No vaccine interference between bovine coronavirus and bovine herpesvirus-1 in a randomized trial when coadministrating two intranasal modified-live viral vaccines to neonatal calves.

OBJECTIVE: Compare immune responses induced by 2 commercial intranasal (IN) modified-live viral (MLV) vaccines given individually or coadministered and evaluate prevention of infection and lung pathology following bovine herpesvirus-1 (BHV-1) challenge. ANIMALS: 36 male Holstein calves (ages, 5 to 12 days). METHODS: In a randomized complete block design, each calf received an IN injection of either vaccine diluent (Placebo), an MLV vaccine containing bovine herpesvirus-1 (BHV-1; N3), bovine coronavirus vaccine (BC), or both N3 and BC (BC + N3) with a booster 4 weeks later. Nasal secretions and blood were collected weekly. Three weeks after the booster, the calves were challenged with BHV-1, sampled for virus shedding, and euthanized 10 days later to quantify lung pathology. The study period was September 7, 2020, to April 6, 2021. RESULTS: Calves were seropositive for BHV-1 and BC before vaccination. No significant difference in BC-specific serum immunoglobin G and nasal immunoglobin A antibody responses in the BC versus BC + N3 group or BHV-1-specific serum immunoglobin G and nasal immunoglobin A antibody responses in the N3 versus BC + N3 group. Cytokine responses to BHV-1 and BC did not differ among groups. BHV-1 shedding after challenge was significantly reduced in N3 groups versus Placebo and BC. There was a significant reduction in lung pathology in the N3 + BC group versus Placebo. CLINICAL RELEVANCE: This study provides evidence an MLV vaccine containing BHV-1 and an MLV BC vaccine can be coadministered to neonatal calves without significantly altering immune responses to the 2 viruses or compromising the prevention of BHV-1 respiratory disease. Calves receiving the BC + N3 vaccine had a significant reduction in lung pathology after BHV-1 aerosol challenge.

Isolation and characterization of eosinophils in bovine blood and small intestine.

An effective method to isolate functional eosinophils from blood and tissues is required to analyze the multiple roles eosinophils play in innate immunity and tissue homeostasis. Highspeed cell sorting was used to isolate bovine eosinophils from blood polymorphonuclear (PMN) cells and from small intestine intraepithelial leukocytes. Eosinophils and neutrophils were purified from bovine blood with highspeed cell sorting after gating on autofluorescence (FL1) high and low PMN subpopulations. Highspeed sorting of intestinal eosinophils was accomplished by using a combination of positive (CD45+, CD11cLow, side scatterHigh) and negative (CD3-) selection parameters. Eosinophils sorted from blood PMNs were 88.6 ± 5.8 % (mean + 1 SD; n = 4) pure and yielded significantly (p < 0.05) more RNA than purified neutrophils. Analysis of Toll-like receptor (TLR) gene expression and TLR ligand-induced pro-inflammatory cytokine (IL-1, IL-6, IL-8, and TNFα) gene expression demonstrated significant (p < 0.01) functional differences between blood eosinophils and neutrophils. Eosinophils varied between 14.7 % to 29.3 % of CD45+ IELs and purity of sorted intestinal eosinophils was 95 + 3.5 % (mean + 1SD; n = 5). A comparison of mucosal and blood eosinophils revealed significant (p < 0.01) differences in TLR gene expression, supporting the hypothesis that functionally distinct eosinophil populations are present in blood and tissues. In conclusion, highspeed cell sorting provides an effective method to isolate viable eosinophils from blood and tissues that can then be used for transcriptome analyses and in vitro function assays.

Innate and acquired immune responses of colostrum-fed neonatal Holstein calves following intranasal vaccination with two commercially available modified-live virus vaccines.

