Acute interdigital phlegmon or bovine foot rot is an infectious disease of cattle caused by mixed anaerobic bacterial colonization of the subcutis of the inter-digital tissues. The condition is characterized by local inflammation, swelling, and development of a necrotic lesion. Although the etiopathogenesis remains unclear, organisms traditionally implicated in foot rot infections include Bacteroides melaninogenicus, Fusobacterium necrophorum,1 and Dichelobacter nodosus.2 We have consistently isolated Porphyromonas levii, Bacteroides fragilis, and Prevotella spp from infected feedlot cattle,3 as determined by recent taxonomic reorganization of the Bacteroides species.4 All are known to be part of the indigenous flora of healthy animals but can become pathogenic when they invade other tissues.
Neutrophils infiltrate the site of infection3 and are a fundamental component of both the innate and adaptive segments of the immune response. Activated PMNs upregulate chemotactic activity, phagocytosis, respiratory burst, degranulation, and cytokine activity directed against foreign agents.5,6 Phagocytosis and destruction of bacteria by PMNs is vital to the clearance of pathogens from infected tissue and promotion of wound healing. Once a bacterium is engulfed, the PMN uses oxidative burst as a primary mechanism for microbial killing, a process that involves production of reactive oxygen species, including superoxide anion (O2−) and hydrogen peroxide (H2 O2).
Bacterial species contributing to foot rot infection have a wide range of virulence factors that enables evasion or alteration of host defenses. Short-chain fatty acids are byproducts of anaerobic bacterial metabolism that have been recognized as potential mediators of disease.7,8 Measurable SCFA concentrations are commonly found at sites of anaerobic bacterial infection.9 In periodontal infections, gingival SCFA concentrations increase in direct proportion to the severity of inflammation.10 It is possible that these small molecules are prime effectors of immune response modulation in bovine foot rot.
Short-chain fatty acids are known to affect PMN function in humans. Incubation of PMNs in high concentrations of succinate at low pH impairs phagocytosis, chemotaxis, and the respiratory burst.11 Exposure of PMNs to propionate induces intracellular calcium release in a manner similar to stimulation by chemotactic factors.12 Acetate, propionate, and butyrate are potential agonists for G-protein receptors, altering normal recruitment and activation processes.13
In addition to specific effects on host immune cell function, SCFAs decrease the ambient pH. The extra-cellular pH of an abscess may vary considerably, depending on the location and species of bacteria involved.14 To investigate the role of SCFAs as potential virulence factors during foot rot infection, we examined the effects of exposure to acetate, butyrate, propionate, and succinate on bovine PMN function in vitro.
Materials and Methods
Buffer and reagent preparation—Bis-Tris buffer (20mM Bis-Tris,a 44μMKH2 PO4, 3.4μMNa2 HPO4, 140mM NaCl, 5.3mM KCl, and 5.6mM glucosea) solution, with and without CaCl2 (1.2mM), MgCl2 (0.5mM), and MgSO4 (0.4mM), was referred to as BTB+ and BTB−, respectively, and was used for all assays. Sodium saltsa for acetate, butyrate, propionate, and succinate were used to prepare SCFA solutions in BTB. Stock solutions for each SCFA were made and adjusted to either pH 6.7 or 5.5. The sodium concentration was adjusted to maintain isotonicity. Working solutions were made as needed at 10, 26, 52, or 78mM for the monoprotic acids and at 4, 10, 21, or 31mM for the succinate. Opsonized zymosan was prepared with zymosan Aa as previously described,15 resuspended in BTB+ at pH 6.7 or 5.5, and stored at −86°C until needed. Phorbol 12-myristate 13-acetatea was dissolved in dimethyl sulfoxidea at a concentration of 5.4 mM and stored in aliquots at −20°C in darkness until needed.
Bacterial growth—Bacterial isolatesP levii (bovine foot rot isolate [BFR]7-5), Prevotella spp, (BFR91-3 and BRF91-5), B fragilis (BFR7-6), and F necrophorum (American Type Culture Collection 27852) were cultured in an anaerobic chamberb at 37°C in 5% CO2, 5% H2, and 90% N2.c All were cultured from frozen samples (stored at −86°C in 20% glycerol brain-heart infusion brothd) on laked blood kanamycin (75μg/mL)-vancomycin (7.5 μg/mL) medium.16 Single colonies were subcultured on anaerobic fastidious agare supplemented with 5% defibrinated ovine blood and incubated for 3 days. When needed, bacterial strains were inoculated in brain-heart infusion broth supplemented with resazurina (1 mg/L), hemina (5 mg/L), and vitamin Ka (1 mg/L).
