A power test was used to determine the number of animals per experimental group. The coefficient of variance (CV %) was 5; the percent difference from the control was decided at 10; and P
0.05 with a power of 90%. The number of replicates per treatment was determined to be 5 mice/experimental group. Assistance in developing the experimental design was through personal communications with M.M. Galyean (TTU, Dept. of Animal & Food Sciences) and with reference to Martin, Meek, and Willeberg. 1987; Veterinary Epidemiology Principals and Methods
; p. 45 Iowa State University Press. Ames, Iowa. In detail the study design is described in Table .
For this study, 23–28 day old BALB/c mice equally divided between male and female, for a total of 410 animals were tested. (Charles River Laboratories, Wilmington, MA). Animals were acclimated for 2 weeks in the Texas Tech University (TTU) Animal Care and Use (ACU) facilities prior to experimentation and animal welfare, housing conditions, and euthanasia were according to protocols established through TTU-ACU (ACUC Approval Number: 07060–12). Five animals per experimental group were housed in sterilized cages with sterilized bedding. Animals were provided with sterile water and mouse chow, ad libitum
. There were a total of 10 experimental groups and four time-points over the course of 180 days, sample collections were conducted at days 45, 90, 135, and 180. At day 0, five-male and five-female mice were euthanized and tissues were collected for histopathology and cryogenic preservation, to evaluate animals prior to experimentation. From day 0 through day 45 animals were fed a diet of: sterile powder chow, sterile powder chow combined with 1×106
CFU/g NP-51, or heat-killed NP-51 at similar concentrations, daily. At day 45, 100 animals from 10 experimental groups were euthanized; animals were sedated with Isoflurane inhalation, followed with cardiac puncture and blood collection. The large (colon) and small intestinal tissues, stomach, and liver from male and female animals (n
4) were preserved for histopathology analysis in 10% formalin solution in phosphate-buffered saline (PBS). Identical tissues collected from male/female mice (n
6) were harvested and flash frozen in liquid nitrogen, followed with long term cryogenic preservation at −80°C. MAP concentrations were determined, from 0.2 g of harvested tissues, using qRT-PCR on large intestine and liver; liver tissues presented granulomas distinct to MAP infection based on histopathology analysis.
MAP cultures and cell harvesting
MAP cultures were originally harvested from cattle at the USDA National Animal Disease Center (NADC), and kindly provided by Judith Stabel (Ames, Iowa). A single culture was shipped to TTU, in Middle Brooks H79 broth with Mycobactin (Allied Monitor, Fayette, MO), at refrigerated conditions. Cultures were grown and harvested according to conditions provided through Stabel et al., at the NADC [39
]. MAP cells were rendered non-viable by boiling cultures for 20 min in a 65°C waterbath [40
NP-51 cultures and chow preparation
Freeze-dried NP-51 with maltodextrin (MD) was shipped directly to TTU by Culture Systems Incorporated (Mishawaka, IN). Twenty-gram packets were made with NP-51, individual packets were used once daily and any remaining material was discarded. Viable cultures of NP-51 were mixed into sterile, powdered mouse chow (7012 Teklad LM-485 Mouse/Rat Sterilizable Diet; Harlan Teklad Diets, Madison WI) using a KitchenAid® 5-Quart Tilt-Head Artisan Series Stand Mixer (Bed Bath & Beyond; Lubbock, TX) at setting 2 or 3, for 15–20 minutes in a BSL-2 safety cabinet (this insured even distribution of NP-51 in the powdered chow). Non-viable NP-51 was prepared by heating samples at 180°C in a dry oven for 20 min (Fisher Scientific Convection Gravity Oven; Fisher Sci, Houston, TX). Non-viable cultures were mixed with an identical mixer system, separately, using sterile bowls and utensils. Each chow was replaced daily with new feed according to experimental conditions. Animal cages and feed containers were handled under a BSL-2 safety cabinet. Feed containers were cleaned and sterilized weekly by autoclaving (121°C for 15 min), new feed containers were replaced along with sterilized cages and bedding every 3rd or 7th day. Utensils for preparing chow including bowls, mixing utensils, and glassware were cleaned daily and sterilized with baking at 180°C in a convection gravity oven at a minimum of 4 hours or overnight, before use.
MAP infection and sampling schedule
On day 46, through intraperitoneal (IP) injection experimental groups were injected with 100 μl of sterile PBS containing 1×107 CFU/ml viable or non-viable MAP. Controls were injected with 100 μl PBS only. Animals were observed closely for 48 h for negative physiological reactions to IP injections. Every 45 days post infection - Days 90, 135, and 180- necropsies were performed.