OBJECTIVE: To compare immune responses induced by 2 commercially available vaccines with a bovine herpesvirus type 1 (BHV1) component following intranasal (IN) administration to colostrum-fed calves. ANIMALS: 90 male Holstein calves (ages, 5 to 14 days). PROCEDURES: In a randomized complete block design, each calf received 2 mL (1 mL/nostril) of vaccine A (n = 30), vaccine B (30), or saline (0.9% NaCl) solution (30) on day 0. Blood samples were collected for determination of serum anti-BHV1 IgG titer, and nasal fluid (NF) samples were collected for determination of interferon (IFN)-α and IFN-γ concentrations and for secretory IgA titers against BHV1, Mannheimia haemolytica, and Pasteurella multocida at predetermined times for 42 days after vaccination. RESULTS: All calves were seropositive for anti-BHV1 IgG, and the mean anti-BHV1 IgG titer did not differ significantly among the 3 groups at any time. Both vaccines induced significant transient increases in NF IFN-α and IFN-γ concentrations. On day 5, mean IFN-α concentration and the proportion of calves with detectable IFN-α concentrations for the vaccine A group were significantly greater than those for the vaccine B and control groups. On day 42, the mean NF anti-P multocida IgA titers for both vaccine groups were significantly greater than that of the control group. CONCLUSIONS AND CLINICAL RELEVANCE: Both vaccines induced innate and acquired immune responses in calves with colostral antibodies. The magnitude of the IFN-α response and proportion of calves with detectable IFN-α differed between the 2 vaccine groups. Both vaccines appeared to enhance the IgA response against P multocida.

EPIphany-A Platform for Analysis and Visualization of Peptide Immunoarray Data.

Antibodies are critical effector molecules of the humoral immune system. Upon infection or vaccination, populations of antibodies are generated which bind to various regions of the invading pathogen or exogenous agent. Defining the reactivity and breadth of this antibody response provides an understanding of the antigenic determinants and enables the rational development and assessment of vaccine candidates. High-resolution analysis of these populations typically requires advanced techniques such as B cell receptor repertoire sequencing, mass spectrometry of isolated immunoglobulins, or phage display libraries that are dependent upon equipment and expertise which are prohibitive for many labs. High-density peptide microarrays representing diverse populations of putative linear epitopes (immunoarrays) are an effective alternative for high-throughput examination of antibody reactivity and diversity. While a promising technology, widespread adoption of immunoarrays has been limited by the need for, and relative absence of, user-friendly tools for consideration and visualization of the emerging data. To address this limitation, we developed EPIphany, a software platform with a simple web-based user interface, aimed at biological users, that provides access to important analysis parameters, data normalization options, and a variety of unique data visualization options. This platform provides researchers the greatest opportunity to extract biologically meaningful information from the immunoarray data, thereby facilitating the discovery and development of novel immuno-therapeutics.

A Bovine Enteric Mycobacterium Infection Model to Analyze Parenteral Vaccine-Induced Mucosal Immunity and Accelerate Vaccine Discovery.

Mycobacterial diseases of cattle are responsible for considerable production losses worldwide. In addition to their importance in animals, these infections offer a nuanced approach to understanding persistent mycobacterial infection in native host species. Mycobacteriumavium ssp. paratuberculosis (MAP) is an enteric pathogen that establishes a persistent, asymptomatic infection in the small intestine. Difficulty in reproducing infection in surrogate animal models and limited understanding of mucosal immune responses that control enteric infection in the natural host have been major barriers to MAP vaccine development. We previously developed a reproducible challenge model to establish a consistent MAP infection using surgically isolated intestinal segments prepared in neonatal calves. In the current study, we evaluated whether intestinal segments could be used to screen parenteral vaccines that alter mucosal immune responses to MAP infection. Using Silirum® - a commercial MAP bacterin - we demonstrate that intestinal segments provide a platform for assessing vaccine efficacy within a relatively rapid period of 28 days post-infection. Significant differences between vaccinates and non-vaccinates could be detected using quantitative metrics including bacterial burden in intestinal tissue, MAP shedding into the intestinal lumen, and vaccine-induced mucosal immune responses. Comparing vaccine-induced responses in mucosal leukocytes isolated from the site of enteric infection versus blood leukocytes revealed substantial inconsistences between these immune compartments. Moreover, parenteral vaccination with Silirum did not induce equal levels of protection throughout the small intestine. Significant control of MAP infection was observed in the continuous but not the discrete Peyer's patches. Analysis of these regional mucosal immune responses revealed novel correlates of immune protection associated with reduced infection that included an increased frequency of CD335+ innate lymphoid cells, and increased expression of IL21 and IL27. Thus, intestinal segments provide a novel model to accelerate vaccine screening and discovery by testing vaccines directly in the natural host and provides a unique opportunity to interrogate mucosal immune responses to mycobacterial infections.

Kinome profiling of peripheral blood mononuclear cells collected prior to vaccination reveals biomarkers and potential mechanisms of vaccine unresponsiveness in pigs.