High-performance liquid chromatography—Broth cultures of every strain were sampled 0, 24, 48, and 72 hours after inoculation, and aqueous SCFAs were extracted with ether, as has previously been described.17 The high-performance liquid chromatography assay was conducted with a 250 × 4.6-mm organic acids columnf with a 25mM KH2 PO4 mobile phase and a 1.0 mL/min flow rate. A dual-absorbance UV detectorg (220 nm) and a refractive index detector were used to identify SCFAs as they eluted. Samples were run at 60-minute intervals, and results were analyzed with commercially available software.h Broths with known amounts of SCFA were also extracted and used to create standard curves for quantification.18
PMN preparation—Blood was collected from mixed-breed beef cattle via jugular venipuncture into 8.5-mL tubesd containing citrate dextrose. The study was conducted according to protocols approved by the University of Calgary Animal Care Committee. After centrifugation at 1,400 × g for 20 minutes at 5°C, the buffy coat and plasma were removed and cold (4°C) Hanks balanced salt solution without calcium or magnesiumi was added to the remaining erythrocytes. Samples were centrifuged again at 1,400 × g for 10 minutes at 5°C, and any remaining buffy coat was removed along with the salt solution. Erythrocytes were hypotonically lysed 3 times by addition of lysing solution (10mM Na2 HPO4 and 2.7mM NaH2 PO4 at pH 7.2) for 1 minute, addition of an equal volume of restoring solution (lysing solution and 0.46M NaCl), and centrifugation in between (1,400 × g for 10 minutes at 5°C), after which PMNs were resuspended in Hanks balanced salt solution, pelleted, washed with BTB–, and resuspended in sterile BTB–. A small sample was removed, centrifugedj (at 200 × g for 10 minutes), and stainedk to assess PMN purity. Concentration was determined by use of a hemocytometer and trypan bluea exclusion staining to assess viability. Only preparations with viability > 95% and PMN cell purity > 95% were used in the study.
O2− and H2O2 evaluation—Two oxidative metabolism assays were used to assess respiratory burst activity in PMNs at pH 5.5 and 6.7. A dihydroethidiuml microfluorometric assay was used to measure O2− production, and the 2′,7′-dichlorofluorescein diacetatel microfluorometric assay was used to measure H2 O2 production.19,20 Black 96-well platesm were used for all fluorometric assays; edge wells were not used. Neutrophils (1 × 106 cells/mL) were exposed to either PMA (10μM) or opsonized zymosan (2.5 mg/mL) in the presence of 1 of the 4 SCFAs. A negative control (no SCFA and no stimulant), a positive control (no SCFA), and a blank well (no PMNs) were included in each assay. Either dihydroethidium (5 mg/mL) or 2′,7′-dichlorofluorescein diacetate (10 mg/mL) were added to each well, and the plates were incubated in darkness at 37°C for 1 hour and read with a fluorescence plate reader.n Ethidium was measured at an excitation wavelength of 490 nm and emission wavelength of 620 nm; dichlorofluorescein was measured at an excitation wavelength of 485 nm and emission wavelength of 530 nm.21 The relative fluorescence unit of each well was recorded, and the blank-well fluorescence was subtracted to account for fluorescence produced by decomposition of the dye. Data were normalized with a stimulation index derived by dividing the relative fluorescence unit of each well by the mean negative control fluorescence value.
Phagocytosis assay—Late logarithmic-phase P levii were opsonized by incubation with normal bovine serum for 30 minutes at 37°C.22 Pelleted cells were washed twice with BTB at the appropriate pH and resuspended to a final concentration of 5 × 107 bacteria/mL. An aliquot was taken from every sample, and the concentration was confirmed by serial dilution and culture. Phagocytosis assays were performed at both pH 6.7 and 5.5 with the same SCFA concentrations used in the oxidative metabolism assays. The opsonized bacterial suspension (1 × 107 cells/mL final volume) was added to the microfuge tubes along with the PMN suspension (1 × 106 cells/mL final volume) for a 1:10 PMN-to-bacterium ratio.22 Cells were incubated together for 1 hour at 37°C on a rotating platform and centrifuged at 600 × g for 5 minutes to pellet the PMNs. The supernatant was removed, and cells were washed with BTB+ and resuspended in BTB+. Aliquots were placed in a cytocentrifuge (200 × g for 10 minutes), and slides were stained and evaluated microscopically. Three hundred cells were counted per treatment group and classified as phagocytosing if they appeared to contain ≥ 1 intracellular bacterium.
