Genome-wide methylation analysis reveals differentially methylated loci that are associated with an age-dependent increase in bovine fibroblast response to LPS.

BACKGROUND: Differences in DNA methylation are known to contribute to the development of immune-related disorders in humans but relatively little is known about how methylation regulates immune function in cattle. Utilizing whole-transcriptome analyses of bovine dermal fibroblasts, we have previously identified an age and breed-dependent up-regulation of genes within the toll-like receptor 4 (TLR4) pathway that correlates with enhanced fibroblast production of IL-8 in response to lipopolysaccharide (LPS). Age-dependent differences in IL-8 production are abolished by treatment with 5-aza-2-deoxycytidine and Trichostatin A (AZA-TSA), suggesting epigenetic regulation of the innate response to LPS. In the current study, we performed reduced representation bisulfite sequencing (RRBS) on fibroblast cultures isolated from the same animals at 5- and 16-months of age to identify genes that exhibit variable methylation with age. To validate the role of methylation in gene expression, six innate response genes that were hyper-methylated in young animals were assessed by RT-qPCR in fibroblasts from animals at different ages and from different breeds. RESULTS: We identified 14,094 differentially methylated CpGs (DMCs) that differed between fibroblast cultures at 5- versus 16-months of age. Of the 5065 DMCs that fell within gene regions, 1117 were located within promoters, 1057 were within gene exons and 2891 were within gene introns and 67% were more methylated in young cultures. Transcription factor enrichment of the promoter regions hyper-methylated in young cultures revealed significant regulation by the key pro-inflammatory regulator, NF-κB. Additionally, five out of six chosen genes (PIK3R1, FES, NFATC1, TNFSF13 and RORA) that were more methylated in young cultures showed a significant reduction in expression post-LPS treatment in comparison with older cultures. Two of these genes, FES and NFATC1, were similarly down-regulated in Angus cultures that also exhibit a low LPS response phenotype. CONCLUSIONS: Our study has identified immune-related loci regulated by DNA methylation in cattle that may contribute to differential cellular response to LPS, two of which exhibit an identical expression profile in both low-responding age and breed phenotypes. Methylation biomarkers of differential immunity may prove useful in developing selection strategies for replacement cows that are less susceptible to severe infections, such as coliform mastitis.

Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome.

BACKGROUND: We have previously found substantial animal-to-animal and age-dependent variation in the response of Holstein fibroblast cultures challenged with LPS. To expand on this finding, fibroblast cultures were established from dairy (Holstein) and beef (Angus) cattle and challenged with LPS to examine breed-dependent differences in the innate immune response. Global gene expression was measured by RNA-Seq, while an epigenetic basis for expression differences was examined by methylated CpG island recovery assay sequencing (MIRA-Seq) analysis. RESULTS: The Holstein breed displayed a more robust response to LPS than the Angus breed based on RNA-Seq analysis of cultures challenged with LPS for 0, 2, and 8 h. Several immune-associated genes were expressed at greater levels (FDR < 0.05) in Holstein cultures including TLR4 at all time points and a number of pro-inflammatory genes such as IL8, CCL20, CCL5, and TNF following LPS exposure. Despite extensive breed differences in the transcriptome, MIRA-Seq unveiled relatively similar patterns of genome-wide DNA methylation between breeds, with an overall hypomethylation of gene promoters. However, by examining the genome in 3Kb windows, 49 regions of differential methylation were discovered between Holstein and Angus fibroblasts, and two of these regions fell within the promoter region (-2500 to +500 bp of the transcription start site) of the genes NTRK2 and ADAMTS5. CONCLUSIONS: Fibroblasts isolated from Holstein cattle display a more robust response to LPS in comparison to cultures from Angus cattle. Different selection strategies and management practices exist between these two breeds that likely give rise to genetic and epigenetic factors contributing to the different immune response phenotypes.

Age dependent changes in the LPS induced transcriptome of bovine dermal fibroblasts occurs without major changes in the methylome.