Inter-individual variance in host immune responses following vaccination can result in failure to develop protective immunity leaving individuals at risk for infection in addition to compromising herd immunity. While developing more efficacious vaccines is one strategy to mitigate this problem, predicting vaccine responsiveness prior to vaccination could inform which individuals require adjunct disease management strategies. To identify biomarkers of vaccine responsiveness, a cohort of pigs (n = 120) were vaccinated and pigs representing the high (n = 6; 90th percentile) and low (n = 6; 10th percentile) responders based on vaccine-specific antibody responses following vaccination were further analyzed. Kinase-mediated phosphorylation events within peripheral blood mononuclear cells collected prior to vaccination identified 53 differentially phosphorylated peptides when comparing low responders with high responders. Functional enrichment analysis revealed pro-inflammatory cytokine signaling pathways as dysregulated, and this was further substantiated by detection of higher (p < 0.01) concentrations of interferon-gamma in plasma of low responders compared to high responders prior to vaccination. In addition, low responder pigs with high plasma interferon-gamma showed lower (p < 0.01) birth weights than high responder pigs. These associations between vaccine responsiveness, cytokine signaling within peripheral immune cells, and body weight in pigs provide both evidence and insight into potential biomarkers for identifying low responders to vaccination.

Regional Dichotomy in Enteric Mucosal Immune Responses to a Persistent Mycobacterium avium ssp. paratuberculosis Infection.

Chronic enteric Mycobacterium avium ssp. paratuberculosis (MAP) infections are endemic in ruminants globally resulting in significant production losses. The mucosal immune responses occurring at the site of infection, specifically in Peyer's patches (PP), are not well-understood. The ruminant small intestine possesses two functionally distinct PPs. Discrete PPs function as mucosal immune induction sites and a single continuous PP, in the terminal small intestine, functions as a primary lymphoid tissue for B cell repertoire diversification. We investigated whether MAP infection of discrete vs. continuous PPs resulted in the induction of significantly different pathogen-specific immune responses and persistence of MAP infection. Surgically isolated intestinal segments in neonatal calves were used to target MAP infection to individual PPs. At 12 months post-infection, MAP persisted in continuous PP (n = 4), but was significantly reduced (p = 0.046) in discrete PP (n = 5). RNA-seq analysis revealed control of MAP infection in discrete PP was associated with extensive transcriptomic changes (1,707 differentially expressed genes) but MAP persistent in continuous PP elicited few host responses (4 differentially expressed genes). Cytokine gene expression in tissue and MAP-specific recall responses by mucosal immune cells isolated from PP, lamina propria and mesenteric lymph node revealed interleukin (IL)22 and IL27 as unique correlates of protection associated with decreased MAP infection in discrete PP. This study provides the first description of mucosal immune responses occurring in bovine discrete jejunal PPs and reveals that a significant reduction in MAP infection is associated with specific cytokine responses. Conversely, MAP infection persists in the continuous ileal PP with minimal perturbation of host immune responses. These data reveal a marked dichotomy in host-MAP interactions within the two functionally distinct PPs of the small intestine and identifies mucosal immune responses associated with the control of a mycobacterial infection in the natural host.

Immune memory induced by intranasal vaccination with a modified-live viral vaccine delivered to colostrum fed neonatal calves.

Bovine respiratory disease (BRD) remains a major health problem despite extensive use of vaccines during the post-weaning period. Apparent vaccine failure is attributed, in part, to primary vaccination during the period of greatest risk for BRD, providing inadequate time for onset of protective immunity. The current study investigated whether intranasal (IN) vaccination of 3-6 week old calves with a modified-live viral (MLV) vaccine induced sufficient immune memory to prevent respiratory disease and accelerate onset of protective immunity 5 months later. Vaccine groups included naïve controls, a single IN vaccination at 3-6 weeks of age, primary IN vaccination at 6 months, and either an IN or subcutaneous (SC) booster vaccination at 6 months (n = 10/group). All calves were challenged with BHV-1 four days after vaccination at 6 months of age. Primary IN vaccination at 6 months did not significantly reduce clinical disease but significantly (P < 0.01) reduced virus shedding. A single IN vaccination at 3-6 weeks of age significantly (P < 0.05) reduced weight loss but did not reduce fever or virus shedding. Both IN and SC booster vaccinations, significantly (P < 0.01) reduced clinical disease but virus shedding was significantly (P < 0.001) reduced only by IN booster vaccination. Reduction in virus shedding was significantly (P < 0.01) greater following booster versus primary IN vaccination at 6 months. All vaccination regimes significantly (P < 0.01) reduced secondary bacterial pneumonia and altered interferon responses relative to naïve controls. Only IN booster vaccination significantly (P < 0.05) increased BHV-1 specific IgA in nasal secretions. These results confirm primary MLV IN vaccination at 3 to 6 weeks of age, when virus neutralizing maternal antibody was present, induced immune memory with a 5 month duration. This immune memory supported rapid onset of protective immunity four days after an IN booster vaccination.

