Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score

Background

During the past ten years many quantitative trait loci (QTL) affecting mastitis incidence and mastitis related traits like somatic cell score (SCS) were identified in cattle. However, little is known about the molecular architecture of QTL affecting mastitis susceptibility and the underlying physiological mechanisms and genes causing mastitis susceptibility. Here, a genome-wide expression analysis was conducted to analyze molecular mechanisms of mastitis susceptibility that are affected by a specific QTL for SCS on Bos taurus autosome 18 (BTA18). Thereby, some first insights were sought into the genetically determined mechanisms of mammary gland epithelial cells influencing the course of infection.

Methods

Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows selected for high and low mastitis susceptibility by applying a marker-assisted selection strategy considering QTL and molecular marker information of a confirmed QTL for SCS in the telomeric region of BTA18. The cells were cultured and subsequently inoculated with heat-inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 h, the cells were harvested and analyzed using the microarray expression chip technology to identify differences in mRNA expression profiles attributed to genetic predisposition, inoculation and cell culture.

Results

Comparative analysis of co-expression profiles clearly showed a faster and stronger response after pathogen challenge in pbMEC from less susceptible animals that inherited the favorable QTL allele ''Q'' than in pbMEC from more susceptible animals that inherited the unfavorable QTL allele ''q''. Furthermore, the results highlighted RELB as a functional and positional candidate gene and related non-canonical Nf-kappaB signaling as a functional mechanism affected by the QTL. However, in both groups, inoculation resulted in up-regulation of genes associated with the Ingenuity pathways ''dendritic cell maturation'' and ''acute phase response signaling'', whereas cell culture affected biological processes involved in ''cellular development''.

Conclusions

The results indicate that the complex expression profiling of pathogen challenged pbMEC sampled from cows inheriting alternative QTL alleles is suitable to study genetically determined molecular mechanisms of mastitis susceptibility in mammary epithelial cells in vitro and to highlight the most likely functional pathways and candidate genes underlying the QTL effect.     

Staphylococcus aureus persistence properties associated with bovine mastitis and alternative therapeutic modalities

Staphylococcus aureus is an important agent of contagious bovine intramammary infections in dairy cattle. Its ability to persist inside the udder is based on the presence of important mechanisms such as its ability to form biofilms, polysaccharide capsules small colony variants, and their ability to invade professional and nonprofessional cells, which will protect S. aureus from the innate and adaptive immune response of the cow, and from antibiotics that are no longer considered to be sufficient against S. aureus bovine mastitis. In this review, we present the recent research outlining S. aureus persistence properties inside the mammary gland, including its regulation mechanisms, and we highlight alternative therapeutic strategies that were tested against S. aureus isolated from bovine mastitis such as the use of probiotic bacteria, bacteriocins and bacteriophages. Overall, the persistence of S. aureus inside the mammary gland remains a pressing veterinary problem. A thorough understanding of staphylococcal persistence mechanisms will elucidate novel ways that can help in the identification of novel treatments.     

Effects of cell culture techniques on gene expression and cholesterol efflux in primary bovine mammary epithelial cells derived from milk and tissue

Primary bovine mammary epithelial cells (pbMEC) are often used in cell culture to study metabolic and inflammatory processes in the udder of dairy cows. The most common source is udder tissue from biopsy or after slaughter. However, it is also possible to culture them from milk, which is non-invasive, repeatable and yields less contamination with fibroblasts. Generally, not much is known about the influence of cell origin and cell culture techniques such as cryopreservation on pbMEC functionality. Cells were extracted from milk and udder tissue to evaluate if milk-derived pbMEC are a suitable alternative to tissue-derived pbMEC and to test what influence cryopreservation has. The cells were cultivated for three passages and stored in liquid nitrogen. The relative gene expression of the five target genes kappa-casein, lingual antimicrobial peptide (LAP), lactoferrin, lysozyme (LYZ1) and the prolactin receptor normalised with keratin 8 showed a tendency to decrease in the tissue cultures, but not in the milk-derived cultures, suggesting a greater influence of the cultivation process on tissue-derived cells, freezing lowered expression levels in both cultures. Overall expression of LAP and LYZ1 tended to be higher in milk cells. Cholesterol efflux was measured to compare passages one to seven in milk-derived cells. Passage number did not alter the efflux rate (p????0.05). We showed for the first time that the extraction of pbMEC from milk can be a suitable alternative to tissue extraction.     

