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.

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.

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 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/.

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.

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.

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.

Two TIR-like domain containing proteins in a newly emerging zoonotic Staphylococcus aureus strain sequence type 398 are potential virulence factors by impacting on the host innate immune response.

Staphylococcus aureus, sequence type (ST) 398, is an emerging pathogen and the leading cause of livestock-associated methicillin-resistant S. aureus infections in Europe and North America. This strain is characterized by high promiscuity in terms of host-species and also lacks several traditional S. aureus virulence factors. This does not, however, explain the apparent ease with which it crosses species-barriers. Recently, TIR-domain containing proteins (Tcps) which inhibit the innate immune response were identified in some Gram-negative bacteria. Here we report the presence of two proteins, S. aureus TIR-like Protein 1 (SaTlp1) and S. aureus TIR-like Protein 2 (SaTlp2), expressed by ST398 which contain domain of unknown function 1863 (DUF1863), similar to the Toll/IL-1 receptor (TIR) domain. In contrast to the Tcps in Gram-negative bacteria, our data suggest that SaTlp1 and SaTlp2 increase activation of the transcription factor NF-κB as well as downstream pro-inflammatory cytokines and immune effectors. To assess the role of both proteins as potential virulence factors knock-out mutants were created. These showed a slightly enhanced survival rate in a murine infectious model compared to the wild-type strain at one dose. Our data suggest that both proteins may act as factors contributing to the enhanced ability of ST398 to cross species-barriers.

Species-specific PAMP recognition by TLR2 and evidence for species-restricted interaction with Dectin-1.

TLRs mediate recognition of a wide range of microbial products, including LPS, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators. In addition to TLRs, many other surface receptors have been proposed to participate in innate immunity and microbial recognition, and signaling through some of these, for example, C-type lectins, is likely to cooperate with TLR signaling in defining inflammatory responses. In the present study, we examined the importance of the ECD and intracellular TIR domain of boTLR2 and huTLR2 to induce a species-specific response by creating a chimeric TLR2 protein. Our results indicate that the strength of the response to any TLR2 ligand tested was dependent on the extracellular, solenoid structure, but not the intracellular TIR domain. Furthermore, we examined whether the recognition of two PAMPs by Dectin-1, a CLR, depends on the interaction with TLR2 from the same species. TLR2 expression seemed to affect the Dectin-1-dependent production of CXCL8 to ß-glucan containing zymosan as well as Listeria monocytogenes. Furthermore, the interaction of Dectin-1 with TLR2 seemed to require that both receptors are from the same species. Our data demonstrate that the differences in the TLR2 response seen between the bovine and human system depend on the ECD of TLR2 and that collaborative recognition of distinct microbial components by different classes of innate-immune receptors is crucial in orchestrating inflammatory responses.

Potential evidence for biotype-specific chemokine profile following BVDV infection of bovine macrophages.

Chemokines play a key role in initiating the innate and subsequently adaptive immune response by recruiting immune cells to the site of an infection. Monocytes/macrophages (MØ) are part of the first line of defence against invading pathogens, and have been shown to release a variety of chemokines in response to infection. Here, we reveal the early transcriptional response of MØ to infection with cytopathogenic (cp) and non-cytopathogenic (ncp) bovine viral diarrhoea strains (BVDV). We demonstrate up-regulation of several key chemokines of the CCL and CXCL families in MØ exposed to cpBVDV, but not ncpBVDV. In contrast, infection of MØ with ncpBVDV led to down-regulation of chemokine mRNA expression compared to uninfected cells. Data suggest that ncpBVDV can shut down production of several key chemokines that play crucial roles in the immune response to infection. This study helps to further our understanding of the pathogenesis of BVDV infection, highlighting biotype-specific cellular responses.

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.

Early response of bovine alveolar macrophages to infection with live and heat-killed Mycobacterium bovis.