A Novel Animal Model for Regional Microbial Dysbiosis of the Pioneer Microbial Community.

Pioneer microbiota colonizing the newborn gastrointestinal tract has long-lasting effects on host health. Restoration of the gut microbial community, following dysbiosis during the neonatal period, may be one strategy to prevent undesirable health outcomes linked to an altered neonatal gut microbiome. Without appropriate animal models that recreate the prolonged human neonatal developmental period it is not possible to effectively analyze interventions designed to restore regional microbial populations. Our study used a lamb model in which intestinal segments were surgically isolated (blind-ended) in fetal lambs to create early microbial dysbiosis by delaying post-natal exposure to intestinal ingesta. Intestinal segments isolated in utero retained blood flow, innervation, and lymphatic drainage through the mesenteric attachment. Continuity of the fetal gastro-intestinal tract was re-established by side-to-side anastomosis of intestine proximal and distal to each isolated intestinal segment. Microbial restoration was then implemented in neonatal lambs by reconnecting a portion of the in utero isolated intestinal segments to adjacent intestinal tract 1 and 7 days after birth. Bacterial communities colonizing the adjacent intestine, in utero isolated intestinal segments, and reconnected intestinal segments were profiled using 16S amplicon sequencing on days 1, 7, and 56 of age. The in utero isolated intestinal segments were colonized 1 day after birth but the density of active bacteria was reduced and community composition altered when compared to adjacent intestine. Proteobacteria dominated the adjacent small intestine at early time points (day 1 and day 7) with a shift to primarily Firmicutes on day 56, consistent with establishment of an anaerobic bacterial community. In contrast, Proteobacteria persisted as the predominant community for 56 days in the in utero isolated intestinal segments. There was, however, almost full restoration of the microbial community composition in the in utero isolated intestinal segments following reconnection to the adjacent intestine. The density of beneficial bacteria, especially Bifidobacterium, remained significantly lower in the reconnected intestinal segments at 56 days when compared to adjacent intestine. Post-natal persistence of a stable pioneer community (Proteobacteria) in the in utero isolated intestinal segments provides a model system to study the temporal effects of regional microbial dysbiosis throughout a prolonged neonatal period.

Taxonomic and Functional Compositions of the Small Intestinal Microbiome in Neonatal Calves Provide a Framework for Understanding Early Life Gut Health.

A lack of information on the intestinal microbiome of neonatal calves prevents the use of microbial intervention strategies to improve calf gut health. This study profiled the taxonomic and functional composition of the small intestinal luminal microbiome of neonatal calves using whole-genome sequencing of the metagenome, aiming to understand the dynamics of microbial establishment during early life. Despite highly individualized microbial communities, we identified two distinct taxonomy-based clusters from the collective luminal microbiomes comprising a high level of either Lactobacillus or Bacteroides Among the clustered microbiomes, Lactobacillus-dominant ileal microbiomes had significantly lower abundances of Bacteroides, Prevotella, Roseburia, Ruminococcus, and Veillonella compared to the Bacteroides-dominated ileal microbiomes. In addition, the upregulated ileal genes of the Lactobacillus-dominant calves were related to leukocyte and lymphocyte chemotaxis, the cytokine/chemokine-mediated signaling pathway, and inflammatory responses, while the upregulated ileal genes of the Bacteroides-dominant calves were related to cell adhesion, response to stimulus, cell communication and regulation of mitogen-activated protein kinase cascades. The functional profiles of the luminal microbiomes also revealed two distinct clusters consisting of functions related to either high protein metabolism or sulfur metabolism. A lower abundance of Bifidobacterium and a higher abundance of sulfur-reducing bacteria (SRB) were observed in the sulfur metabolism-dominant cluster (0.2% ± 0.1%) compared to the protein metabolism-dominant cluster (12.6% ± 5.7%), suggesting an antagonistic relationship between SRB and Bifidobacterium, which both compete for cysteine. These distinct taxonomic and functional clusters may provide a framework to further analyze interactions between the intestinal microbiome and the immune function and health of neonatal calves.IMPORTANCE Dietary interventions to manipulate neonatal gut microbiota have been proposed to generate long-term impacts on hosts. Currently, our understanding of the early gut microbiome of neonatal calves is limited to 16S rRNA gene amplicon based microbial profiling, which is a barrier to developing dietary interventions to improve calf gut health. The use of a metagenome sequencing-based approach in the present study revealed high individual animal variation in taxonomic and functional abundance of intestinal microbiome and potential impacts of early microbiome on mucosal immune responses during the preweaning period. During this developmental period, age- and diet-related changes in microbial diversity, richness, density, and the abundance of taxa and functions were observed. A correlation-based approach to further explore the individual animal variation revealed potential enterotypes that can be linked to calf gut health, which may pave the way to developing strategies to manipulate the microbiome and improve calf health.