Cellular assessment—The centrifuged preparations of PMNs were photographed with a digital camerao after the phagocytosis assays. Cytologic assessment was performed by use of double-blinded methodology, and 4 categories were defined on the basis of the severity of necrotic changes observed in PMNs, including fragmentation and swelling of nuclear chromatin, loss of membrane integrity, and cytoplasmic vacuolization. Category A PMNs underwent no necrotic change, whereas cells in categories B, C, and D had mild, moderate, and severe necrotic changes, respectively.
Statistical analysis— Data were analyzed with the Kruskal-Wallis multifactorial ANOVA. The Dunn test for multiple comparisons was used to determine significance. Values of P < 0.05 were considered significant.
Results
SCFA production—To determine whether foot rot–causing bacteria produced SCFAs in clinically relevant quantities, pure cultures of P levii, Prevotella spp, B fragilis, andF necrophorum were analyzed for acetate, butyrate, propionate, and succinate. All 5 isolates produced ≥ 3 of the SCFAs evaluated at concentrations as high as 25.3 ± 8.6mM, 25.4 ± 2mM, 10.4 ± 2.2mM, and 3.8 ± 0.5mM for acetate, butyrate, propionate, and succinate, respectively, after 72 hours of incubation (Table 1). Starting at an initial pH of 7.5, medium pH decreased by a minimum of 0.8 units (for F necrophorum) to a low of pH 5.8 (for B fragilis).
Mean ± SEM SCFA concentrations and pH in broth cultures of bacteria associated with foot rot in cattle prior to inoculation (0 hours) and after 72 hours of incubation with 4 SCFAs.
| Bacterial isolate | SCFA (mM) | ||||
|---|---|---|---|---|---|
| Acetate | Butyrate | Propionate | Succinate | pH | |
| Porphyromonas levii (BFR7-5) | |||||
| 0 hours | 1.0 ± 0.12 | 0 | 0 | 0.6 ± 0.35 | 7.5 |
| 72 hours | 25.3 ± 8.60 | 8.3 ± 5.30 | 1.5 ± 0.07 | 2.4 ± 0.08 | 6.2 |
| Prevotella sp (BFR91-3) | |||||
| 0 hours | 1.0 ± 0.14 | 0 | 0 | 0.3 | 7.5 |
| 72 hours | 6.1 ± 0.03 | 0 | 3.8 ± 1.33 | 2.3 ± 1.31 | 6.2 |
| Prevotella sp (BFR91-5) | |||||
| 0 hours | 1.0 ± 0.08 | 0 | 0 | 0.4 ± 0.04 | 7.5 |
| 72 hours | 7.8 ± 0.97 | 0 | 8.7 ± 1.50 | 1.2 ± 0.20 | 6.7 |
| Fusobacterium necrophorum (ATCC 27852) | |||||
| 0 hours | 1.1 ± 0.11 | 0 | 0 | 0.4 ± 0.02 | 7.5 |
| 72 hours | 12.9 ± 0.43 | 25.4 ± 2.03 | 10.4 ± 2.22 | 0.4 ± 0.09 | 6.7 |
| Bacteroides fragilis (BFR7-6) | |||||
| 0 hours | 0.7 ± 0.36 | 0 | 0 | 0.1 ± 0.10 | 7.5 |
| 72 hours | 14.0 ± 0.63 | 0 | 8.7 ± 0.30 | 3.8 ± 0.51 | 5.8 |
BFR = Bovine foot rot isolate. ATCC = American Type Culture Collection.
Data were obtained from samples of each bacterial supernatant that were analyzed independently on 2 occasions.
Oxidative burst and phagocytosis—Results for the oxidative burst assays were adjusted by use of the calculated stimulation index to normalize the data and account for variation among PMN isolations. Assay results for all SCFAs examined were similar; therefore, results described here apply to all 4 SCFAs unless otherwise noted. Superoxide anion production in PMA-activated PMNs incubated with SCFAs at pH 6.7 did not change significantly from that in positive controls at any concentration. However, under the same conditions at pH 5.5, O2− decreased by a mean of 85% as SCFA concentration increased (Figure 1). These concentrations were significant (P < 0.05) and approached values near those of unstimulated cells.