BACKGROUND: By comparing fibroblasts collected from animals at 5-months or 16-months of age we have previously found that the cultures from older animals produce much more IL-8 in response to lipopolysaccharide (LPS) stimulation. We now expand this finding by examining whole transcriptome differences in the LPS response between cultures from the same animals at different ages, and also investigate the contribution of DNA methylation to the epigenetic basis for the age-dependent increases in responsiveness. RESULTS: Age-dependent differences in IL-8 production by fibroblasts in response to LPS exposure for 24 h were abolished by pretreatment of cultures with a DNA demethylation agent, 5-aza-2'deoxycytidine (AZA). RNA-Seq analysis of fibroblasts collected from the same individuals at either 5 or 16 months of age and exposed in parallel to LPS for 0, 2, and 8 h revealed a robust response to LPS that was much greater in the cultures from older animals. Pro-inflammatory genes including IL-8, IL-6, TNF-α, and CCL20 (among many other immune associated genes), were more highly expressed (FDR < 0.05) in the 16-month old cultures following LPS exposure. Methylated CpG island recovery assay sequencing (MIRA-Seq) revealed numerous methylation peaks spread across the genome, combined with an overall hypomethylation of gene promoter regions, and a remarkable similarity, except for 20 regions along the genome, between the fibroblasts collected at the two ages from the same animals. CONCLUSIONS: The fibroblast pro-inflammatory response to LPS increases dramatically from 5 to 16 months of age within individual animals. A better understanding of the mechanisms underlying this process could illuminate the physiological processes by which the innate immune response develops and possibly individual variation in innate immune response arises. In addition, although relatively unchanged by age, our data presents a general overview of the bovine fibroblast methylome as a guide for future studies in cattle epigenetics utilizing this cell type.

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC official method 2005.04 assurance GdS E. coli 0157:H7 method to the reference culture method: 375 gram sample size.

The Assurance GDS Escherichia coli (E. col) O157:H7, AOAC Official Method 2005.04, has been modified to include a larger sample size of 375 g. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Ninety samples and controls, representing three foods, were analyzed. Results show no statistically detectable difference between the Assurance GDS E. coli O157:H7 assay and the reference culture methods for the detection of E. coli O157:H7, other than the low level of inoculation for leaf lettuce, for which the GDS gave noticeably higher recovery [difference in probability of detection between candidate methods (dPODc = +0.45)]. There were also suggestions of moderate differences (dPODc = +0.15 to +0.20) for ground beef and the high level of leaf lettuce, but the study size was too small to detect differences of this size. Results showed that the Assurance GDS E. coli O157:H7 method is equivalent to reference culture methods for the detection of E. coli O157:H7.

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Official Method 2005.05 Assurance GDS shiga Toxin Genes (O157) method to the reference culture method: 375 gram sample size.

The Assurance GDS Shiga Toxin Genes (0157), AOAC Official MethodsM 2005.05, has been modified to include a larger sample size of 375 g. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Ninety samples and controls, representing three foods, were analyzed. Results show no statistically detectable difference between the Assurance GDS Escherichia coli O157:H7 assay and the reference culture methods for the detection of E. coli O157:H7, other than the low level of inoculation for leaf lettuce for which the GDS gave noticeably higher recovery [difference in Probability of Detection between candidate methods (dPODc = +0.45)]. There were also suggestions of moderate differences (dPODc = +0.15 to +0.20) for ground beef and the high level of leaf lettuce, but the study size was too small to detect differences of this size. Results showed that the Assurance GDS Shiga Toxin Genes (0157) method is equivalent to the reference culture methods for the detection of Shiga toxigenic E. coli O157:H7.

Evaluation of the Assurance GDS for Salmonella method in foods and environmental surfaces: multilaboratory collaborative study.

A multilaboratory collaborative study was conducted to compare the detection of Salmonella by the Assurance GDS for Salmonella method and the Reference culture methods. Six foods, representing a variety of low microbial and high microbial load foods were analyzed. Seventeen laboratories in the United States and Canada participated in this study. No statistical differences (P < 0.05) were observed between the Assurance GDS for Salmonella and the Reference culture methods for any inoculation level of any food type or naturally contaminated food, except for pasta, for which the Assurance GDS method had a higher number of confirmed test portions for Salmonella compared to the Reference method.

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Method 996.09 Visual Immunoprecipitate (VIP) for E. coli O157:H7 method to the reference culture method.

The Visual Immunoprecipitate (VIP) for the Detection of Escherichia coli 0157:H7 in Foods, AOAC Official Method 996.09, has been modified to change the color of the test and control lines of the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Three foods were analyzed. In total, there were valid results from 225 samples and controls. Results showed that the VIP Gold for E. coli O157:H7 is equivalent to the reference culture methods for the detection of E. coli O157:H7.

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Method 999.09 Visual Immunoprecipitate (VIP) for Salmonella method to the reference culture method.