Fetal environment and fetal intestine are sterile during the third trimester of pregnancy.

Recent next generation sequencing studies on host-associated microbiomes generated debatable conclusions regarding the central dogma of fetal gut sterility. These observations challenge the concepts that microbial colonization of the gut begins during and after birth as well as the concept of antigen-independent prenatal maturation of mucosal-associated lymphoid tissue in ruminants and humans. The placental barrier varies markedly among mammalian species with mice and humans having haemochorial placentas (fetal tissue in direct contact with maternal blood) versus epitheliochorial placentation (maternal and fetal blood separated by six tissue layers) in ruminants. Therefore, this study re-examined the question of fetal gut sterility using the fetal lamb as a model ruminant species with the most complete placental barrier. Use of PCR and quantitative real-time PCR with three different pairs of universal bacterial primers (27 F and 1492R, HDA1 and HDA2, U2F and U2R) to amplify 16S rRNA gene did not generate detectable PCR products from samples collected from the fetal environment (placenta, amniotic fluid) and fetal intestine during the third trimester of pregnancy. Procedures to further enrich microbial DNA from total extracted DNA also resulted in no detectable genomic DNA. Moreover, use of 16S amplicon sequencing confirmed the absence of bacteria in the fetal environment during the third trimester of pregnancy. A 'No Template' control containing only PCR reagents generated sequences that could be clustered into OTUs at 97% similarity and assigned to bacterial genera, including Staphylococcus, Lactobacillus and Escherichia-Shigella. Use of multiple molecular-based approaches to profile fetal environment-associated microbiota supports the conclusion that the fetal environment and fetal intestine remain sterile during the third trimester of pregnancy. The use of appropriate controls, both positive and no template, revealed inherent contamination in reagents and that variations in the data analysis pipeline can produce artificial microbial profiles from host tissues containing low microbial biomass. Finally, these findings confirm that extensive development of gut-associated lymphoid tissue in the ruminant fetal intestine, characterized by active B cell proliferation and immunoglobulin V gene somatic mutation, is not associated with exposure to bacterial DNA and antigens.

Lambda display phage as a mucosal vaccine delivery vehicle for peptide antigens.

Bacteriophage are structurally stable in the gastro-intestinal tract and have favorable traits of safety, stability, ease of production, and immunogenicity. These attributes make them potential candidates as oral vaccine delivery vehicles but little is known about their capacity to induce mucosal immune responses in the small intestine. Whole body imaging of mice confirmed lambda bacteriophage (LP) were distributed throughout the gastro-intestinal tract 24 h after oral delivery. In newborn calves, targeted delivery of LP within the small intestine confirmed LP were immunogenic in a dose-dependent manner and were taken up by Peyer's patches. LP-specific IgA responses were induced within both Peyer's patches and draining mesenteric lymph nodes. A lambda display phage (LDP) was constructed to present three immunogenic disease specific epitopes (DSE) from cervid prion protein (amino acids 130-140 [YML]; 163-170 [YRR]; and 171-178[YRR]) fused to phage capsid head protein D (LDP-DSE). Targeted delivery of purified LDP-DSE to intestinal segments induced IgA responses to all three peptide epitopes. Further, delivery of bacteria expressing soluble D-DSE also induced epitope-specific IgA responses in the targeted Peyer's patches. These are the first studies to report use of LDP to induce epitope-specific IgA responses in the small intestine andconfirm Peyer's patchesfunction as a site for LP uptake. Furthermore, IgA responses to peptide epitopes on LDP were observed in the absence of a mucosal adjuvant. These observations confirm LDP have the capacity to function as a mucosal delivery vehicle with protein D as an effective carrier for peptide epitopes.