Differential expression of immune response genes associated with subclinical mastitis in dairy buffaloes

Buffalo milk production has become of significant importance on the world scale, however, there are few studies involving biotechnological tools specifically for buffalo. To verify the effects caused by subclinical mastitis on the components of milk and to study the innate immune system in the udder of dairy buffaloes with subclinical mastitis, we evaluated the levels of expression of the lactoferrin (LTF), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-8 (IL-8), and toll-like receptors 2 (TLR-2) and 4 (TLR-4) genes in buffaloes with and without subclinical mastitis. Milk samples were collected for the determination of milk components: somatic cell score (SCS), fat, protein, lactose, total solids and solids-not-fat (SNF), as well as for RNA extraction of milk cells, complementary DNA synthesis, and expression profile quantification by quantitative real-time PCR. For gene expression, the ΔΔCt was estimated using contrasts of the target genes expression adjusted for the expression of the housekeeping genes between both groups. Linear regression analysis was performed to determine the relationship between the genes studied and the milk components. Subclinical mastitis induced changes in the fat, lactose and SNF in milk of buffaloes, and the messenger RNA abundance was upregulated for TLR-2, TLR-4, TNF-α, IL-1β and IL-8 genes in milk cells of buffaloes with subclinical mastitis, whereas the LTF gene was not differentially expressed. Results of linear regression analysis showed that TLR-2 gene expression most explains the variation in SCS, and the change in a unit of ΔCt of the TNF-α gene would result in a higher increase in SCS. The study of these immune function genes that are active in the mammary gland is important to characterize the action mechanism of the innate immunity that occurs in subclinical mastitis in dairy buffaloes and may aid the development of strategies to preserve the health of the udder.     

Molecular Identification of Staphylococcus aureus in Airway Samples from Children with Cystic Fibrosis

Background

Staphylococcus aureus is a common and significant pathogen in cystic fibrosis. We sought to determine if quantitative PCR (qPCR) and 16S rRNA gene sequencing could provide a rapid, culture-independent approach to the identification of S. aureus airway infections.

Methods

We examined the sensitivity and specificity of two qPCR assays, targeting the femA and 16S rRNA gene, using culture as the gold standard. In addition, 16S rRNA gene sequencing to identify S. aureus directly from airway samples was evaluated. DNA extraction was performed with and without prior enzymatic digestion.

Results

87 samples [42 oropharyngeal (OP) and 45 expectorated sputum (ES)] were analyzed. 59 samples (68%) cultured positive for S. aureus. Using standard extraction techniques, sequencing had the highest sensitivity for S. aureus detection (85%), followed by FemA qPCR (52%) and 16SrRNA qPCR (34%). For all assays, sensitivity was higher from ES samples compared to OP swabs. Specificity of the qPCR assays was 100%, but 21.4% for sequencing due to detection of S. aureus in low relative abundance from culture negative samples. Enzymatic digestion increased the sensitivity of qPCR assays, particularly for OP swabs.

Conclusion

Sequencing had a high sensitivity for S. aureus, but low specificity. While femA qPCR had higher sensitivity than 16S qPCR for detection of S. aureus, neither assay was as sensitive as sequencing. The significance of S. aureus detection with low relative abundance by sequencing in culture-negative specimens is not clear.     

Engineering Disease Resistant Cattle

Mastitis is a disease of the mammary gland caused by pathogens that find their way into the lumen of the gland through the teat canal. Mammary gland infections cost the US dairy industry approximately $2 billion dollars annually and have a similar impact in Europe. In the absence of effective treatments or breeding strategies to enhance mastitis resistance, we have created transgenic dairy cows that express lysostaphin in their mammary epithelium and secrete the antimicrobial peptide into milk. Staphylococcus aureus, a major mastitis pathogen, is exquisitely sensitive to lysostaphin. The transgenic cattle resist S. aureus mammary gland challenges, and their milk kills the bacteria, in a dose dependent manner. This first step in protecting cattle against mastitis will be followed by introduction of other genes to deal with potential resistance issues and other mastitis causing organisms. Care will be taken to avoid altering milk’s nutritional and manufacturing properties. Multi-cistronic constructs may be required to achieve our goals as will other strategies possibly involving RNAi and gene targeting technology. This work demonstrates the possibility of using transgenic technology to address disease problems in agriculturally important species. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Biofilm Formation on Biotic and Abiotic Surfaces in the Presence of Antimicrobials by Escherichia coli Isolates from Cases of Bovine Mastitis

