Diversity of rumen bacterial urease gene
Rumen digesta samples were collected from four rumen-fistulated Chinese Holstein dairy cows before morning feeding. Total microbial DNA was extracted using the RBB + C method . A degenerate primer set specific for the ureC gene (ureC forward:5′-TGGGCCTTAARMTHCAYGARGAYTGGG-3′, and ureC reverse:5′-GTGRTGRCAMACCATNANCATRTC-3′)  was used in PCR amplification of the UreC in the rumen samples. A 25 μL PCR reaction contained 2.5 μL PCR buffer (Invitrogen, Carlsbad, CA), 0.75 μL MgCl2 (50 mM), 0.5 μL dNTP (10 mM), 1.5 μL each forward and reverse primer (10 μm), 0.3 μL Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), 1 μL rumen microbial DNA (~100 ng μL−1), and 16.95 μL sterile ddH2O. The PCR cycling included 94°C for 5 min; 30 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s; 72°C for 15 min; and 10°C for 30 min. The expected PCR amplicons of about 324 bp were visualized on agarose (2%) gel and then purified using a Gel Purification Kit (Qiagen, Valencia, CA), cloned into the pMD19-T vector (TaKaRa, Dalian, LN, China), and then transformed into competent E.coli JM109 cells (TaKaRa, Dalian, LN). Random clones were sequenced with the T7 primer using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Inc., Foster, CA). All sequences were trimmed to remove the vector regions and low-quality ends using the PREGAP4 program of the STADEN software package . The sequences were then compared to GenBank sequences using Blastx, and the most similar UreC sequences derived from known bacterial species were downloaded and combined with the UreC protein sequences recovered in this study. A phylogenetic tree was constructed from the combined UreC sequences using the MEGA software .
Expression and purification of urease alpha subunit (UreC)
The gene encoding the UreC of H. pylori was amplified from the genomic DNA of H. pylori UMAB41 using a forward primer (5′-AAAACATATGAAAAAGATTAGCAGGAAAG-3′) with a NdeI cutting site (italic) and a reverse primer (5′-CCGCTCGAGCTACCGCGCCATCTTCCACCAG-3′) with a XhoI cutting site (italic). Following double digestion with NdeI and XhoI, the purified full-length gene was ligated into correspondingly double-digested pET-30a(+) (Novagen, Madison, WI). The recombinant plasmids were then transformed into competent E.coli BL21 (DE3) (Promega, Madison, WI). The transformants were grown until an OD600 of 0.6, and over expression of the cloned UreC was induced at 30°C by IPTG (1.0 mM). The E. coli cells were harvested by centrifugation, and total cell protein was isolated using the BugBuster® Protein Extraction Reagent (Novagen, Madison, WI). Purification of the overexpressed UreC was achieved using a Ni-NTA kit (Novagen, Madison, WI) per manufacturer’s instructions. The UreC protein was analyzed by SDS-PAGE and visualized after staining with Coomassie Blue R-250. The UreC protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA).
Purification of rumen urease and western blotting using UreC of H. pylori
Rumen fluid was collected fistulated dairy cows fed a total mixed ration (TMR) (see Additional file 3). The bacterial cells were isolated by gradient centrifugation and then disrupted by ultrasonication (300 W, 15 min). The cellular proteins were concentrated using ultrafiltration (50 kDa) and then a Hi Trap Capto Q ion exchange column (GE Healthcare, Little Chalfont, UK) that was pre-equilibrated with a Tris–HCl buffer (20 mm, pH 8.0). Gradient elution was used to separate urease protein using the same Tris–HCl buffer with a NaCl concentration ranging from 0 to 1 M at a flow rate of 1 mL min−1. The fractions with positive urease activities were pooled and concentrated by lyophilization .
The urease protein was separated on SDS-PAGE and transferred to a nitrocellulose membrane (Sigma, St Louis, MO, USA) for immune blotting analysis. The membrane was blocked with 5% low-fat dry milk dissolved in TBS-T buffer for 1 h at room temperature and then stained overnight with the bovine anti-urease serum (1:1000). Following three washes for 10 min each in TBS-T buffer, the membrane was incubated in diluted (1:1000 diluted) horseradish peroxidase-conjugated sheep anti-bovine antibody (Bethyl Laboratories, Montgomery, TX) for 1 h at room temperature before incubation in 6 mL of chemiluminescence reagent (Sigma, St Louis, MO, USA) for 1 min. Positive immunostaining was determined based on the presence of a visible band corresponding to the expected UreC protein.
