Potential for epidemic take-off from the primary outbreak farm via livestock movements

Background

We consider the potential for infection to spread in a farm population from the primary outbreak farm via livestock movements prior to disease detection. We analyse how this depends on the time of the year infection occurs, the species transmitting, the length of infectious period on the primary outbreak farm, location of the primary outbreak, and whether a livestock market becomes involved. We consider short infectious periods of 1 week, 2 weeks and 4 weeks, characteristic of acute contagious livestock diseases. The analysis is based on farms in Scotland from 1 January 2003 to 31 July 2007.

Results

The proportion of primary outbreaks from which an acute contagious disease would spread via movement of livestock is generally low, but exhibits distinct annual cyclicity with peaks in May and August. The distance that livestock are moved varies similarly: at the time of the year when the potential for spread via movements is highest, the geographical spread via movements is largest. The seasonal patterns for cattle differ from those for sheep whilst there is no obvious seasonality for pigs. When spread via movements does occur, there is a high risk of infection reaching a livestock market; infection of markets can amplify disease spread. The proportion of primary outbreaks that would spread infection via livestock movements varies significantly between geographical regions.

Conclusions

In this paper we introduce a set-up for analysis of movement data that allows for a generalized assessment of the risk associated with infection spreading from a primary outbreak farm via livestock movements, applying this to Scotland, we assess how this risk depends upon the time of the year, species transmitting, location of the farm and other factors.     

Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation

A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.     

Na+/H+ exchanger activity in the pig kidney epithelial cell line, LLC-PK1: inhibition by amiloride and its derivatives

Rapidly growing pig-kidney-derived epithelial cells, LLC-PK1, lack detectable amiloride-sensitive Na+/H+ exchange activity when assayed directly. A large 22Na uptake is induced when the cells are acid-loaded prior to assay by incubation with buffer containing ammonium chloride or nigericin. The acid-stimulated sodium uptake is sensitive to amiloride, with half-maximal inhibition at 3.5-4.5 microM in buffer containing 15 mM sodium ion. There is simple competitive interaction between amiloride and sodium ion when the amiloride concentration is below 25 microM and the sodium ion concentration is above 20 mM. Derivatives of amiloride which carry substituents on the 5-amino group are 35- to 175-fold more inhibitory than amiloride itself.     

The canine isolate Lactobacillus acidophilus LAB20 adheres to intestinal epithelium and attenuates LPS-induced IL-8 secretion of enterocytes in vitro

Background

For a good probiotic candidate, the abilities to adhere to intestinal epithelium and to fortify barrier function are considered to be crucial for colonization and functionality of the strain. The strain Lactobacillus acidophilus LAB20 was isolated from the jejunum of a healthy dog, where it was found to be the most pre-dominant lactobacilli. In this study, the adhesion ability of LAB20 to intestinal epithelial cell (IECs) lines, IECs isolated from canine intestinal biopsies, and to canine, porcine and human intestinal mucus was investigated. Further, we studied the ability of LAB20 to fortify the epithelial cell monolayer and to reduce LPS-induced interleukin (IL-8) release from enterocytes.

Results

We found that LAB20 presented higher adhesion to canine colonic mucus as compared to mucus isolated from porcine colon. LAB20 showed adhesion to HT-29 and Caco-2 cell lines, and importantly also to canine IECs isolated from canine intestinal biopsies. In addition, LAB20 increased the transepithelial electrical resistance (TER) of enterocyte monolayers and thus strengthened the intestinal barrier function. The strain showed also anti-inflammatory capacity in being able to attenuate the LPS-induced IL-8 production of HT-29 cells.

Conclusion

In conclusion, canine indigenous strain LAB20 is a potential probiotic candidate for dogs adhering to the host epithelium and showing intestinal barrier fortifying and anti-inflammatory effects.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-014-0337-9) contains supplementary material, which is available to authorized users.     

Species-specific Mn2+/Mg2+ antiport from Mg2(+)-loaded erythrocytes.

Mg2(+)-loaded rat erythrocytes performed Mn2+/Mg2+ antiport, which was nonspecifically stimulated by anions and cations. Mn2+/Mg2+ antiport was shown to operate via the Na+/Mg2+ antiporter because extracellular Na+ and Mn2+ inhibited the intracellular uptake of each other's ions competitively. Furthermore, Mn2+/Mg2+ antiport and Na+/Mg2+ antiport were identically inhibited by various amiloride derivatives. Na+/Mg2+ antiport of chicken and human erythrocytes cannot perform Mn2+/Mg2+ antiport although chicken erythrocytes took up more Mn2+ than rat erythrocytes.     

