Antimicrobial efficacy of an innovative emulsion of medium chain triglycerides against canine and feline periodontopathogens.

OBJECTIVES: To test the in vitro antimicrobial efficacy of a non-toxic emulsion of free fatty acids against clinically relevant canine and feline periodontopathogens METHODS: Antimicrobial kill kinetics were established utilising an alamarBlue(®) viability assay against 10 species of canine and feline periodontopathogens in the biofilm mode of growth at a concentration of 0·125% v/v medium chain triglyceride (ML:8) emulsion. The results were compared with 0·12% v/v chlorhexidine digluconate and a xylitol-containing dental formulation. Mammalian cellular cytotoxicity was also investigated for both the ML:8 emulsion and chlorhexidine digluconate (0·25 to 0·0625% v/v) using in vitro tissue culture techniques. RESULTS: No statistically significant difference was observed in the antimicrobial activity of the ML:8 emulsion and chlorhexidine digluconate; a high percentage kill rate (>70%) was achieved within 5 minutes of exposure and was maintained at subsequent time points. A statistically significant improvement in antibiofilm activity was observed with the ML:8 emulsion compared with the xylitol-containing formulation. The ML:8 emulsion possessed a significantly lower (P < 0·001) toxicity profile compared with the chlorhexidine digluconate in mammalian cellular cytotoxicity assays. CLINICAL SIGNIFICANCE: The ML:8 emulsion exhibited significant potential as a putative effective antimicrobial alternative to chlorhexidine- and xylitol- based products for the reduction of canine and feline periodontopathogens.

Ultrastructure and electrolyte transport of the epithelium of coprodeum, colon and the proctodeal diverticulum of Rhea americana.

The structure and function of the lower intestinal tract of Rhea americana were characterized to evaluate the evolutionary relationship to other struthioniform and avian species. In 5 rheas the gross anatomy and the light and transmission electron microscopy were studied in parallel to in vitro electrophysiological measurements of ion transport. The mucosa in the colon was amplified with villi, often branched, and in the coprodeum with folds. In both tissues the epithelium was a monolayer composed of columnar absorptive cells, goblet cells and mitochondria-rich cells. Colon and coprodeum appeared to produce large amounts of mucus. The proctodeal diverticulum was rich in lymphoid tissue arranged into lobuli bursales, and it was concluded that this structure is a modified bursa of Fabricius. The sparse interlobular epithelium of the diverticulum resembled that of colon and coprodeum. Baseline short circuit currents (I(SC)) averaged 114.5+/-13.8 microA/cm(2) in colon, 193.1+/-30.3 microA/cm(2) in coprodeum and 60.4+/-9.1 microA/cm(2) in the diverticulum. Amiloride sensitive Na+-transport amounted to 31, 88 and 38% of the baseline I(SC) in these three tissues, respectively. In all tissues, there was also a modest, theophylline activated chloride secretion response, and ouabain, the Na+/K+-ATPase inhibitor, abolished most of the I(SC). The transepithelial resistance (TER) of the diverticulum was much higher than the other tissues. Upon dissection, urate from ureteral urine was observed in the lower third of the colon and to a lesser extent in the proctodeal diverticulum, indicating retrograde peristalsis of the urine. Thus, unlike the ostrich, there is no sphincter separating colon and coprodeum. On the other hand, a thick mucus layer was seen overlying the mucosa in both colon and coprodeum, as in the ostrich. This may help to prevent osmotic water loss, despite the presence of hyperosmotic urine (up to 800 mOsm) in the lower intestine. Both morphological and electrophysiological data from the rhea support the hypothesis that the rhea lower intestine contributes to post-renal modification of ureteral urine and to the regulation of osmotic balance, as also seen in domestic fowl and other avian species. The proctodeal diverticulum functions mainly as an immune organ, with only limited transport capability.

Aldosterone suppresses expression of an avian colonic sodium-glucose cotransporter.

Transport in the colon of the domestic fowl switches from sodium-linked hexose and amino acid cotransport on high-salt intake to amiloride-sensitive sodium channel expression on low-salt (LS) diets. The present experiments were designed to investigate the role of aldosterone in suppression of the colonic sodium-glucose luminal cotransporter (SGLT). LS-adapted hens were resalinated with or without simultaneous aldosterone treatment. Changes in the electrophysiological responses and SGLT protein expression levels were examined at 1, 3, and 7 days of treatment. Serum aldosterone levels fell from approximately 400 pmol/l in LS-adapted hens to values below the detection limit (<44 pmol/l) after 1 day of resalination. At the same time, glucose-stimulated short circuit current (I(SC)) increased from 20.9 +/- 8.7 to 56.3 +/- 15.5 microA/cm(2), whereas amiloride-sensitive I(SC) decreased from -68.9 +/- 12.7 microA/cm(2) on LS to +0.6 +/- 12.0 microA/cm(2). Glucose-stimulated I(SC) increased further at 3 and 7 days of resalination, whereas amiloride-sensitive I(SC) remained suppressed. When resalinated birds were simultaneously treated with aldosterone, the LS pattern of high amiloride-sensitive I(SC) and low glucose-stimulated I(SC) was maintained. Immunoblotting results from the same tissues demonstrated that SGLT-like protein expression increased following resalination. Aldosterone treatment completely blocked this effect. These results demonstrate that aldosterone suppresses both activity and protein expression of hen colonic SGLT. Resalination either through decreased aldosterone or other factors may be able to activate SGLT activity independently of increases in protein expression.