Box-and-whisker plots depicting the stimulation index for the effects of incubating PMA-stimulated bovine PMNs with various concentrations of butyrate at pH 5.5 and 6.7 on superoxide anion (O2−) production. Data were derived from samples collected from 3 cows on 5 occasions. The stimulation index was derived by dividing the relative fluorescence unit for each well by the mean fluorescence in negative control wells. Boxes indicate interquartile range, horizontal lines indicate median, and whiskers indicate range. *Significantly (P < 0.05) different from negative controls. †Significantly (P < 0.05) different from positive controls. aSignificantly (P < 0.05) different from values obtained with 78mM butyrate at pH 6.7. bSignificantly (P < 0.05) different from values obtained with 52mM butyrate at pH 6.7. cSignificantly (P < 0.05) different from values obtained with 26mM butyrate at pH 6.7.dSignificantly (P < 0.05) different from values obtained with 10mM butyrate at pH 6.7. eSignificantly (P < 0.05) different from values obtained with 78mM butyrate at pH 5.5.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Box-and-whisker plots depicting the stimulation index for the effects of incubating PMA-stimulated bovine PMNs with various concentrations of butyrate at pH 5.5 and 6.7 on superoxide anion (O2−) production. Data were derived from samples collected from 3 cows on 5 occasions. The stimulation index was derived by dividing the relative fluorescence unit for each well by the mean fluorescence in negative control wells. Boxes indicate interquartile range, horizontal lines indicate median, and whiskers indicate range. *Significantly (P < 0.05) different from negative controls. †Significantly (P < 0.05) different from positive controls. aSignificantly (P < 0.05) different from values obtained with 78mM butyrate at pH 6.7. bSignificantly (P < 0.05) different from values obtained with 52mM butyrate at pH 6.7. cSignificantly (P < 0.05) different from values obtained with 26mM butyrate at pH 6.7.dSignificantly (P < 0.05) different from values obtained with 10mM butyrate at pH 6.7. eSignificantly (P < 0.05) different from values obtained with 78mM butyrate at pH 5.5.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Box-and-whisker plots depicting the stimulation index for the effects of incubating PMA-stimulated bovine PMNs with various concentrations of butyrate at pH 5.5 and 6.7 on superoxide anion (O2−) production. Data were derived from samples collected from 3 cows on 5 occasions. The stimulation index was derived by dividing the relative fluorescence unit for each well by the mean fluorescence in negative control wells. Boxes indicate interquartile range, horizontal lines indicate median, and whiskers indicate range. *Significantly (P < 0.05) different from negative controls. †Significantly (P < 0.05) different from positive controls. aSignificantly (P < 0.05) different from values obtained with 78mM butyrate at pH 6.7. bSignificantly (P < 0.05) different from values obtained with 52mM butyrate at pH 6.7. cSignificantly (P < 0.05) different from values obtained with 26mM butyrate at pH 6.7.dSignificantly (P < 0.05) different from values obtained with 10mM butyrate at pH 6.7. eSignificantly (P < 0.05) different from values obtained with 78mM butyrate at pH 5.5.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Similar results were obtained when H2 O2 production by PMA-stimulated PMNs was assayed. At pH 6.7, SCFAs had no effect on the ability of PMNs to respond to the positive control. However, at the lower pH, cells' production of H2 O2 was impaired, declining by a mean of 80% as SCFA concentration increased (Figure 2).
Box-and-whisker plots depicting the stimulation index for the effects of incubating PMA-stimulated bovine PMNs with various concentrations of succinate at pH 5.5 and 6.7 on H2 O2 production. *Significantly (P < 0.05) different from negative controls. †Significantly (P < 0.05) different from the positive controls and from values obtained with 4 and 10mM butyrate at pH 6.7.aSignificantly (P < 0.05) different from values obtained with 21 and 31mM butyrate at pH 6.7
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Box-and-whisker plots depicting the stimulation index for the effects of incubating PMA-stimulated bovine PMNs with various concentrations of succinate at pH 5.5 and 6.7 on H2 O2 production. *Significantly (P < 0.05) different from negative controls. †Significantly (P < 0.05) different from the positive controls and from values obtained with 4 and 10mM butyrate at pH 6.7.aSignificantly (P < 0.05) different from values obtained with 21 and 31mM butyrate at pH 6.7
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Box-and-whisker plots depicting the stimulation index for the effects of incubating PMA-stimulated bovine PMNs with various concentrations of succinate at pH 5.5 and 6.7 on H2 O2 production. *Significantly (P < 0.05) different from negative controls. †Significantly (P < 0.05) different from the positive controls and from values obtained with 4 and 10mM butyrate at pH 6.7.aSignificantly (P < 0.05) different from values obtained with 21 and 31mM butyrate at pH 6.7
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Opsonized zymosan was the second stimulant used to assess the oxidative burst response. At pH 6.7, O2− production was similar to that of PMA-activated PMNs in that no meaningful influence on function was observed. The only exception was observed with exposure to succinate, which caused a significant dose-dependent decrease in O2− production at pH 6.7, decreasing nearly 85% (Figure 3). At pH 5.5, the ability of opsonized zymosan to activate PMNs was substantially reduced with regard to O2− production and no meaningful trends could be measured for any treatment.