Bovine tuberculosis (TB) is a disease of economic importance and a significant animal health and welfare issue. The alveolar macrophage (AlvMϕ) plays a vital role in the immune response to TB and recent studies provide insights into the interactions between Mϕ and Mycobacterium bovis. Here we reveal the early transcriptional response of bovine AlvMϕ to M. bovis infection. We demonstrate up-regulation of immune response genes, including chemokines, members of the NF-κB pathway which may be involved in their transcription and also pro- and anti-apoptotic genes. M. bovis may therefore induce multiple mechanisms to manipulate the host immune response. We compared the response of AlvMϕ to infection with live and heat-killed M. bovis to determine transcriptional differences dependent on the viable pathogen. Several chemokines up-regulated following live M. bovis infection were not up-regulated after heat-killed M. bovis stimulation; hence the Mϕ seems to differentiate between the two stimuli.

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.

Characterisation of antibodies to bovine Toll-like receptor (TLR)-2 and cross-reactivity with ovine TLR2.

Host recognition of conserved pathogen-associated molecular patterns (PAMPs) and their interactions with pattern-recognition receptors, including the Toll-like receptors (TLR) is essential for innate immune response induction. The TLR1 family (TLR1, 2, 6 and 10) is involved in the recognition of gram-positive and gram-negative bacteria and heterodimers of TLR1 or TLR6 with TLR2 are crucial for the identification of several PAMPs. Studies on cell surface expression of TLR in ruminants are hampered by the lack of specific antibodies and no convincingly cross-reactive anti-human antibodies have been described so far. We describe herein four antibodies which recognise bovine TLR2. Differences in TLR2 expression were evident on bovine antigen presenting cells with high level expression on peripheral blood monocytes and monocyte-derived macrophages. Lower levels of expression were evident on dendritic cell populations derived in vitro and ex vivo, and on alveolar macrophages. One of the antibodies recognised TLR2 expression on ovine peripheral blood monocytes. The identification of antibodies specific for bovine and ovine TLR2 will facilitate studies of the role of this important PRR in the initiation of immune responses to important pathogens.

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.

Sortase anchored proteins of Streptococcus uberis play major roles in the pathogenesis of bovine mastitis in dairy cattle.

Streptococcus uberis, strain 0140J, contains a single copy sortase A (srtA), encoding a transamidase capable of covalently anchoring specific proteins to peptidoglycan. Unlike the wild-type, an isogenic mutant carrying an inactivating ISS1 insertion within srtA was only able to infect the bovine mammary gland in a transient fashion. For the first 24 h post challenge, the srtA mutant colonised at a similar rate and number to the wild type strain, but unlike the wild type did not subsequently colonise in higher numbers. Similar levels of host cell infiltration were detected in response to infection with both strains, but only in those mammary quarters infected with the wild type strain were clinical signs of disease evident. Mutants that failed to express individual sortase substrate proteins (sub0135, sub0145, sub0207, sub0241, sub0826, sub0888, sub1095, sub1154, sub1370, and sub1730) were isolated and their virulence determined in the same challenge model. This revealed that mutants lacking sub0145, sub1095 and sub1154 were attenuated in cattle. These data demonstrate that a number of sortase anchored proteins each play a distinct, non-redundant and important role in pathogenesis of S. uberis infection within the lactating bovine mammary gland.

Correlation between lymph node pathology and chemokine expression during bovine tuberculosis.

Bovine tuberculosis is a disease of worldwide importance yet comparatively little is known about chemokine responses to infection. We report on the levels of chemokine expression within lymph nodes of cattle infected with Mycobacterium bovis when infection would be well established. Expression levels of a number of chemokines were increased in infected cattle and could be correlated to levels of respective chemokine receptors. Several chemokines were significantly correlated to pathology within the lymph node, indicating a direct relationship between chemokine expression and disease. Vaccinated animals challenged with M. bovis had lower levels of chemokine expression than unvaccinated, challenged animals, correlating with lower levels of disease in vaccinated animals. The chemokine expression profile correlated with previous evidence for a pro-inflammatory bias within the lymph node. At this stage of infection we suggest there is on-going chemokine expression by cells associated with the granuloma and continual recruitment of cells to control infection.