Escherichia coli is a highly adaptive microorganism, and its ability to form biofilms under certain conditions can be critical for antimicrobial resistance. The adhesion of four E. coli isolates from bovine mastitis to bovine mammary alveolar (MAC-T) cells, biofilm production on a polystyrene surface, and the expression profiles of the genes fliC, csgA, fimA, and luxS in the presence of enrofloxacin, gentamicin, co-trimoxazole, and ampicillin at half of the MIC were investigated. Increased adhesion of E. coli isolates in the presence of antimicrobials was not observed; however, increased internalization of some isolates was observed by confocal microscopy. All of the antimicrobials induced the formation of biofilms by at least one isolate, whereas enrofloxacin and co-trimoxazole decreased biofilm formation by at least one isolate. Quantitative PCR analysis revealed that all four genes were differentially expressed when bacteria were exposed to subinhibitory concentrations of antimicrobials, with expression altered on the order of 1.5- to 22-fold. However, it was not possible to associate gene expression with induction or reduction of biofilm formation in the presence of the antimicrobials. Taken together, the results demonstrate that antimicrobials could induce biofilm formation by some isolates, in addition to inducing MAC-T cell invasion, a situation that might occur in vivo, potentially resulting in a bacterial reservoir in the udder, which might explain some cases of persistent mastitis in herds.     

Adhesion of Staphylococcus aureus to Bovine Mammary Epithelial Cells Induced by Growth in Milk Whey

Staphylococcus aureus strains isolated from bovine intramammary infection (mastitis) were tested for adhesion to bovine mammary epithelial cells after growth in milk whey or TSB. Bacteria grown in milk whey adhered more efficiently to mammary gland epithelial cells in vitro than the corresponding homologous bacteria grown in TSB. Trypsin treatment of milk whey-grown S. aureus had no effect on their adherence. Whereas, pretreatment with periodate significantly decreased bacterial adherence capacity. Periodate treatment of TSB-grown bacteria had no effect on adhesion to the mammary gland epithelial cells.     

Secondary Staphylococcus aureus intramammary colonization is reduced by non-aureus staphylococci exoproducts

Non-aureus staphylococci (NAS) and Staphylococcus aureus are pathogens that cause bovine mastitis, a costly disease for dairy farmers, however; many NAS are considered part of the normal udder microbiota. It has been suggested that through a mechanism that remains to be elucidated, NAS intramammary colonization can prevent subsequent infection with other bacterial pathogens. This study shows that in a murine mastitis model, secondary Staph. aureus intramammary colonization is reduced by exoproducts from Staph. chromogenes and Staph. simulans, both NAS, while Streptococcus spp. exoproducts have much less ability to affect the course of the infection caused by S. aureus.     

Solexa sequencing and custom microRNA chip reveal repertoire of microRNAs in mammary gland of bovine suffering from natural infectious mastitis

Identification of microRNAs (miRNAs), target genes and regulatory networks associated with innate immune and inflammatory responses and tissue damage is essential to elucidate the molecular and genetic mechanisms for resistance to mastitis. In this study, a combination of Solexa sequencing and custom miRNA chip approaches was used to profile the expression of miRNAs in bovine mammary gland at the late stage of natural infection with Staphylococcus aureus, a widespread mastitis pathogen. We found 383 loci corresponding to 277 known and 49 putative novel miRNAs, two potential mitrons and 266 differentially expressed miRNAs in the healthy and mastitic cows’ mammary glands. Several interaction networks and regulators involved in mastitis susceptibility, such as ALCAM, COL1A1, APOP4, ITIH4, CRP and fibrinogen alpha (FGA), were highlighted. Significant down‐regulation and location of bta‐miR‐26a, which targets FGA in the mastitic mammary glands, were validated using quantitative real‐time PCR, in situ hybridization and dual‐luciferase reporter assays. We propose that the observed miRNA variations in mammary glands of mastitic cows are related to the maintenance of immune and defense responses, cell proliferation and apoptosis, and tissue injury and healing during the late stage of infection. Furthermore, the effect of bta‐miR‐26a in mastitis, mediated at least in part by enhancing FGA expression, involves host defense, inflammation and tissue damage.     