Box-and-whisker plots depicting the stimulation index for the effects of incubating opsonized zymosan–stimulated bovine PMNs with various concentrations of succinate at pH 6.7 on O2− production. *Significantly (P < 0.05) different from negative controls. †Significantly (P < 0.05) different from positive controls. aSignificantly (P < 0.05) different from values obtained with 31mM succinate.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Box-and-whisker plots depicting the stimulation index for the effects of incubating opsonized zymosan–stimulated bovine PMNs with various concentrations of succinate at pH 6.7 on O2− production. *Significantly (P < 0.05) different from negative controls. †Significantly (P < 0.05) different from positive controls. aSignificantly (P < 0.05) different from values obtained with 31mM succinate.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Box-and-whisker plots depicting the stimulation index for the effects of incubating opsonized zymosan–stimulated bovine PMNs with various concentrations of succinate at pH 6.7 on O2− production. *Significantly (P < 0.05) different from negative controls. †Significantly (P < 0.05) different from positive controls. aSignificantly (P < 0.05) different from values obtained with 31mM succinate.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
When H2 O2 production was measured with opsonized zymosan as the stimulant, the PMN response differed depending on the SCFA tested. At pH 6.7, as acetate, butyrate, and propionate concentrations increased, H2 O2 production increased significantly, by a mean of > 25 times (Figure 4). Conversely, succinate exposure decreased H2 O2 production by 40% under the same conditions. In contrast, at pH 5.5, monoprotic SCFAs caused a mean 65% decrease in PMN H2 O2 production (Figure 5), a change that was significant. Succinate exposure had no significant effect on H2 O2 production at either pH in zymosan-stimulated PMNs.
Box-and-whisker plots depicting the stimulation index for the effects of incubating opsonized zymosan–stimulated bovine PMNs with various concentrations of acetate at pH 6.7 on H2 O2 production. See Figure 3 for key.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Box-and-whisker plots depicting the stimulation index for the effects of incubating opsonized zymosan–stimulated bovine PMNs with various concentrations of acetate at pH 6.7 on H2 O2 production. See Figure 3 for key.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Box-and-whisker plots depicting the stimulation index for the effects of incubating opsonized zymosan–stimulated bovine PMNs with various concentrations of acetate at pH 6.7 on H2 O2 production. See Figure 3 for key.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Box-and-whisker plots depicting the stimulation index for the effects of incubating opsonized zymosan–stimulated bovine PMNs with various concentrations of butyrate at pH 5.5 on H2 O2 production. aSignificantly (P < 0.05) different from values obtained with 10mM butyrate. bSignificantly (P < 0.05) different from values obtained with 26mM butyrate. See Figure 3 for remainder of key.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Box-and-whisker plots depicting the stimulation index for the effects of incubating opsonized zymosan–stimulated bovine PMNs with various concentrations of butyrate at pH 5.5 on H2 O2 production. aSignificantly (P < 0.05) different from values obtained with 10mM butyrate. bSignificantly (P < 0.05) different from values obtained with 26mM butyrate. See Figure 3 for remainder of key.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Box-and-whisker plots depicting the stimulation index for the effects of incubating opsonized zymosan–stimulated bovine PMNs with various concentrations of butyrate at pH 5.5 on H2 O2 production. aSignificantly (P < 0.05) different from values obtained with 10mM butyrate. bSignificantly (P < 0.05) different from values obtained with 26mM butyrate. See Figure 3 for remainder of key.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Regardless of the stimulant used, extracellular pH had a marked effect on activation, independent of SCFA: when PMNs were stimulated with opsonized zymosan, there was a roughly 6-fold increase in O2− and H2 O2 production at the 2 pHs across all assays. Conversely, when cells were stimulated with PMA, there was no difference in O2− production at pH 6.7 or 5.5, but H2 O2 production was a mean of 20 times less at pH 5.5.
At pH 6.7, there was a significant dose-dependent increase in phagocytosis of P levii when cells were exposed to monoprotic SCFA, with the mean number of phagocytes more than doubling (Figure 6). Succinate increased phagocytosis at pH 6.7 but not significantly. In contrast, at the lower pH, SCFA exposure inhibited phagocytosis by a mean of 50%. Phagocytosis activity had the same variability in response to extracellular pH, independent of exposure to SCFAs. At pH 6.7, the mean number of phagocytosing PMNs was 56 per 300 cells counted. This was significantly different from mean phagocytosis at pH 5.5 of 37 per 300 cells counted, a 34% decrease.