The Visual Immunoprecipitate (VIP) for the Detection of Salmonella in Foods, AOAC Official Method 999.09, has been modified to change the color of the test and control lines of the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Three foods were analyzed. In total, there were valid results from 155 samples and controls. Results showed that the modified VIP Gold for Salmonella is equivalent to the reference culture methods for the detection of Salmonella.

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Method 997.03 Visual Immunoprecipitate (VIP) for Listeria to the reference culture method.

The Visual Immunoprecipitate (VIP) for the Detection of Listeria in Foods and Environmental Surfaces, AOAC Official Method 997.03, has been modified to change the color of the test and control lines of the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Two food matrixes and one environmental surface were analyzed. In total, there were valid results from 100 samples and controls. Results showed that the modified VIP Gold for Listeria spp. is equivalent to the reference culture methods for the detection of Listeria.

Evaluation of the TRANSIA® PLATE Staphylococcal Enterotoxins Kit for the Detection of Staphylococcal Enterotoxins in Selected Foods.

The TRANSIA® PLATE Staphylococcal Enterotoxins enzyme immunoassay (EIA) was validated according to AOAC INTERNATIONAL guidelines for validating qualitative binary chemistry and microbiological methods. Five food matrixes were analyzed to determine the probability of detection (POD) for staphylococcal enterotoxins (SE), including SEA, SEB, SEC1, SEC2, SEC3, SED, and SEE, by the TRANSIA ^{PLATE Staphylococcal Enterotoxins EIA. The food matrixes tested were food types implicated in staphylococcal enterotoxin outbreaks and included raw milk cheese, liquid infant formula, eclairs, ready-to-eat ham, and canned mushrooms. Cheese and infant formula were tested with and without dialysis/concentration. The infant formula was also tested by an independent laboratory. Each food matrix was inoculated with specific toxins at low levels to yield fractional recoveries (0.015-0.20 ng/g of food) for POD analysis. One hundred percent recovery was achieved at concentrations ranging from <0.10 ng/g to 0.25 ng/g of toxin in the various food matrixes. At the same time, 50 Staphylococcus aureus strains known to produce toxins and 30 non-toxin producing bacteria (including 22 Staphylococcus strains) were grown and tested. All the SE toxin-producing strains yielded positive results and all of the exclusivity strains were negative. Robustness studies showed that changes in sample volume, sample pH, and EIA assay temperature had no significant effect on performance. Stability studies showed that kits stored for 12+ months performed as well as newly made kits. This assay has been approved as a Performance Tested MethodSM.}

Comparative Validation Study to Demonstrate the Equivalence of an Alternate Next-Day Enrichment Protocol for the TRANSIA® PLATE Salmonella Gold Method to Culture Methods for the Detection of Salmonella in Selected Foods and Environmental Surfaces.

TRANSIA® PLATE Salmonella Gold is an ELISA that was validated by Association Française de Normalisation (AFNOR) in 2001 and as a Performance Tested MethodSM (PTM) by AOAC in 2006 (PTM No. 010602) as a two-step enrichment protocol requiring 48 h. A simple next-day enrichment protocol using modified Enterohemorrhagic Escherichia coli media was developed for the TRANSIA PLATE Salmonella Gold to improve the time-to-results and laboratory work flow. We tested 128 Salmonella strains, representing all serotypes from A though Z and 51-66. TRANSIA PLATE Salmonella Gold detected all 128 of these strains. None of the 50 non-Salmonella strains were detected by TRANSIA PLATE Salmonella Gold. Performance of TRANSIA PLATE Salmonella Gold using the new enrichment protocol was compared with U.S. Department of Agriculture Microbiology Laboratory Guidebook reference culture procedure for the detection of Salmonella in ready-to-eat poultry, ready-to-eat beef, and chicken carcass rinsate. In addition, TRANSIA PLATE Salmonella Gold performance was compared with U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) for the detection of Salmonella from raw spinach, raw almonds, raw pasta, and environmental surfaces (stainless steel, rubber, and plastic). There was no statistically significant difference in the numbers of positive results TRANSIA PLATE Salmonella Gold protocol compared with the appropriate U.S. Department of Agriculture Food Safety and Inspection Service or FDA-BAM reference methods for any of these matrixes. Robustness testing demonstrated that the introduction of small changes in the normal assay parameters had no impact on the method performance. This new enrichment protocol has been approved as a Third Level modification to Performance Tested Method 010602.

Comparative Validation Study to Demonstrate the Equivalence of an Alternate Next-Day Enrichment Protocol for VIP® Gold for Salmonella Method to Culture Methods for the Detection of Salmonella in Selected Foods and Environmental Surfaces.