Induction of interferon and interferon-induced antiviral effector genes following a primary bovine herpesvirus-1 (BHV-1) respiratory infection.

Invitro investigations have identified a variety of mechanisms by which herpesviruses evade interferon-stimulated antiviral effector mechanisms. However, these immune evasion mechanisms have not been evaluated during a bovine herpesvirus-1 (BHV-1) infection. This study investigated the transcription and secretion of type I and II interferons (IFNs) and the transcription of IFN-stimulated genes (ISGs) during a primary BHV-1 infection of the upper respiratory tract (URT) in naïve calves. IFN-α, -ß and -γ transcription in nasal turbinates and protein levels in nasal secretions increased following infection. Increased IFN type I and II secretion was detected 3 days post-infection (p.i.) and IFN production increased in parallel with virus shedding. Expression of ISGs, including Mx1, OAS and BST-2, also increased significantly (P<0.05) in nasal turbinates on day 3 p.i. and elevated ISG expression persisted throughout the period of viral shedding. In contrast, RNAase L gene expression was not induced during the BHV-1 infection in the nasal turbinates, but was induced on day 10 p.i. in the trachea. In vitro studies confirmed that recombinant bovine (rBo)IFN-α, -ß and -γ induced expression of Mx1, OAS and BST-2, but decreased RNAse L transcript in bovine epithelial cells. Relative to vesicular stomatitisvirus (VSV), BHV-1 was resistant to the antiviral activity of rBoIFN-α and -γ, but treatment of epithelial cells with 10 ng rBoIFN-ß ml-1 effected an 80 % inhibition of BHV-1 replication and complete inhibition of VSV replication. These observations confirm that the transcription and translation of type I and II IFNs increase during BHV-1 infection, while the transcription of some ISGs is not inhibited.

Transcriptome analysis reveals regional and temporal differences in mucosal immune system development in the small intestine of neonatal calves.

BACKGROUND: Postnatal development of the mammalian mucosal immune system is crucial for responding to the rapid colonization by commensal bacteria and possible exposure to pathogens. This study analyzed expression patterns for mRNAs and their relationship with microRNAs (miRNAs) in the bovine small intestine during the critical neonatal period (0 to 42 days). This analysis revealed molecular mechanisms regulating the postnatal development of the intestinal mucosal immune system. RESULTS: Small intestine samples (jejunum and ileum) were collected from newborn male, Holstein calves immediately post-partum (n = 3) and at 7 (n = 5), 21 (n = 5), and 42 (n = 5) days of age and the transcriptomes were profiled using RNA-Seq. When analyzing all time points collectively, greater expression of genes encoding the complement functional pathway, as well as lower expression of genes encoding Toll-like receptors and NOD-like receptors were observed in the jejunum when compared to the ileum. In addition, significant changes in the expression of immune-related genes were detected within the first week post-partum in both jejunum and ileum. For example, increased expression of genes encoding tight junction proteins (claudin 1, claudin 4 and occludin), an antimicrobial peptide (Regenerating Islet-Derived 3-γ), NOD-like receptors (NACHT, LRR and PYD domain-containing protein 3), regulatory T cell marker (forkhead box P3), and both anti-inflammatory (interleukin 10) and pro-inflammatory (interleukin 8) cytokines was observed throughout the small intestine of 7-day-old calves when compared to newborn calves. Moreover, the expression of mucosal immune-related genes were either positively or negatively correlated with total bacterial population depending on both intestinal region and age. The integrated analysis of miRNAs and mRNAs supported the conclusion that miRNAs may regulate temporal changes in the expression of genes encoding tight junction proteins (miR-335), cytokines (miR-335) and bacterial recognition (miR-100) during the first week of small intestine development. CONCLUSION: The rapid development of transcriptional differences between jejunum and ileum reveal that these two intestinal regions make distinct contributions to the intestinal mucosal immune system during the early neonatal period. In addition, transcriptome analysis indicates that the first week after birth is a very dynamic developmental period for the intestinal mucosal immune system and these changes may be regulated by both miRNAs and microbial colonization. Findings from this study indicate that a detailed analysis of both the abundance and diversity of the colonizing microbiome may be necessary to understand factors regulating the rapid development of the mucosal immune system during the first week of life.