Characterization of a primary cell culture model of the avian renal proximal tubule.

Methods have been developed for producing functional, transporting monolayers of avian proximal tubule (PT) cells. A highly homogenous fraction of PT fragments was prepared by enzymatic digestion (collagenase + Dispase) of chick (3- to 5-day-old) kidneys, followed by Percoll gradient centrifugation. The PT fraction was enriched in glucose-6-phosphatase, a proximal enzyme marker, and reduced in specific activity of hexokinase, a distal marker. PT fragments were grown to confluence in serum-free media on collagen-coated permeable filter supports. Electron microscopy of confluent monolayers revealed numerous microvilli and mitochondria, central cilia, and tight junctions, all characteristic of PT cells. gamma-Glutamyltranspeptidase, a proximal brush-border enzyme, showed threefold higher activity on apical than on basolateral sides of the monolayer. The electrophysiological characteristics of monolayers were investigated by voltage-clamp techniques. Monolayers displayed low transepithelial resistances (40-60 Omega . cm2), lumen-negative potentials, and baseline currents of 6-12 microA/cm2 (with or without 5 mM glucose). Both alpha-methyl-D-glucose (2 mM), a nonmetabolizable hexose, and phenylalanine (2 mM) significantly stimulated short-circuit current when added to the mucosal side of glucose-free monolayers. Phloridzin, a specific inhibitor of Na+-coupled glucose transport, significantly inhibited short-circuit current, as did 10(-5) M amiloride. Monolayers also expressed net secretory transport of urate. This cell culture preparation may provide a useful working model for the study of avian PT transport.

Transport characteristics of the colonic epithelium of the Japanese quail (Coturnix coturnix).

The colon of the domestic fowl sustains a reabsorptive Na+ current on both high- and low-sodium diets. However, there is a marked shift in the apical transport step under these two extreme conditions, from amino acid/hexose cotransport on high-salt diets to amiloride-sensitive Na+ channels on low-salt diets. The present experiments were performed to study colonic Na+ transport in another galliform species, the Japanese quail (Coturnix coturnix). Birds were maintained on a commercial game feed containing 0.18% Na+ (78 mumoles/g), an intermediate level of salt intake. Experiments were performed on unstripped colons in standard Ussing chambers with bicarbonate/CO2 buffer solution on both sides. Baseline values (n = 11) for PD (3.13 +/- 0.68 mV) and short circuit current (SCC, 30.87 +/- 7.79 microA/cm2) were lower than those reported for chickens on a similar diet, whereas tissue resistance (76.06 +/- 4.19 omega.cm2) was similar. Addition of amino acids (4 mM leucine + lysine) increased SCC by 10.85 +/- 1.97 microA/cm2. Both phloridzin (1 mM) and amiloride (10(-5) M) decreased SCC, by 7.05 +/- 1.26 and 9.64 +/- 2.68 microA/cm2, respectively. Thus, on this diet the quail colonic epithelium maintains both amino acid/hexose cotransporter activity and amiloride sensitive channel activity. Arginine vasotocin (10(-6) M) caused a small, but consistent decrease in SCC, while acetazolamide increased SCC. Aldosterone (128 micrograms/kg), given 4 hr prior to the experiment (n = 4) significantly reduced the amino acid stimulated SCC. These results confirm, for the Japanese quail, the presence of multiple apical Na+ entry mechanisms in colonic epithelium. Amino acid cotransporter activity, in particular, appears to be highly sensitive to aldosterone suppression.

Mucosal acidification and an acid microclimate in the hen colon in vitro.