Immunization of dairy cows
Eight rumen-fistulated lactating Chinese Holstein dairy cows, with a body weight of 556 ± 19 kg, were randomly allocated to two treatment groups (n = 4), with one group (control) mock vaccinated with physiological saline containing the Freund’s adjuvant only, while the other group (vaccinated) was vaccinated with the overexpressed UreC of H. pylori. Briefly, an UreC protein solution (0.4 mg mL−1 UreC protein) was combined with an equal volume of Freund’s complete adjuvant. The mixture was emulsified, resulting in an UreC vaccine containing 0.2 mg mL−1 UreC protein. Each cow in the vaccinated group was injected subcutaneously on the neck and intramuscularly on the buttock  with 0.5 mL UreC vaccine at day 0. The injections were repeated at days 14, 28 and 42 as boosters, but with the Freund’s complete adjuvant being replaced by Freund’s incomplete adjuvant. The control group received the same injection procedures in parallel but with physiological saline containing Freund’s adjuvant only. All cows were housed under identical conditions and fed the same TMR (see Additional file 3) thrice daily. The animals were strictly cared for following the standard protocols approved specifically for this study by the Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China (Permit Number: RNL10/08).
Animal sample collection and analysis
At days 0, 7, 21, 35, and 49, samples of blood, saliva, and rumen fluid were collected about 2 h after morning feeding. Blood samples were collected from the caudal vein of each cow into evacuated tubes and centrifuged at 3000 × g for 10 min to separate the serum. Saliva samples were collected from the oral cavity of the cows using a suction tube and then centrifuged at 10000 × g for 15 min to collect the supernatant. Rumen fluid samples were collected through rumen fistula and filtered through four layers of cheesecloth. Subsamples of rumen fluid were also collected for analysis of urease activity. At day 56, 60 g urea was infused directly into the rumen of the cows of both groups through the rumen fistula after morning feeding. Rumen fluid was then collected at 0, 1, 2, 4, 6 and 8 h post infusion and analyzed for pH, ammonia concentration, and VFA profile.
The titers of specific anti-urease IgG and IgA in the serum and the saliva samples were determined using a modified ELISA protocol . Briefly, the plates were coated with 100 μL well−1 UreC solution (40 μg mL−1) and incubated overnight at 4°C. After washing, the plates were blocked with 150 μL well−1 of 1% (vol./vol.) chicken serum for 90 min at 37°C. An aliquot of 100 μL well−1 serially diluted serum (1:400 to 1:25600 for IgG, 1:500 to 1:32000 for IgA) and saliva (1:640 to 1:10240 for IgG, 1:20 to 1:640 for IgA) from either the vaccinated cows or the cows in the control group was added. Fetal calf serum was used as a negative control. The plates were incubated at 37°C for 2 h. Then, 100 μL donkey anti-bovine IgG or IgA alkaline phosphatase conjugate (Promega, Madison, WI) (1:10000 diluted) was added to each well and incubated at 37°C for 2 h. Following dilution to a final concentration of 1.5 mg mL−1 in a buffer containing 1 M diethanolamine and 0.5 mM MgCl2, the substrate chromogen tetramethylbenzidine (100 μL) was added to each well. After incubation for 30 min at 37°C, the reaction was terminated by adding 50 μL of 2 N NaOH to each well. The absorbance was recorded at 405 nm using an ELISA plate-reader (Infinite F200; Tecan, Mannedorf Switzerland). The reaction was defined as positive when the absorbance exceeded twice that of the negative control. Antibody titers were expressed as the highest dilution that gave a positive reaction.
Urease activity was determined by measuring the amount of ammonia released from urea . One unit of urease activity was defined as one μmol ammonia released per min per mL rumen fluid or mg microbial protein. Ammonia concentration was determined by the phenol-hypochlorite reaction as described by Weatherburn et al. . Concentrations of VFA were analyzed by gas chromatography (model 6890, Series II; Hewlett Packard Co., Avondale, PA) as described by Mohammed et al. .
Evaluation of the anti-urease antibody on urea hydrolysis in rumen fluid in vitro
The TMR diet, which was the same as that fed to the cows, was weighed into serum bottles (0.25 g bottle−1) containing 20 mL McDougall’s buffer, 10 mL of strained fresh rumen fluid, and urea (final concentration of 1 g L−1). To each bottle, bovine serum from either the control group or the vaccinated group was added. Two bovine serum concentrations from the vaccinated group was used in two treatments: Trt1 (IgG titer, 1:40000) and Trt2 (IgG titer, 1:80000). Three replicates were used both the control and the anti-urease serum treatments. The bottles were gassed with CO2, sealed with rubber stoppers, and incubated in a 39°C shaking water bath. Subsamples were collected at 0, 1, 2, 4, 8 and 12 h post incubation, and the pH was measured immediately. Ammonia concentrations were determined colorimetrically as described above. Urea concentration was determined with the diacetyl monoxime method of Marsh et al. . Rates of urea disappearance and concomitant ammonia N formation were computed as the slope of regression of the natural logarithm of urea and ammonia N concentration, respectively, over the course of the incubation.
All data were subjected to analysis of variance using the MIXED procedure of SAS (version 9.0, SAS Institute Inc., Cary, NC). The REPEATED statement was used for variables measured over days (titers of IgA and IgG, and urease activity) or times (pH, VFA, NH3-N). Tukey multiple comparison test was used to separate the means when significant differences were indicated by the MIXED procedure. Differences were considered significant at P < 0.05.
Nucleotide sequence accession numbers
The ureC sequences obtained in this study were deposited in the GenBank database under accession numbers JQ611755 to JQ612071.