Serum/ tissue collection & cytokine analysis
At each necropsy, blood was collected into serum separation tubes, and serum was pooled from each experimental group (n =5) (13×100 mm, SST™ Serum Separation Tubes; Beckton- Dickinson; San Jose, CA). Blood samples were refrigerated for 24-48 h after collection, followed by centrifugation at 5,000 × g for 5 min (Marathon 2100R, Thermo-Fisher Scientific; Houston, TX). Serum was transferred, using disposable, sterile serological pipettes to sterile, 2 ml cryogenic tubes and stored at −20°C (Fisher Scientific; Houston, TX).
Two-hundred microliters of serum from each experimental condition and for all collection time points were shipped to TTUHSC, at El Paso and analyzed using a Mouse Cytokine 20-Plex Panel for the Luminex® platform - according to manufacturer protocol (Invitrogen/Life Technologies; Carlsbad, CA). Serum was analyzed in triplicate wells and compared to standards.
Tissue RNA/DNA extractions, cDNA synthesis, & cDNA analysis
Colon tissues were ground with mortar and pestle in liquid nitrogen to preserve RNA/DNA and prevent nuclease activity in tissues. Approximately, 100 mg of tissue were extracted for RNA using a Trizol® kit (Invitrogen, Carlsbad, CA). Co-purification of DNA from these extractions were preformed from the separated organic layer, using a DNeasy® Blood & Tissue Kit according to protocols for total bacterial DNA extractions (Qiagen, Valencia, CA). Purified DNA were kept in 1x Tris-EDTA Buffer and concentrations were measured spectrophotometerically at a ratio of 260/280 nm (Nanodrop 1000, Wilmington, DE). DNA at concentrations of 40–50 ng/μl in 50 μl of water was provided for sequencing. High throughput sequencing was conducted using 454 ®pyrosequencing technology (Roche Laboratories, Branford, CT) at Research and Testing Laboratories, LLC (Lubbock, TX).
Duplicate samples of RNA, collected from triplicate animals from each sex for each experimental condition were prepared for quantitative Real Time- PCR (qRT-PCR). High- Capacity® cDNA Reverse Transcription kit was used (ABI, Foster City, CA). For RNA samples with concentrations below 60 ng/μl a High® Capacity RNA-to-cDNA Master Mix kit was used for cDNA synthesis (ABI; Foster City, CA). cDNA were analyzed using SYBR green probes for genes of interest for Open® Array platform (Life Technologies Inc.; Carlsbad, CA). Probes for all genes were selected from array panels and customized for our study- 9 plates were used in the analysis. Assays were performed by The University of Texas, Southwestern at Dallas. Analysis of data was conducted using Open® Array Real Time qPCR Analysis Software Version 1.0.4. Each cDNA sample was analyzed in duplicate, from triplicate animals and both sexes.
qRT-PCR analysis of MAP concentrations from tissues
The template DNA used for construction of standards was extracted from MAP culture. Briefly, 10 ml of the MAP culture was pelleted using centrifugation (Marathon 2100R, Thermo-Fisher Scientific, Houston, TX) at 5000 × g for 15 minutes. The cells were washed twice with HPLC-grade water (Ricca Chemical Company; Arlington, TX) and again suspended in new HPLC-grade water. DNA was extracted by heating 50 μl of cell suspension in PCR tubes (VWR Int, Westchester PA) at 99°C for 15 minutes in Gene Amp PCR system 2700 Thermocycler (Applied Biosystems, Foster City, CA). The heated sample was centrifuged to pellet the cell debris and the supernatant was used as template for successive experiments.