Marked Differences in Mucosal Immune Responses Induced in Ileal versus Jejunal Peyer's Patches to Mycobacterium avium subsp. paratuberculosis Secreted Proteins following Targeted Enteric Infection in Young Calves.

In cattle, Mycobacterium avium subsp. paratuberculosis infection is primarily mediated through M cells overlying Peyer's patches (PP) in the ileum. The capacity of M. avium subsp. paratuberculosis to invade ileal PP (IPP) versus discrete PP in the jejunum (JPP) and subsequent differences in mucosal immune responses were investigated. Intestinal segments were surgically prepared in both mid-jejunum, containing two JPPs, and in terminal small intestine containing continuous IPP. M. avium subsp. paratuberculosis (109 CFU) was injected into the lumen of half of each intestinal segment when calves were 10-14 days-old and infection confirmed 1-2 months later by PCR and immunohistochemistry. Thirteen recombinant M. avium subsp. paratuberculosis proteins, previously identified as immunogenic, were used to analyze pathogen-specific B- and T-cell responses in PP and mesenteric lymph nodes. IgA plasma cell responses to 9 of 13 recombinant proteins were detected in JPP but not in IPP. Secretory IgA reacting in ELISA with 9 of the 13 recombinant proteins was detected in luminal contents from both jejunal and ileal segments. These observations support the conclusion that pathogen-specific IgA B cells were induced in JPP but not IPP early after a primary infection. The presence of secretory IgA in intestinal contents is consistent with dissemination of IgA plasma cells from the identified mucosa-associated immune induction sites. This is the first direct evidence for M. avium subsp. paratuberculosis uptake by bovine JPP and for local induction of pathogen-specific IgA plasma cell responses after enteric infection. We also provide evidence that bacterial invasion of IPP, a primary B lymphoid tissue, provides a novel strategy to evade induction of mucosal immune responses. Over 60% of PPs in the newborn calf small intestine is primary lymphoid tissue, which has significant implications when designing oral vaccines or diagnostic tests to detect early M. avium subsp. paratuberculosis infections.

Altered microRNA expression and pre-mRNA splicing events reveal new mechanisms associated with early stage Mycobacterium avium subspecies paratuberculosis infection.

The molecular regulatory mechanisms of host responses to Mycobacterium avium subsp. paratuberculosis (MAP) infection during the early subclinical stage are still not clear. In this study, surgically isolated ileal segments in newborn calves (n = 5) were used to establish in vivo MAP infection adjacent to an uninfected control intestinal compartment. RNA-Seq was used to profile the whole transcriptome (mRNAs) and the microRNAome (miRNAs) of ileal tissues collected at one-month post-infection. The most related function of the differentially expressed mRNAs between infected and uninfected tissues was "proliferation of endothelial cells", indicating that MAP infection may lead to the over-proliferation of endothelial cells. In addition, 46.2% of detected mRNAs displayed alternative splicing events. The pre-mRNA of two genes related to macrophage maturation (monocyte to macrophage differentiation-associated) and lysosome function (adenosine deaminase) showed differential alternative splicing events, suggesting that specific changes in the pre-mRNA splicing sites may be a mechanism by which MAP escapes host immune responses. Moreover, 9 miRNAs were differentially expressed after MAP infection. The integrated analysis of microRNAome and transcriptome revealed that these miRNAs might regulate host responses to MAP infection, such as "proliferation of endothelial cells" (bta-miR-196 b), "bacteria recognition" (bta-miR-146 b), and "regulation of the inflammatory response" (bta-miR-146 b).

Tissue- and age-dependent expression of the bovine DEFB103 gene and protein.

Beta-defensin 103 (DEFB103) shares little homology with 8 other members of the bovine beta-defensin family and in other species DEFB103 protein has diverse functions, including antimicrobial activity, a chemoattractant for dendritic cells, enhancing epithelial wound repair and regulating hair colour. Expression of the bovine DEFB103 gene was surveyed in 27 tissues and transcript was most abundant in tissues with stratified squamous epithelium. Oral cavity epithelial tissues and nictitating membrane consistently expressed high levels of DEFB103 gene transcript. An age-dependent decrease (P < 0.05) in DEFB103 gene expression was only observed for buccal epithelium when comparing healthy 10- to 14-day-old and 10- to 12-month-old calves. A bovine herpesvirus-1 respiratory infection did, however, significantly (P < 0.05) up-regulate DEFB103 gene expression in the buccal epithelium of 6- to 8-month-old calves. Finally, DEFB103 transcript was low in lymph nodes draining the skin and at the limit of detection in other internal organs such as lung, intestine and kidney. Affinity-purified rabbit antisera to bovine DEFB103 was used to identify cells expressing DEFB103 protein within tissues with stratified squamous epitheliums. DEFB103 protein was most abundant in basal epithelial cells and was present in these cells prior to birth. Beta-defensins have been identified as regulators of dendritic cell (DC) chemokine responses and we observed a close association between DCs and epithelial cells expressing DEFB103 in both the fetus and newborn calf. In conclusion, bovine DEFB103 gene expression is most abundant in stratified squamous epithelium with DEFB103 protein localised to basal epithelial cells. These observations are consistent with proposed roles for DEFB103 in DC recruitment and repair of stratified squamous epithelium.

