Evaluation of immune responses of cattle as a means to identify high or low responders and use of a human microarray to differentiate gene expression

An immune response (IR) index to identify cows with high (H) and low (L) antibody-mediated immune responses (AMIR) had been previously devised. High AMIR associated with decreased mastitis and improved response to vaccination. Measurement of cell-mediated immune response (CMIR) was not included in the index; therefore various antigen/adjuvant combinations were evaluated as inducers of DTH to be added to the IR-index. The Bacillus Calmette Guérin (BCG)-induced/purified protein derivative (PPD)-elicited tuberculin skin test is a reliable measure of DTH; however, its use to identify livestock with high CMIR may be confounded due to previous exposure to Mycobacteria tuberculosis. DTH to BCG/PPD was therefore compared with that induced by Mycobacteria phlei (saprophyte) and its derivative phlein as the test antigen. Antibody to OVA was also evaluated. The results indicated that BCG/PPD and M. phlei/phlein induced similar DTH, but cross reaction to PPD was evident following induction of DTH using M. phlei making it a less than ideal alternative for testing livestock. Nonetheless, cows could be ranked for both AMIR and CMIR. RNA from two cows with the highest and lowest IR ranks was then used to probe a human 1.7 kD microarray to determine the ability of a human array to provide information on bovine genes associated with H and L

Changes in Holstein cow milk and serum proteins during intramammary infection with three different strains of Staphylococcus aureus

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>is one of the most prevalent pathogens to cause mastitis in dairy cattle. Intramammary infection of dairy cows with <it>S. aureus </it>is often subclinical, due to the pathogen's ability to evade the innate defense mechanisms, but this can lead to chronic infection. A sub-population of <it>S. aureus</it>, known as small colony variant (SCV), displays atypical phenotypic characteristics, causes persistent infections, and is more resistant to antibiotics than parent strains. Therefore, it was hypothesized that the host immune response will be different for SCV than its parental or typical strains of <it>S. aureus</it>. In this study, the local and systemic immune protein responses to intramammary infection with three strains of <it>S. aureus</it>, including a naturally occurring bovine SCV strain (SCV Heba3231), were characterized. Serum and casein-depleted milk cytokine levels (interleukin-8, interferon-γ, and transforming growth factor-β1), as well as serum haptoglobin concentrations were monitored over time after intramammary infection with each of the three <it>S. aureus </it>strains. Furthermore, comparative proteomics was used to evaluate milk proteome profiles during acute and chronic phases of <it>S. aureus </it>intramammary infection.</p> <p>Results</p> <p>Serum IL-8, IFN-γ, and TGF-β1 responses differed in dairy cows challenged with different strains of <it>S. aureus</it>. Changes in overall serum haptoglobin concentrations were observed for each <it>S. aureus </it>challenge group, but there were no significant differences observed between groups. In casein-depleted milk, strain-specific differences in the host IFN-γ response were observed, but inducible IL-8 and TGF-β1 concentrations were not different between groups. Proteomic analysis of the milk following intramammary infection revealed unique host protein expression profiles that were dependent on the infecting strain as well as phase of infection. Notably, the protein, component-3 of the proteose peptone (CPP3), was differentially expressed between the <it>S. aureus </it>treatment groups, implicating it as a potential antimicrobial peptide involved in host defense against <it>S. aureus </it>intramammary infection.</p> <p>Conclusions</p> <p>Intramammary infection of dairy cattle with <it>S. aureus </it>causes an up-regulation of serum and milk immune-related proteins, and these responses vary depending on the infecting strain.</p

Characterization of the bovine salivary gland transcriptome associated with Mycobacterium avium subsp. paratuberculosis experimental challenge

peer-reviewedBackground Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of Johne’s disease is spread between cattle via the fecal-oral route, yet the functional changes in the salivary gland associated with infection remain uncharacterized. In this study, we hypothesized that experimental challenge with MAP would induce stable changes in gene expression patterns in the salivary gland that may shed light on the mucosal immune response as well as the regional variation in immune capacity of this extensive gland. Holstein-Friesian cattle were euthanized 33 months’ post oral challenge with MAP strain CIT003 and both the parotid and mandibular salivary glands were collected from healthy control (n = 5) and MAP exposed cattle (n = 5) for histopathological and transcriptomic analysis. Results A total of 205, 21, 61, and 135 genes were significantly differentially expressed between control and MAP exposed cattle in dorsal mandibular (M1), ventral mandibular (M2), dorsal parotid (P1) and ventral parotid salivary glands (P2), respectively. Expression profiles varied between the structurally divergent parotid and mandibular gland sections which was also reflected in the enriched biological pathways identified. Changes in gene expression associated with MAP exposure were detected with significantly elevated expression of BoLA DR-ALPHA, BOLA-DRB3 and complement factors in MAP exposed cattle. In contrast, reduced expression of genes such as polymeric immunoglobin receptor (PIGR), TNFSF13, and the antimicrobial genes lactoferrin (LF) and lactoperoxidase (LPO) was detected in MAP exposed animals. Conclusions This first analysis of the transcriptomic profile of salivary glands in cattle adds an important layer to our understanding of salivary gland immune function. Transcriptomic changes associated with MAP exposure have been identified including reduced LF and LPO. These critical antimicrobial and immunoregulatory proteins are known to be secreted into saliva and their downregulation may contribute to disease susceptibility. Future work will focus on the validation of their expression levels in saliva from additional cattle of known infection status as a potential strategy to augment disease diagnosis

