infection on IL-1ß; antibiotics positively influenced IL-17A as higher levels were found in cultures with antibiotics. Infection of primary fibroblasts resulted in lower plaque formation when fibroblasts were primed with IL-17A. Our results indicate a role for IL-17A in reducing C. parvum-dependent host cell damage.

Subset-Specific Expression of Toll-Like Receptors by Bovine Afferent Lymph Dendritic Cells.

Within the ruminant system, several possibilities exist to generate dendritic cells migrating out from the tissue into the regional draining lymph nodes as afferent lymph dendritic cells (ALDCs). Here, we analyzed toll-like receptor (TLR) 1-10 mRNA expression by using quantitative real-time PCR in highly purified subsets of bovine ALDC. As TLR expression may be influenced by pathogens or vaccines and their adjuvant, it is necessary to understand what TLRs are expressed in a steady-state system to elucidate specific differences and to potentially optimize targeted vaccines. In this study, we have assessed the TLR expression profiles of the four main bovine ALDC subsets [cDC1 and cDC2 (subsets 2-4)]. We demonstrate differences in TLR expression between the four subsets that may reflect the ability of these cells to respond to different pathogens or to respond to adjuvants.

Expression of TOLL-like receptors (TLR) by bovine antigen-presenting cells-potential role in pathogen discrimination?

Invading pathogens are controlled by the innate and adaptive arms of the immune system. Adaptive immunity, mediated by B and T lymphocytes, recognises pathogens via high affinity receptors. However, the establishment of a primary adaptive immune response is not rapid enough to eradicate invading microorganisms as it involves cell proliferation, gene activation and protein synthesis. More rapid defence mechanisms are provided by innate immunity, which recognises invading pathogens by germ-line-encoded pattern recognition receptors. Recent evidence shows that this recognition can mainly be attributed to the family of TOLL-like receptors (TLR). Binding of pathogen-associated molecular patterns to TLR induces the production of reactive oxygen and nitrogen intermediates, pro-inflammatory cytokines, and up-regulates expression of co-stimulatory molecules, subsequently initiating the adaptive immunity. In this paper, we will discuss the current knowledge with regards to the TLR, and in particular the bovine family of TLR. In addition, we will show the expression of TLR mRNA in bovine antigen-presenting cell subsets, summarise the discovery and the critical roles of TLR2 in host defence against Mycobacteria, and provide evidence for a mycobacteria species-specific response of bovine macrophages.