Experiments were performed on isolated, stripped colonic epithelia of low-salt-adapted hens (Gallus domesticus) in order to characterize acid secretion by this tissue. With symmetric, weak buffer solutions, colonic epithelia acidified both mucosal and serosal sides. Titration measurements of the mucosal acidification rate (pH-stat technique) averaged 1.63 +/- 0.25 microEq.cm-2.h-1. Mucosal acidification was also evident in colons from high-salt-adapted birds and in low-salt-adapted coprodeum, but was completely abolished in the high-salt coprodeum. Mucosal acidification by low-salt-adapted colonic epithelium was unaffected by sodium replacement, mucosal amiloride (10(-3) mol.l-1), and serosal ouabain (5 x 10(-4) mol.l-1), although all three treatments significantly reduced or reversed the short-circuit current. Acetazolamide (10(-3) mol.l-1, serosal) reduced mucosal acidification by 15% and simultaneously increased short-circuit current by a similar amount. Colonic epithelia incubated in glucose-free solutions had significantly lower acidification rates (0.59 +/- 0.13 microEq.cm-2.h-1, P < 0.002 versus controls) and addition of glucose (15 mmol.l-1), but not galactose, partially restored acidification to control levels. Anoxia (N2 gassing) completely inhibited short-circuit current, but reduced acidification by only 30%. A surface microclimate pH, nearly 2 pH units more acidic than the bath pH of 7.1-7.4, was measured in low-salt-adapted colon and coprodeum. The acid microclimate of both tissues was partially attenuated by adaptation to a high-salt diet. Colonic microclimate pH was dependent on the presence of glucose and sensitive to the bath pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the parathyroid hormone antagonist [Tyr34]bPTH-(7-34)amide on avian renal phosphate transport.

The in vivo parathyroid hormone antagonist, [Tyr34]bPTH-(7-34)amide, was tested for its ability to block endogenous parathyroid hormone (PTH) activity in parathyroid-intact European starlings. The inhibitor was infused at two dose levels, 0.005 and 0.05 mg/hr. A third group received a vehicle/control infusion. High-dose infusion led to rapid and significant decreases in plasma calcium and in both absolute and relative phosphate (Pi) excretion. Low-dose infusion significantly reduced plasma calcium, but had no effect on Pi excretion. Plasma Pi tended to increase with time in all three groups. Removal of the antagonist after 140 min of high-dose infusion led to a rapid recovery of relative Pi excretion, and a subsequent overshoot to more than twice the original control level. To further test the ability of the antagonist to block endogenous PTH activity, an EGTA challenge was used to induce hypocalcemia and subsequent elevation of circulating PTH levels. In vehicle-infused birds, EGTA challenge led to a doubling of relative Pi excretion, but in birds receiving the high-dose infusion, no effect of EGTA was observed. These results demonstrate that [Tyr34]bPTH-(7-34)amide can completely block endogenous PTH activity in birds at the receptor level, including the hormone response of the renal Pi secretory transport mechanism.

Micropuncture study of proximal tubule pH in avian kidney.

Transepithelial potentials (PD) and pH gradients were measured by in vivo micropuncture in superficial proximal tubules of anesthetized European starlings. The average PD for 46 tubules was 2.24 +/- 1.17 mV (mean +/- SD), lumen negative. Only a single lumen-positive potential was recorded, even though late proximal segments are accessible to micropuncture. Proximal luminal pH, measured with single-barrel pH-sensitive microelectrodes, averaged 7.62 +/- 0.26. The pH of peritubular blood, also measured with microelectrodes, was 7.58 +/- 0.15. Correction of the luminal pH for the average PD resulted in a value of 7.58. A few measurements made with double-barrel microelectrodes indicated a pH in proximal tubules of 7.65 +/- 0.08 with a PD of -3.32 +/- 1.47 mV. Thus there appears to be no transepithelial pH gradient across these tubules. On the other hand, pH measurements of cortical collecting ducts averaged 6.40 +/- 0.37, indicating significant acidification in more distal segments. Starlings made acutely acidotic by HCl infusion had significantly reduced arterial and renal cortical pH values, but there was still no significant pH gradient across the proximal tubule. In conclusion, these superficial "reptilian-type" proximal tubules do not appear to have the capacity to maintain a pH gradient. Based on this and on the lack of lumen-positive chloride diffusion potentials, there appears to be no early proximal preferential bicarbonate reabsorption by these nephrons.

Kidney function in domestic fowl with chronic occlusion of the ureter and caudal renal vein.

A previously published technique for producing renal insufficiency in chicks involves ligating the caudal renal vein together with the ureter near the midpoint of the kidney. This ligation occludes the normal route (caudal renal vein) by which blood exits the medial and posterior kidney divisions. The present study was conducted to evaluate kidney function following chronic caudal renal vein and ureter occlusion. The ureters and caudal renal veins of 2 to 3-week-old chicks were clamped with hemostatic clips (Clamp Group), and kidney function evaluations were conducted when the birds reached 14 weeks of age. Plasma values [osmolality, Na, K, Ca, inorganic P (Pi)] of control, sham, and clamp groups did not differ significantly. Plasma uric acid concentrations did not differ when the control and clamp groups were compared. Clamping the ureter near the midpoint of the kidney caused a significant reduction in the number of filtering glomeruli per kidney, but due to compensatory hypertrophy the kidney weights of the groups did not differ significantly. Kidney function comparisons (urine flow rates, glomerular filtration rates, renal plasma flow rates, urine pH, fractional excretion of Na, K, Ca, Pi, and uric acid) revealed a significant reduction in Na and K excretion by the clamp group, but other renal function parameters did not differ significantly. These experiments demonstrate that chickens have a remarkable capacity to survive significant reductions in renal mass and to adapt to major disruptions of blood flow patterns.