Técnicas de Proteção e Reparação de Estruturas de Betão Armado contra a Oxidação Causadas pela Água do Mar Estudo de Caso – Residencial Áustria – Calheta São Miguel

A degradação de estruturas de betão armado em larga escala, seja nos grandes centros urbanos, no meio rural, industrial, nas proximidades ou não dos ambientes marítimos, atualmente, deixa observar problemas patológicos relacionados com a oxidação e a degradação do betão. Muitas vezes esses problemas aparecem de uma forma precoce, cuja recuperação envolve custos elevados, outras vezes é de tal forma que se torna difícil a sua recuperação. Nesta óptica, a aplicação de técnicas de intervenção inovadoras e destinadas a solucionar estes problemas tem cada vez mais uma importância primordial. Baseando na Norma Europeia EN 1504 (2004), por ainda não existir uma Norma Caboverdiana, pretende-se fazer um estudo da aplicação das técnicas, bem como dos métodos de intervenção a elas associadas. A alternativa de intervenção para o edifício em estudo é apresentada como proposta, por forma a melhorar o entendimento sobre a aplicabilidade das técnicas de proteção e de reparação. As escolhas dessas técnicas baseiam-se na visita de inspeção visual, levantamento fotográfico e realização de ensaios para o diagnóstico. O LEC (Laboratório de Engenharia Civil), foi o colaborador para a realização dos ensaios

Osteopontin: an early innate immune marker of Escherichia coli mastitis harbors genetic polymorphisms with possible links with resistance to mastitis

<p>Abstract</p> <p>Background</p> <p>Mastitis is the most important disease in dairy cows and it causes significant lost of profit to producers. Identification of the genes, and their variants, involved in innate immune responses is essential for the understanding of this inflammatory disease and to identify potential genetic markers for resistance to mastitis. The progeny of dairy cows would benefit from receiving favourable alleles that support greater resistance to infection, thus reducing antibiotic use. This study aims to identify a key gene in the innate immune response to mastitis, led us to evaluate its genetic association with somatic cell score (SCS), which is an indicator of clinical mastitis, and to evaluate its impact on other traits related to milk production.</p> <p>Results</p> <p>The osteopontin transcript (<it>SPP1</it>) was identified in the somatic cells from cows experimentally infected with <it>Escherichia coli</it>. By selecting bulls with extreme estimated breeding values (EBVs) for SCS, which is an indicator of mammary gland health, four DNA polymorphisms in the <it>SPP1 </it>genomic sequence were found. Statistical analysis revealed that the SNP <it>SPP1c.-1301G>A </it>has an impact on EBV for SCS (<it>P </it>< 0.001) Using an allele substitution model, <it>SPP1c.-1251C>T</it>, <it>SPP1c.-430G>A</it>, and <it>SPP1c.*40A>C </it>have an impact on SCS whereas <it>SPP1c.-1301G>A </it>has an effect on the EBVs for milk yield (second and third lactations), fat and protein percentages (all three lactations). Analysis revealed statistically significant differences between haplotype groups at a comparison-wise level with sire EBVS for SCS for the first (<it>P </it>= 0.012), second (<it>P </it>< 0.001), and third (<it>P </it>< 0.001) lactations.</p> <p>Conclusion</p> <p>This study reports the link between DNA polymorphisms of <it>SPP1</it>, the number of milk immune cells and, potentially, the susceptibility to mastitis. These SNPs were identified by <it>in silico </it>search to be located in transcription factor recognition sites which factors are presumably involved in the Th1 immune response and in the Th2 regulation pathway. Indeed, one SNP abolished the SP1 recognition site, whereas another SNP affected the transcription binding factor IKAROS. All together, these findings support the genetic potential of these variants in terms of selection for the improvement of mastitis resistance in dairy cows.</p

Characterization of ovine hepatic gene expression profiles in response to Escherichia coli lipopolysaccharide using a bovine cDNA microarray