Box-and-whisker plots depicting the number of bacteria phagocytosed for the effects of incubating bovine PMNs with various concentrations of acetate at pH 6.7 and 5.5 on phagocytosis (n = 45 for pH 6.7 positive controls, n = 29 for pH 5.5 positive controls, and n = 9 for treatment groups). Data were derived from samples collected from 3 cows on 3 occasions. †Significantly (P< 0.05) different from positive controls. aSignificantly (P < 0.05) different from values obtained with 10mM acetate at pH 6.7.bSignificantly (P < 0.05) different from values obtained with 26mM acetate at pH 6.7.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Box-and-whisker plots depicting the number of bacteria phagocytosed for the effects of incubating bovine PMNs with various concentrations of acetate at pH 6.7 and 5.5 on phagocytosis (n = 45 for pH 6.7 positive controls, n = 29 for pH 5.5 positive controls, and n = 9 for treatment groups). Data were derived from samples collected from 3 cows on 3 occasions. †Significantly (P< 0.05) different from positive controls. aSignificantly (P < 0.05) different from values obtained with 10mM acetate at pH 6.7.bSignificantly (P < 0.05) different from values obtained with 26mM acetate at pH 6.7.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Box-and-whisker plots depicting the number of bacteria phagocytosed for the effects of incubating bovine PMNs with various concentrations of acetate at pH 6.7 and 5.5 on phagocytosis (n = 45 for pH 6.7 positive controls, n = 29 for pH 5.5 positive controls, and n = 9 for treatment groups). Data were derived from samples collected from 3 cows on 3 occasions. †Significantly (P< 0.05) different from positive controls. aSignificantly (P < 0.05) different from values obtained with 10mM acetate at pH 6.7.bSignificantly (P < 0.05) different from values obtained with 26mM acetate at pH 6.7.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Cytologic assessment—The appearance of phagocytosing PMNs incubated at pH 5.5 was noticeably different from those incubated at pH 6.7 (Figure 7). All PMNs incubated at pH 6.7 as well as the pH 5.5 controls were classified category A. Of the SCFA-treated PMNs at pH 5.5, category B encompassed PMNs incubated with 4mM succinate or 10mM butyrate, propionate, or acetate. Cells incubated with 26mM butyrate, propionate, or acetate and 21 or 31mM succinate were classified in category C. Only PMNs exposed to the 2 highest concentrations of butyrate, propionate, or acetate had characteristics of those in category D.
Photomicrographs of PMNs from centrifuged preparations representing 4 categories of severity of changes associated with cell necrosis after incubation with 4 SCFAs at pH 5.5 and pH 6.7. A—Typical cell in category A with no changes. Notice that nuclei are well defined and segmented, with heterogeneous staining of the chromatin. Cytoplasmic membranes are also well defined and the margin is regular. B—Typical cell in category B with mild changes. Notice that the nucleus is swollen and fragmented with hyperchromatic chromatin (white arrowhead) at the margins of the fragments. The nuclear membrane is mostly intact. The cytoplasmic membrane is irregular, and the cytoplasm is noticeably vacuolated (black arrow). C—Typical cell in category C. Notice that the nucleus is swollen and fragmented with hyperchromatic chromatin at the margin of the fragments, and there is loss of integrity of the nuclear membrane in some areas (asterisk). The cytoplasmic membrane is irregular, and the cytoplasm is vacuolated. D—Typical cell in category D. Notice that the nuclear membrane is almost completely disrupted with dispersal of chromatin in the cells. The cytoplasmic membrane is irregular or disrupted (double arrowheads), and the cytoplasm is vacuolated. H&E stain; bar = 3 μm.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Photomicrographs of PMNs from centrifuged preparations representing 4 categories of severity of changes associated with cell necrosis after incubation with 4 SCFAs at pH 5.5 and pH 6.7. A—Typical cell in category A with no changes. Notice that nuclei are well defined and segmented, with heterogeneous staining of the chromatin. Cytoplasmic membranes are also well defined and the margin is regular. B—Typical cell in category B with mild changes. Notice that the nucleus is swollen and fragmented with hyperchromatic chromatin (white arrowhead) at the margins of the fragments. The nuclear membrane is mostly intact. The cytoplasmic membrane is irregular, and the cytoplasm is noticeably vacuolated (black arrow). C—Typical cell in category C. Notice that the nucleus is swollen and fragmented with hyperchromatic chromatin at the margin of the fragments, and there is loss of integrity of the nuclear membrane in some areas (asterisk). The cytoplasmic membrane is irregular, and the cytoplasm is vacuolated. D—Typical cell in category D. Notice that the nuclear membrane is almost completely disrupted with dispersal of chromatin in the cells. The cytoplasmic membrane is irregular or disrupted (double arrowheads), and the cytoplasm is vacuolated. H&E stain; bar = 3 μm.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Photomicrographs of PMNs from centrifuged preparations representing 4 categories of severity of changes associated with cell necrosis after incubation with 4 SCFAs at pH 5.5 and pH 6.7. A—Typical cell in category A with no changes. Notice that nuclei are well defined and segmented, with heterogeneous staining of the chromatin. Cytoplasmic membranes are also well defined and the margin is regular. B—Typical cell in category B with mild changes. Notice that the nucleus is swollen and fragmented with hyperchromatic chromatin (white arrowhead) at the margins of the fragments. The nuclear membrane is mostly intact. The cytoplasmic membrane is irregular, and the cytoplasm is noticeably vacuolated (black arrow). C—Typical cell in category C. Notice that the nucleus is swollen and fragmented with hyperchromatic chromatin at the margin of the fragments, and there is loss of integrity of the nuclear membrane in some areas (asterisk). The cytoplasmic membrane is irregular, and the cytoplasm is vacuolated. D—Typical cell in category D. Notice that the nuclear membrane is almost completely disrupted with dispersal of chromatin in the cells. The cytoplasmic membrane is irregular or disrupted (double arrowheads), and the cytoplasm is vacuolated. H&E stain; bar = 3 μm.