Avian renal responses to oxidized and nonoxidized bPTH(1-34).

The hypotensive, hypercalcemic, and hyperphosphaturic responses to bovine synthetic 1-34 amino acid parathyroid hormone [bPTH(1-34)] were simultaneously evaluated in anesthetized laying hens, and compared to the same responses to oxidized (0.3% hydrogen peroxide) bPTH(1-34) prepared from the same commercial batch of hormone. The oxidized and nonoxidized hormones were administered intravenously into separate groups of animals, which were also receiving a constant intravenous infusion of inulin and p-aminohippuric acid (PAH) to serve as renal clearance test markers. bPTH(1-34) caused a 33.25 +/- 7.05 mm Hg decrease in mean arterial blood pressure. The corresponding change in response to the oxidized hormone preparation was a 10.75 +/- 1.49 mm Hg decrease, a significant reduction in hypotensive activity compared to the intact hormone. In contrast, oxidation had no influence on the hypercalcemic or hyperphosphaturic (relative clearance of phosphate, CPO4/CIn) responses to bPTH(1-34), both preparations causing significant and parallel increases in these variables. Both hormone preparations also caused increases in urine flow rate, GFR, CPAH, CCa/CIn, CK/CIn, CNa/CIn, and urine pH. However, on the basis of percentage change from the respective control periods, only CNa/CIn and urine pH showed significant differences between the two hormone preparations, with the intact hormone causing greater natriuresis and urine alkalinization than the oxidized hormone. The failure of peroxide oxidation to significantly affect the renal hyperphosphaturic response to bPTH(1-34) in chickens contrasts with results reported on mammalian species, and may be related to differences in the mechanism of renal phosphate transport between these groups.

Micropuncture study of urate transport by superficial nephrons in avian (Sturnus vulgaris) kidney.

Tubule fluid samples from proximal tubules of superficial, reptilian-type nephrons of the European starling (Sturnus vulgaris) were obtained by micropuncture and, along with plasma samples, were analyzed for urate by high performance liquid chromatography with electrochemical detection, as well as for the elements sodium, chloride, calcium, magnesium, phosphorus and potassium by electron microprobe analysis. All proximal tubules sampled showed net secretion of urate, but the magnitude of transport was highly variable. Mean fractional delivery of urate to the point of micropuncture was 4.04 +/- 0.37 (mean +/- SE, n = 40 tubules). Fractional delivery of urate could not be correlated with fractional delivery of any of the measured elements or with relative fluid absorption. Samples collected from surface segments of cortical collecting ducts suggest that, although much net secretion of urate occurs early within the proximal tubule of superficial nephrons, some additional secretion may occur in more distal segments. Fractional excretion of urate at the level of the cloaca averaged 4.72 +/- 0.97 for nine birds, confirming the result of overall net secretion of urate previously reported for other avian species. These results clearly demonstrate the presence of significant secretory transport of urate in the proximal tubule of superficial reptilian-type nephrons, but do not rule out the possibility of additional secretory or reabsorptive fluxes at other sites.

Micropuncture of superficial nephrons in avian (Sturnus vulgaris) kidney.

Single nephron glomerular filtration rate (SNGFR), fluid absorption, and tubule transport of sodium, chloride, calcium, magnesium, potassium, and phosphate were evaluated under control (2.5% mannitol infusion) conditions by micropuncture of superficial, reptilian-type nephrons of European starlings (Sturnus vulgaris). SNGFR averaged 0.36 nl . min-1 . Sodium, chloride, and water were transported at equivalent rates with about 24% of the filtered load absorbed up to the point of micropuncture. Calcium was absorbed continuously along these tubules in excess of sodium and fluid, whereas magnesium appeared to undergo net secretion early and net absorption late in the tubules. Potassium transport varied considerably, with all tubules from a single bird generally showing either net potassium absorption or no net transport. In the latter group, data for a few tubules suggested net potassium secretion. For phosphate, individual tubules from a single bird were capable of either net absorption or net secretion. Mean fractional delivery of phosphate for all tubules was 1.62, but secretion rates apparently can exceed filtration rates for phosphate by as much as 6 times.