Background: VIP® Gold for Salmonella is a lateral flow immunodetection device that was validated by AOAC in 1999 as Official Method of Analysis 999.09. It was improved upon in 2009 by introducing gold colloid as the detection method. Objective: A simple next-day enrichment protocol using modified enterohemorrhagic Escherichia coli media was developed for the VIP Gold for Salmonella to improve the time-to-results and laboratory work flow. Methods: We tested 128 Salmonella strains, representing all serotypes from A to Z and 51 to 66 as well as 50 non-Salmonella strains for inclusivity/exclusivity. Performance of the VIP using the new enrichment protocol was compared with the U.S. Department of Agriculture (USDA) Microbiology Laboratory Guidebook reference culture procedure for the detection of Salmonella in ready-to-eat poultry, roast beef, and chicken carcass rinsate. VIP performance was also compared with the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) for the detection of Salmonella from raw spinach, raw almonds, raw pasta, and environmental surfaces (stainless steel, rubber, and plastic). Results: The VIP detected all 128 of Salmonella strains and none of the 50 non-Salmonella strains. There was no statistically significant difference in the numbers of positive results with VIP Gold for Salmonella protocol compared with appropriate USDA-Food Safety and Inspection Service or FDA-BAM reference methods for any of these matrixes. Conclusions: This new enrichment protocol has met all the requirements to be approved as a Performance Tested MethodSM. Highlights: The new enrichment protocol will improve the time-to-results and allow quicker decisions about the contamination of food products.

Characterization of a bacteriophage lysin (Ply700) from Streptococcus uberis.

The antibacterial properties of bacteriophage lytic enzymes may be of importance in future mastitis control programs. A prophage was isolated from a strain of Streptococcus uberis (ATCC 700407) following exposure to mitomycin C. Partial sequencing of the phage DNA revealed a putative lysin based on sequence similarity to other streptococcal phage lysins. The putative lysin (Ply700) was recombinantly expressed in Escherichia coli, and chromatographically purified. Addition of the purified Ply700 to bacterial suspensions of S. uberis, Streptococcus pyogenes, and Streptococcus dysgalactiae caused a rapid, calcium-dependent lysis while there was little activity against Streptococcus agalactiae, Staphylococcus aureus, or E. coli. Killing of S. uberis in milk by Ply700 (50 microg/ml) was confirmed by plate count assay. Activity was related to the initial concentration of bacteria in that 31% killing (P<0.05) was observed with an inoculating dose of approximately 4500 cfu/ml, while 81% killing (P<0.01) was observed when the inoculum was reduced to approximately 600 cfu/ml. In contrast, complete sterilization was observed in parallel cultures suspended in assay buffer indicating that factors in milk are able to neutralize the lysin. Functional characterization of the C-terminal domain, as a component of a GFP fusion protein, revealed its calcium-dependent ability to bind to S. uberis. The C-terminal domain may have utility in targeting S. uberis while it remains to be determined if the lysin by itself has sufficient potency in milk for effective use in the control of S. uberis mastitis.

Genetically enhanced cows resist intramammary Staphylococcus aureus infection.

Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 micrograms/ml [corrected] in their milk were produced. In vitro assays demonstrated the milk's ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 micrograms/ml [corrected] of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.

Validation study to demonstrate the equivalence of a minor modification of Assurance Enzyme Immunoassay method, Official Method 996.14, for detection of Listeria in foods and environmental surfaces to the reference culture methods.

The Assurance Enzyme Immunoassay (EIA) for the Detection of Listeria in Foods and Environmental Surfaces, AOAC Official Method 996.14, has been modified to combine the separate antibody and conjugate addition steps into one. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture methods. Two food matrixes and one environmental surface were analyzed. In total, there were valid results from 145 samples and controls. Results showed that the Assurance EIA for Listeria spp. is equivalent to the reference culture methods for the detection of Listeria.

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Method 996.09 [Visual Immunoprecipitate (VIP) for E. coli O157:H7] to the reference culture methods.

The Visual Immunoprecipitate (VIP) for the Detection of E. coli O157:H7 in Foods, AOAC Official Method 996.09, has been modified to use a simplified plastic housing for the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Three foods were analyzed. In total, valid results were obtained from 240 samples and controls. Results showed that the VIP for E. coli O157:H7 is equivalent to the reference culture methods for the detection of E. coli O157:H7.

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Method 997.03 [Visual Immunoprecipitate (VIP) for Listeria] to the reference culture methods.