BACKGROUND: During systemic gram-negative bacterial infections, lipopolysaccharide (LPS) ligation to the hepatic Toll-like receptor-4 complex induces the production of hepatic acute phase proteins that are involved in the host response to infection and limit the associated inflammatory process. Identifying the genes that regulate this hepatic response to LPS in ruminants may provide insight into the pathogenesis of bacterial diseases and eventually facilitate breeding of more disease resistant animals. The objective of this research was to profile the expression of ovine hepatic genes in response to Escherichia coli LPS challenge (0, 200, 400 ng/kg) using a bovine cDNA microarray and quantitative real-time PCR (qRT-PCR). RESULTS: Twelve yearling ewes were challenged iv with E. coli LPS (0, 200, 400 ng/kg) and liver biopsies were collected 4–5 hours post-challenge to assess hepatic gene expression profiles by bovine cDNA microarray and qRT-PCR analyses. The expression of CD14, C3, IL12R, NRAMP1, SOD and IGFBP3 genes was down regulated, whereas the expression of ACTHR, IFNαR, CD1, MCP-1 and GH was increased during LPS challenge. With the exception of C3, qRT-PCR analysis of 7 of these genes confirmed the microarray results and demonstrated that GAPDH is not a suitable housekeeping gene in LPS challenged sheep. CONCLUSION: We have identified several potentially important genes by bovine cDNA microarray and qRT-PCR analyses that are differentially expressed during the ovine hepatic response to systemic LPS challenge. Their potential role in regulating the inflammatory response to LPS warrants further investigation

Identification of single nucleotide polymorphisms in bovine CARD15 and their associations with health and production traits in Canadian Holsteins

<p>Abstract</p> <p>Background</p> <p>Toll-like receptor-2 (TLR2) and Caspase Recruitment Domain 15 (CARD15) are important pattern recognition receptors that play a role in the initiation of the inflammatory and subsequent immune response. They have been previously identified as susceptibility loci for inflammatory bowel diseases in humans and are, therefore, suitable candidate genes for inflammatory disease resistance in cattle. The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the bovine <it>TLR2 </it>and <it>CARD15 </it>and evaluate the association of these SNPs with health and production traits in a population of Canadian Holstein bulls.</p> <p>Results</p> <p>A selective DNA pool was constructed based on the estimated breeding values (EBVs) for SCS. Gene segments were amplified from this pool in PCR reactions and the amplicons sequenced to reveal polymorphisms. A total of four SNPs, including one in intron 10 (c.2886-14A>G) and three in the exon 12 (c.3020A>T, c.4500A>C and c.4950C>T) were identified in <it>CARD15</it>; none were identified in <it>TLR2</it>. Canadian Holstein bulls (n = 338) were genotyped and haplotypes were reconstructed. Two SNPs, c.3020A>T and c.4500A>C, were associated with EBVs for health and production traits. The SNP, c.3020A>T, for example, was associated with SCS EBVs (p = 0.0097) with an allele substitution effect of 0.07 score. When compared to the most frequent haplotype Hap12(AC), Hap22(TC) was associated with increased milk (p < 0.0001) and protein (p = 0.0007) yield EBVs, and hap21(TA) was significantly associated with increased SCS EBV(p = 0.0120). All significant comparison-wise associations retained significance at 8% experimental-wise level by permutation test.</p> <p>Conclusion</p> <p>This study indicates that SNP c.3020A>T might play a role in the host response against mastitis and further detailed studies are needed to understand its functional mechanisms.</p

The role of interleukin-10 receptor alpha (IL10Rα) in Mycobacterium avium subsp. paratuberculosis infection of a mammary epithelial cell line

BackgroundJohne’s disease is a chronic wasting disease caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). Johne’s disease is highly contagious and MAP infection in dairy cattle can eventually lead to death. With no available treatment for Johne’s disease, genetic selection and improvements in management practices could help reduce its prevalence. In a previous study, the gene coding interleukin-10 receptor subunit alpha (IL10Rα) was associated with Johne’s disease in dairy cattle. Our objective was to determine how IL10Rα affects the pathogenesis of MAP by examining the effect of a live MAP challenge on a mammary epithelial cell line (MAC-T) that had IL10Rα knocked out using CRISPR/cas9. The wild type and the IL10Rα knockout MAC-T cell lines were exposed to live MAP bacteria for 72 h. Thereafter, mRNA was extracted from infected and uninfected cells. Differentially expressed genes were compared between the wild type and the IL10Rα knockout cell lines. Gene ontology was performed based on the differentially expressed genes to determine which biological pathways were involved.ResultsImmune system processes pathways were targeted to determine the effect of IL10Rα on the response to MAP infection. There was a difference in immune response between the wild type and IL10Rα knockout MAC-T cell lines, and less difference in immune response between infected and not infected IL10Rα knockout MAC-T cells, indicating IL10Rα plays an important role in the progression of MAP infection. Additionally, these comparisons allowed us to identify other genes involved in inflammation-mediated chemokine and cytokine signalling, interleukin signalling and toll-like receptor pathways.ConclusionsIdentifying differentially expressed genes in wild type and ILR10α knockout MAC-T cells infected with live MAP bacteria provided further evidence that IL10Rα contributes to mounting an immune response to MAP infection and allowed us to identify additional potential candidate genes involved in this process. We found there was a complex immune response during MAP infection that is controlled by many genes