The primers used for this assay amplifies a 163 bp region of the IS-Mav region in the MAP genome. Various primer pairs were tested before selecting the ISMav2 primers [3
]. By using plasmids with the 163 bp fragment DNA insertion as standards, serial dilutions were tested to develop a standard curve and then enumerate the number of MAP cells in the experimental samples by plotting the Ct values on the curve. This was confirmed using the melting curve analysis of the PCR product which showed only one peak for ISMav2; thus the amplicon was very specific for MAP. Using recombinant plasmids at specific concentrations as standards we tried to do an absolute quantification of MAP but based on the standard curve generated using these plasmids the minimum number of cells that can be detected was 10. PCR reactions were carried out in 50 μl containing primer ISMav2 (Forward seq 5′
-CGG CAA AAT CGA GCA GTT TC-3′
; Reverse seq 5′
-TGA GCC GGT GTG ATC TTT-3′
), 10 μl of template DNA, using Qiagen Hot®-Start PCR kit (Qiagen Sciences, MD) following manufacturer protocols [3
]. The PCR products were run on 2% agarose gel stained with EtBr in 1X TAE buffer to check for a single amplicon. The PCR product was purified using Qiagen® PCR-Purification Kit (Qiagen Sciences, MD) and used for direct cloning using pGEM-T® Easy vector system (Promega Corporation, Madison, WI) in HB101® competent E. coli
cells (Promega Corporation, Madison, WI) following manufacturer’s protocol. The recombinant plasmids were purified using Quick ®Plasmid mini- prep kit (Invitrogen, Carlsbad, CA) following manufacturer’s methods and were sequenced at the Biotech Core Facility (Texas Tech University, Lubbock, TX). The sequence data was analyzed using BLAST to confirm its uniqueness to MAP. These recombinant plasmids were used as standards for RT-PCR.
The plasmid concentration was measured at 260 nm at a ratio of 260/280 nm using ND®-1000 spectrophotometer in the TTU Biotech Core Facility. Based on the concentration and the length of the recombinant plasmids, the number of plasmids in the solution was calculated and dilutions of 10, 100, 1000, and 10000 plasmids per microliter were prepared in 1X TE buffer.
These plasmid dilutions were used for constructing a standard curve for the quantification of MAP cells from mouse colon and liver tissue using RT- PCR. A 16 s rRNA sequence present in bacteria was used as the reference gene. The primer pair used for amplification of that sequence were universal primers (Forward 5′ CCA TGA AGT CGG AAT CGC TAG-3′; Reverse 5′- ACT CCC ATG GTG TGA CGG-3′).
PCR reactions were carried out in 25 μl using SuperScript® III Platinum Two step qRT- PCR kit with SYBR Green (Invitrogen; Carlsbad, CA). The reaction set up and the thermal cycling parameters were according to manufacturer’s instructions. The 7500 Real-Time PCR system (Applied Biosystems; Foster City, CA) at the TTU, Biotech Core Facility was used for real time detection of amplified dsDNA with SYBR Green. Melting curve analysis was also performed according to the instrument protocol. The experimental samples were divided into 4, 96 well plates. Every sample was run in triplicate. Each plate had non-template controls for ISMav2 primers and universal primers; quantification standards were recombinant plasmids with ISMav2 representative of cell numbers (1×105, 1×103, 1×102, and 1×101), experimental samples were evaluated with ISMav2 primers or universal primers. Specific amplification of target DNA was monitored by comparing the normalized reporter signal (SYBR Green) for a threshold cycle (Ct) and the signal obtained for controls.
Real time PCR analysis
Data were normalized and a One Way Analysis of Variance (ANOVA) was conducted; if the mean values among the treatment groups were greater than what would be expected by chance there was a significant difference (P < 0.05). A post-hoc, all pairwise multiple comparison procedure (Tukey Test) was performed for statistical analysis of significance.
Tissue cytokine transcript analysis
Files from the Luminex® and Open® Array analyses were parsed and organized into tab-delimited files using custom perl scripts. Values across multiple days and sexes were averaged to result in one value for each of 6 experimental conditions (Control, L-MAP, K-MAP, L-NP-51, K-MAP
L-NP-51 and L-MAP
L-NP-51). Targets (cytokines or transcripts) that gave reliable results above background were included in the final analysis. All values were normalized to control values and expressed as log base 2.
Gut microbiota analysis
For microbiota analysis, .sff files generated from 454 sequencing were demultiplexed, converted to .fastq files and resulting sequences were trimmed and mapped to 16S ribosomal DNA intergenic regions to classify the origin of the sequence. The methodology associated with 454 sequencing were conducted by Research and Laboratory Testing (Lubbock, TX) according to protocols previously developed and described by Dowd et al., [44
]. Sequencing data were deposited to GenBank short reads archive (SRA056455). The percent of sequences from each organism in each sample was normalized across all samples and final values were normalized to control and values were expressed as log base 2 of the difference between each sample and the control. A custom R script was written to perform a Pearson correlation between the relative abundance of each genus and relative abundance of each cytokine; geni with p-values of <0.05 in the Pearson and at least one cytokine from the Luminex® analysis were included in the final table, separated based on whether the r-value was positive (positive correlation) or negative (negative correlation).