Non-Classical ProIL-1beta Activation during Mammary Gland Infection Is Pathogen-Dependent but Caspase-1 Independent

Infection of the mammary gland with live bacteria elicits a pathogen-specific host inflammatory response. To study these host-pathogen interactions wild type mice, NF-kappaB reporter mice as well as caspase-1 and IL-1beta knockout mice were intramammarily challenged with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The murine mastitis model allowed to compare the kinetics of the induced cytokine protein profiles and their underlying pathways. In vivo and ex vivo imaging showed that E. coli rapidly induced NF-kappaB inflammatory signaling concomitant with high mammary levels of TNF-alpha, IL-1 alpha and MCP-1 as determined by multiplex analysis. In contrast, an equal number of S. aureus bacteria induced a low NF-kappaB activity concomitant with high mammary levels of the classical IL-1beta fragment. These quantitative and qualitative differences in local inflammatory mediators resulted in an earlier neutrophil influx and in a more extensive alveolar damage post-infection with E. coli compared to S. aureus. Western blot analysis revealed that the inactive proIL-1beta precursor was processed into pathogen-specific IL-1beta fragmentation patterns as confirmed with IL-1beta knockout animals. Additionally, caspase-1 knockout animals allowed to investigate whether IL-1beta maturation depended on the conventional inflammasome pathway. The lack of caspase-1 did not prevent extensive proIL-1beta fragmentation by either of S. aureus or E. coli. These non-classical IL-1beta patterns were likely caused by different proteases and suggest a sentinel function of IL-1beta during mammary gland infection. Thus, a key signaling nodule can be defined in the differential host innate immune defense upon E. coli versus S. aureus mammary gland infection, which is independent of caspase-1.     

Microbial Diversity of Bovine Mastitic Milk as Described by Pyrosequencing of Metagenomic 16s rDNA

Dairy cow mastitis is an important disease in the dairy industry. Different microbial species have been identified as causative agents in mastitis, and are traditionally diagnosed by bacterial culture. The objective of this study was to use metagenomic pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture. One hundred and thirty-six milk samples were collected from cows showing signs of mastitis and used for microbiological culture. Additionally, 20 milk samples were collected from healthy quarters. Bacterial DNA was isolated from the same milk samples and the 16S rRNA genes were individually amplified and pyrosequenced. Discriminant analysis showed that the groups of samples that were most clearly different from the rest and thus easily discriminated were the normal milk samples from healthy cows and those characterised by culture as Trueperella pyogenes and Streptococcus spp. The mastitis pathogens identified by culture were generally among the most frequent organisms detected by pyrosequencing, and in some cases (Escherichia coli, Klebsiella spp. and Streptococcus uberis mastitis) the single most prevalent microorganism. Trueperella pyogenes sequences were the second most prevalent sequences in mastitis cases diagnosed as Trueperella pyogenes by culture, Streptococcus dysgalactiae sequences were the second most prevalent sequences in mastitis cases diagnosed as Streptococcus dysgalactiae by culture, and Staphyloccocus aureus sequences were the third most prevalent in mastitis cases diagnosed as Staphylococcus aureus by culture. In samples that were aerobic culture negative, pyrosequencing identified DNA of bacteria that are known to cause mastitis, DNA of bacteria that are known pathogens but have so far not been associated with mastitis, and DNA of bacteria that are currently not known to be pathogens. A possible role of anaerobic pathogens in bovine mastitis is also suggested.     

Incidence of Staphylococci and Streptococci During Winter in Mastitic Milk of Sahiwal Cow and Murrah Buffaloes

Mastitis is a serious problem in dairy sector and among various aetiological agents, the incidence of staphylococci and streptococci remains high in milking animal. The present study was focused on detection of staphylococci and streptococci in winter season. Milk samples (117) of mastitic animals were tested for presence of staphylococci and streptococci using biochemical and PCR based assays. The testing revealed majority of animals (90.6%) were infected with more than one causative agent. Amongst 117 sample, 109 and 90 comprised of staphylococci and 90 streptococci, respectively. Distribution proportion of S. aureus, S. epidermidis, S. agalactiae, S. uberis and S. dysgalactiae among the mastitic cases was found as 64.9, 7.7, 5.1, 1.7, 48.7, 65.8 and 0.8%, respectively. Streptococci and staphylococci were observed in different combinations and the frequent were S. aureus/S. agalactiae/S. uberis, S. aureus/S. uberis, S. aureus/S. agalactiae and S. agalactiae/S. uberis which were accounted for 23.9, 19.7, 5.9 and 2.6%, respectively. Approximately half of the (52.1%) cases were observed for reoccurrence of mastitis. Reoccurrence of mastitis in winter season among these cases was significantly low as compared to summer (cattle-5 cases; buffaloes-2 cases). In addition, prevalence of S. aureus, S. agalactiae, S. uberis, and S. epidermidis in reoccurring mastitic cases was 73.7, 63.9, 45.9 and 6.6%, respectively. The observations revealed mastitis causing pathogens remains in hidden phase in winter season; however, cannot be neglected. The observation might be helpful in culling or segregation of cows for mastitis reduction programmes.     