Citation: American Journal of Veterinary Research 67, 11; 10.2460/ajvr.67.11.1901
Discussion
Short-chain fatty acids produced by bacteria such as P levii, Prevotella spp,F necrophorum, and B fragilis could be important factors in preventing efficient clearance of infection by PMNs in some cattle. Results of previous studies indicate that certain SCFAs alter PMN function in vitro in humans, but bovine PMNs can differ substantially from human PMNs.23 The impact of SCFAs on bovine PMN microbicidal function, such as O2− or H2 O2 production and phagocytosis, has not previously been examined to our knowledge.
Because information regarding in vivo concentrations of SCFAs in affected bovine tissue is unavailable, production of SCFAs in broth culture by anaerobes isolated from foot rot infections or commonly associated with foot rot was measured after 72 hours of incubation, along with associated changes in pH. Observed values were not unexpected because several in vivo and in vitro studies have revealed comparable concentrations. Concentrations of 8mM for butyrate,9 10mM for propionate,24 31 to 69mM for succinate,9,25 and 872mM for acetate25 have been reported at sites of anaerobic bacterial infection in humans.Bacteroides fragilis cultured in vitro produces SCFAs in concentrations from 0.1 to 46mM, although differences in observed SCFA concentrations in vitro can be attributed, at least in part, to different culture media.26 Moreover, the pH of in vitro culture supernatants does not necessarily reflect the pH at sites of infection, which may vary considerably depending on the site of infection, nutrients available, and bacterial species involved.27 However, it is reasonable to infer that bacteria associated with foot rot are capable of generating SCFAs in millimolar concentrations at sites of infection and that the pH of the milieu is subsequently decreased.
Two discrepancies were observed in the PMN responses relating to the stimulant used for activation. First, SCFA solutions with a pH of 5.5 caused no mean reduction in PMN O2− production relative to solutions of pH 6.7 when PMA was used as the stimulant. However, when opsonized zymosan was used under the same conditions, the reduction was so substantial at pH 5.5 that it could not be quantified meaningfully. Secondly, although SCFA concentration had no effect on H2 O2 production at pH 6.7 in PMA-stimulated cells, SCFA exposure markedly increased H2 O2 production in zymosan-stimulated cells under the same conditions. These results reflect profound differences in the stimulatory mechanisms of these substances23 and highlight the importance of experimental design and use of positive controls when conducting this type of in vitro study.
Bovine PMN function was impaired under conditions of extracellular acidosis, indicated by decreases in the stimulation index and number of bacteria phagocytosed at pH 5.5, compared with those variables at pH 6.7. Indeed, at pH 5.5, inhibition of respiratory burst activity may overshadow any dose-dependent effects of SCFAs, as the PMN response in those assays was markedly suppressed, and at high SCFA concentrations, H2 O2 production decreased below that of negative controls. Multiple pH-dependent effects have been reported in human PMNs,28 including modulation of the oxidative burst and phagocytosis. Most effects can be attributed to a shift in intracellular pH. Undissociated SCFAs can traverse plasma membranes and dissociate in the cytoplasm, thereby lowering intracellular pH and inhibiting PMN function more than would a decrease in extracellular pH alone.24,29 At pH 6.7, most SCFAs in solution are in an ionized state; however, at pH 5.5, SCFAs in solution would be nearly 50% undissociated.