The Visual Immunoprecipitate (VIP) for the Detection of Listeria in Foods and Environmental Surfaces, AOAC Official Method 997.03, has been modified to use a simplified housing for the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture methods. Two food matrixes and one environmental surface were analyzed. In total, valid results were obtained from 145 samples and controls. Results showed that the modified VIP for Listeria spp. is equivalent to the reference culture methods for the detection of Listeria.

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Method 999.09 [Visual Immunoprecipitate (VIP) for Salmonella] to the reference culture methods.

The Visual Immunoprecipitate (VIP) for the Detection of Salmonella in Foods, AOAC Official Method 999.09, has been modified to use a simplified housing for the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Three foods were analyzed. In total, valid results were obtained from 125 samples and controls. Results showed that the modified VIP for Salmonella is equivalent to the reference culture methods for the detection of Salmonella.

In utero exposure to LPS alters the postnatal acute-phase response in beef heifers.

The potential effect of prenatal LPS exposure on the postnatal acute phase response (APR) to an LPS challenge in heifers was determined. Pregnant crossbred cows were separated into prenatal immune stimulation (PIS) and saline groups (Control). From these treatments, heifer calves were identified at weaning to subsequently receive an exogenous LPS challenge. Sickness behavior scores (SBS) were recorded and blood samples were collected at 30-min intervals from -2 to 8 h and again at 24 h relative to the LPS challenge. There was a treatment × time interaction for the change in vaginal temperature (VT) such that the change in VT was greater in Control than PIS from 150 to 250 min, yet it was greater in PIS than Control from 355 to 440 min and from 570 to 1145 min. There was also a treatment × time interaction for SBS such that scores were greater in Control than PIS at 0.5 h, yet were greater in PIS than Control from 2.5 to 4 h post-LPS. There was a tendency for a treatment × time interaction for serum concentrations of IL-6, which were greater in PIS than Control heifers from 5.5 to 6 h and from 7 to 8 h post-challenge. Thus, a single exposure to LPS during gestation can alter the postnatal APR to LPS in heifer calves.

Comparison of Assurance GDS(®) MPX ID for Top STEC with Reference Culture Methods for the Detection of E. coli Top 6 STEC; Direct Confirmation of Top 6 STEC from Isolation Plates and Determination of Equivalence of PickPen(®) and FSIS OctoMACS™ Concentration Protocols.

Assurance GDS(®) MPX ID for Top Shiga toxin-producing Escherichia coli (STEC; MPX ID) was validated according to the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Foods and Environmental Surfaces as (1) a secondary screening method for specific detection of the Top 6 STEC serogroups (O26, O45, O103, O111, O121, and O145) in raw beef trim, raw ground beef, raw spinach, and on stainless steel; and (2) as a confirmatory method for the identification of pure culture isolates as Top 6 STEC. MPX ID is used in conjunction with the upfront BCS Assurance GDS MPX Top 7 STEC assay. This Performance Tested Method(SM) validation has two main parts: Method Developer studies and the Independent Laboratory study. A total of 180 samples and controls were analyzed. Results showed that MPX ID had no statistically significant differences with the reference culture methods for the detection of Top 6 STEC in the food matrixes (raw beef trim, raw ground beef, and raw spinach) and environmental sponges (stainless steel) studied. Inclusivity/exclusivity studies were also conducted. One hundred percent inclusivity among the 50 Top 6 STEC serovars tested and 100% exclusivity for the 30 non-Top 6 STEC organisms tested were demonstrated. For validation of MPX ID as a confirmatory method for isolated colonies, all inclusivity and exclusivity organisms were streaked for isolation onto five STEC plating media: modified rainbow agar, Levine's eosin-methylene blue (L-EMB) agar, rainbow agar with novobiocin and cefixime, and enterohemolysin agar with selective agents as well as trypticase soy agar with yeast extract. These isolated colonies were suspended and analyzed by Assurance GDS MPX Top 7 STEC and MPX ID. MPX ID was able to correctly confirm all inclusivity organisms from all plate types, except two STEC isolates from L-EMB agar plates only in the Independent Laboratory study. All exclusivity organisms were correctly determined by MPX ID as non-Top 6 STEC from the STEC plating media. An additional but separate part of these studies was a comparison of immunomagnetic separation (IMS) efficiency using the Assurance GDS procedure with a PickPen(®) device and the U.S. Department of Agriculture procedure using the OctoMACS™ Separator device for plating onto chromogenic agar. Results demonstrated the equivalence of the two IMS procedures for plate confirmation of Top 7 STEC.