Intramammary Immunization of Pregnant Mice with Staphylococcal Protein A Reduces the Post-Challenge Mammary Gland Bacterial Load but Not Pathology

Protein A, encoded by the spa gene, is one of the major immune evading MSCRAMM of S. aureus, demonstrated to be prevalent in a significant percentage of clinical bovine mastitis isolates in Australia. Given its’ reported significance in biofilm formation and the superior performance of S. aureus biofilm versus planktonic vaccine in the mouse mastitis model, it was of interest to determine the immunogenicity and protective potential of Protein A as a potential vaccine candidate against bovine mastitis using the mouse mastitis model. Pregnant Balb/c mice were immunised with Protein A emulsified in an alum-based adjuvant by subcutaneous (s/c) or intramammary (i/mam) routes. While humoral immune response of mice post-immunization were determined using indirect ELISA, cell-mediated immune response was assessed by estimation of interferon-gamma (IFN-γ) produced by protein A-stimulated splenocyte supernatants. Protective potential of Protein A against experimental mastitis was determined by challenge of immunized versus sham-vaccinated mice by i/mam route, based upon manifestation of clinical symptoms, total bacterial load and histopathological damage to mammary glands. Significantly (p<0.05) higher levels of IgG1 isotype were produced in mice immunized by the s/c route. In contrast, significantly higher levels of the antibody isotype IgG2a were produced in mice immunized by the i/mam route (p<0.05). There was significant reduction (p<0.05) in bacterial loads of the mammary glands of mice immunized by Protein A regardless of the route of immunization, with medium level of clinical symptoms observed up to day 3 post-challenge. However, Protein A vaccine failed to protect immunized mice post-challenge with biofilm producing encapsulated S. aureus via i/mam route, regardless of the route of immunization, as measured by the level of mammary tissue damage. It was concluded that, Protein A in its’ native state was apparently not a suitable candidate for inclusion in a cell-free vaccine formulation against mastitis.     

Multiplex PCR assay for species identification of bovine mastitis pathogens

Aim: To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk. Methods and Results: A two‐tube mPCR assay was developed. The accuracy of the mPCR was evaluated using 56 standard reference strains and 705 strains comprising of E. coli (n = 99), staphylococci (n = 522) and streptococci (n = 84). The threshold of detection of the mPCR assay was 10 fg of genomic DNA and <10³ CFU ml^?1. A comparative evaluation of mPCR with culture method using 115 milk samples from subclinical mastitis showed mPCR to be more efficacious. Subsequently, the mPCR showed successful detection of target bacteria, when applied directly for the assessment of 36 bulk milk samples. Conclusion: The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time. Significance and Impact of the Study: The assay will be useful for the detection of mastitis, testing bacteriological safety of milk and for species level differentiation. The assay will be of value in the dairy sector for diagnosis and research. The early and accurate identification of pathogens will enable timely interventions for the treatment and control of bovine mastitis.     

Immunorelevant proteins for the diagnosis of bovine staphylococcal mastitis

Staphylococcus aureus is a leading cause of bovine mastitis, a condition in which the udder of the cow is inflamed, reducing the quality and quantity of milk produced. Staphylococcal mastitis is a common infection that can develop into a chronic form. The segregation of infected animals is an important preventive practice but relies on an effective diagnostic method. For this purpose, we constructed a genomic library of S. aureus, and a screening step was conducted with antiserum produced using the total protein extract of the pathogen. The nucleotide sequences of the immunoselected clones were aligned with the genome of bovine S. aureus RF122, which enabled the identification of 65 different loci, including proteins related to metabolism, adhesion and cell wall production, toxins, regulatory proteins, and hypothetical proteins. The subcellular location of the immunoreactive polypeptides was also determined. Fifty-two percent were cytoplasmic, 34 % were located in areas exposed to the host’s immune system, and for 14 %, the location could not be determined. In silico analysis of the presence of these proteins in mastitis pathogens showed that Fib, ClfA, and the hypothetical protein SAB0166 were the only proteins specific for S. aureus. Therefore, these proteins are promising candidates for the serodiagnosis of staphylococcal mastitis.