Neutrophils not only had dose-dependent variation in the scope of microbicidal activity when exposed to SCFAs but the trends were different from and sometimes opposite to those observed at the other pH. A distinct difference between the effects of monoprotic and diprotic SCFAs also emerged. Whereas the monoprotic SCFAs had no inhibitory effect on O2− or H2 O2 production at pH 6.7, succinate did inhibit their production when PMNs were stimulated with opsonized zymosan. Succinate may inhibit both O2− and H2 O2 production independently or only O2− production, thereby limiting H2 O2 production. Either way, results suggested that there is a PMN inhibitory role specific to succinate, supporting previous findings29–31 by other investigators working with human PMNs.
As concentrations of acetate, propionate, or butyrate increased, there was an increase in H2 O2 production when PMNs were stimulated with opsonized zymosan. In human PMNs stimulated with either PMA or opsonized zymosan, butyrate increased H2 O2 production.19 This was attributed to inhibition of H2 O2-degrading enzymes such as myeloperoxidase because there was no concomitant increase in O2− production. If these findings held true for bovine PMNs (that in the presence of SCFAs, H2 O2 is not easily converted to hypochlorous acid), they could explain the increase in H2 O2 observed in the present study. Acetate, butyrate, and propionate may adversely affect PMN function by restricting destructive activities because H2 O2 is not as toxic to bacteria as hypochlorous acid.32
At pH 6.7, monoprotic SCFAs appeared to stimulate PMN phagocytosis in vitro, whereas all 4 SCFAs inhibited PMN phagocytosis at pH 5.5. This finding contradicted results of a previous study33 in which SCFA exposure decreased phagocytosis by human PMNs. However, it is difficult to draw conclusions because factors such as solution tonicity and pH were not controlled in the earlier study.
It was apparent that SCFAs affected PMN cell morphology dramatically when incubated at pH 5.5, compared with the effects at pH 6.7. Necrosis, characterized by uncontrolled loss of membrane integrity and release of cellular contents, can injure host tissue secondary to exposure to cytotoxic molecules that are normally reserved for destroying bacteria.5 The ability of SCFAs to induce necrotic changes in bovine PMNs, most likely through their effects on intracellular pH, may increase tissue necrosis as well as exacerbate and prolong foot rot infection.34
In the present study, acetate, butyrate, and propionate affected bovine PMN function at pH 6.7 by increasing H2 O2 concentration and phagocytosis of the foot rot isolate P levii. Succinate appeared to inhibit both O2− and H2 O2 production in bovine PMNs at pH 6.7. At pH 5.5, the PMN response to SCFAs was much different; all 4 SCFAs evaluated inhibited phagocytosis and respiratory burst activity in that environment. In addition to decreasing the pH of the microenvironment at the site of foot rot infection, SCFAs may also impair bovine PMN function through mechanisms such as decreasing PMN intracellular pH or inhibiting myeloperoxidase activity. Even if the pH in the abscess is not extremely low, monoprotic acids may suppress PMN function by inhibiting efficient killing or, if succinate is present, by suppressing production of O2− and H2 O2. Whether acting directly or indirectly via their effect on pH in the microenvironment, SCFA-mediated PMN dysfunction could contribute to the persistence and proliferation of anaerobic bacteria in foot rot infections. Production of SCFAs in SC interdigital tissue in cattle may, under certain conditions, allow anaerobic bacteria to establish microcolonies that persist because of impaired detection and elimination functions in innate immune cells.
ABBREVIATIONS
| PMN | Polymorphonucleocyte |
| SCFA | Short-chain fatty acid |
| BTB | Bis-Tris buffer |
| PMA | Phorbol 12-myristate 13-acetate |
Sigma-Aldrich Co, St Louis, Mo.
Bactron II anaerobic chamber, Sheldon Manufacturing Inc, Cornelius, Ore.
Praxair Products Inc, Mississauga, ON, Canada.
Becton, Dickinson and Co, Sparks, Md.
Med-Ox Diagnostics Inc, Ottawa, ON, Canada.
Prevail Organic Acid 5u, Alltech Associates Inc, Deerfield, Ill.
Waters 2487 UV detector, Waters Corp, Milford, Mass.
GraphPad Prism, version 4, GraphPad Software Inc, San Diego, Calif.
Mediatech Inc, Herndon, Va.
Shandon Cytospin, Thermo Electron Corp, Waltham, Mass.
Dade Behring Inc, Newark, Del.
Molecular Probes, Invitrogen Canada Inc, Burlington, ON, Canada.
E&K Scientific, Santa Clara, Calif.
SpectraMax Gemini, Molecular Devices Corp, Sunnyvale, Calif.
DFC320 digital camera, Leica Microsystems (Canada) Inc, Richmond Hill, ON, Canada.
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