The Gut Microbiome and Its Potential Role in the Development and Function of Newborn Calf Gastrointestinal Tract.

A diverse microbial population colonizes the sterile mammalian gastrointestinal tract during and after the birth. There is increasing evidence that this complex microbiome plays a crucial role in the development of the mucosal immune system and influences newborn health. Microbial colonization is a complex process influenced by a two-way interaction between host and microbes and a variety of external factors, including maternal microbiota, birth process, diet, and antibiotics. Following this initial colonization, continuous exposure to host-specific microbes is not only essential for development and maturation of the mucosal immune system but also the nutrition and health of the animal. Thus, it is important to understand host-microbiome interactions within the context of individual animal species and specific management practices. Data is now being generated revealing significant associations between the early microbiome, development of the mucosal immune system, and the growth and health of newborn calves. The current review focuses on recent information and discusses the limitation of current data and the potential challenges to better characterizing key host-specific microbial interactions. We also discuss potential strategies that may be used to manipulate the early microbiome to improve production and health during the time when newborn calves are most susceptible to enteric disease.

BVDV vaccination in North America: risks versus benefits.

The control and prevention of bovine viral diarrhea virus (BVDV) infections has provided substantial challenges. Viral genetic variation, persistent infections, and viral tropism for immune cells have complicated disease control strategies. Vaccination has, however, provided an effective tool to prevent acute systemic infections and increase reproductive efficiency through fetal protection. There has been substantial controversy about the safety and efficacy of BVDV vaccines, especially when comparing killed versus modified-live viral (MLV) vaccines. Furthermore, numerous vaccination protocols have been proposed to protect the fetus and ensure maternal antibody transfer to the calf. These issues have been further complicated by reports of immune suppression during natural infections and following vaccination. While killed BVDV vaccines provide the greatest safety, their limited immunogenicity makes multiple vaccinations necessary. In contrast, MLV BVDV vaccines induce a broader range of immune responses with a longer duration of immunity, but require strategic vaccination to minimize potential risks. Vaccination strategies for breeding females and young calves, in the face of maternal antibody, are discussed. With intranasal vaccination of young calves it is possible to avoid maternal antibody interference and induce immune memory that persists for 6-8 months. Thus, with an integrated vaccination protocol for both breeding cows and calves it is possible to maximize disease protection while minimizing vaccine risks.

Methanogen prevalence throughout the gastrointestinal tract of pre-weaned dairy calves.

The methanogenic community throughout the gastrointestinal tract (GIT) of pre-weaned calves has not been well studied. The current study firstly investigated the distribution and composition of the methanogenic community in the rumen, ileum, and colon of 3-4 week-old milk-fed dairy calves (n = 4) using 16S rRNA gene clone library analysis. The occurrence of methanogens in the GIT of pre-weaned calves was further validated by using PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and quantitative real-time PCR (qPCR) was applied to quantify the methanogenic community in the rumen, jejunum, ileum, cecum, colon and rectum of 8 3-4 week old animals. Both cloning libraries and PCR-DGGE revealed that phylotypes close to Methanobrevibacter were the main taxon along the GIT in pre-weaned sucking calves. The composition and abundance of methanogens varied significantly among individual animals, suggesting that host conditions may influence the composition of the symbiotic microbiota. Segregation of methanogenic communities throughout the GIT was also observed within individual animals, suggesting possible functional differences among methanogens residing in different GIT regions. This is the first study to analyze methanogenic communities throughout the GIT of milk-fed newborn dairy calves and reveal both their diversity and abundance. The identification of methanogens in the lower GIT of pre-weaned dairy calves warrants further investigation to better define methanogen roles in GIT function and their impact on host metabolism and health.