Regulation of human neutrophil chemotaxis by intracellular pH

The relationship of N-formyl-methionyl-leucyl-phenylalanine-stimulated Na+/H+ exchange to the chemotactic responsiveness of human neutrophils was investigated. The pHi changes, measured from the equilibrium distribution of 5,5-dimethyloxazolidine-2,4-dione, were correlated with the migratory behavior of the cells as assessed by the leading front method. Exposure of cells to 10 nM FMLP caused activation of Na+/H+ exchange, leading to a rise in pHi from approximately 7.25 to approximately 7.75. This intracellular alkalinization was inhibited by amiloride and by three more potent analogues. All four compounds reduced the chemotactic response to FMLP with apparent Ki values similar to those for inhibition of the pHi transients, thereby suggesting that the blocking effect of the drugs on directed cell migration was related to inhibition of Na+/H+ exchange. The effect was specific for stimulated cell locomotion: FMLP-induced chemotaxis and chemokinesis were inhibited in parallel, whereas random motility was unimpaired. The relationship of pHi to function was also studied as the pHi of FMLP-activated cells was varied between 6.8 and 8.6 by altering the chemical gradients for Na+ and H+ across the cell membrane. There was a direct, positive correlation between the pHi value attained following FMLP-stimulation and the locomotor response to a chemotactic gradient. These results indicate that the motile functions of human neutrophils can be regulated by their pHi.     

Activation of the Na+/H+ antiport is not required for lectin-induced proliferation of human T lymphocytes

Interaction of some mitogenic lectins and growth factors with the cell surface leads to activation of the Na+/H+ antiport and a resultant cytoplasmic alkalinization. Because amiloride inhibits both Na+/H+ exchange and cell proliferation, it has been hypothesized that activation of the antiport is an obligatory requirement and may, perhaps, be the "trigger" for proliferation. However, concentrations of amiloride which inhibit the antiport also inhibit several other intracellular processes, including protein synthesis and phosphorylation. To determine whether activation of the Na+/H+ antiport is necessary for lectin-induced proliferation, we examined the inhibitory activity of a series of potent amiloride analogs by measuring [3H]thymidine incorporation, cell cycle progression, and induction of the interleukin 2 (IL 2) receptor on human lymphocytes. In medium containing bicarbonate, and at concentrations at least 10 times higher than required to inhibit the antiport, these drugs did not inhibit the proliferative response of human peripheral blood T cells to the mitogen phytohemagglutinin. The amiloride analogs also failed to inhibit induction of the IL 2 receptor. Similarly, with human thymocytes, the amiloride analogs did not inhibit the co-mitogenic effects of the lectins phytohemagglutinin and concanavalin A together with IL 2 or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. This finding suggests that Na+/H+ exchange through the antiport is not an obligatory requirement for activation or proliferation of human lymphocytes or thymocytes.     

Role of Na+/H+ exchange in thrombin-induced platelet-activating factor production by human endothelial cells

Thrombin-stimulated endothelial cells produce platelet-activating factor (PAF) in a dose-dependent manner: the activation of a Ca2+-dependent lyso-PAF acetyltransferase is the rate-limiting step in this process. The present study shows that acetyltransferase activation and consequent PAF production induced by thrombin in human endothelial cells are markedly inhibited in Na+-free media or after addition of the amiloride analog 5-(N-ethyl-N-isopropyl)amiloride, suggesting that a Na+/H+ antiport system is present in endothelial cells and plays a prominent role in thrombin-induced PAF synthesis. Accordingly, thrombin elicits a sustained alkalinization in 6-carboxyfluorescein-loaded endothelial cells, that is abolished in either Na+-free or 5-(N-ethyl-N-isopropyl)amiloride-containing medium. Extracellular Ca2+ influx induced by thrombin (as measured by quin2 and 45Ca methods) is completely blocked in the same experimental conditions, and monensin, a Na+/H+ ionophore mimicking the effects of the antiporter activation, evokes a dose-dependent PAF synthesis and a marked Ca2+ influx, which are abolished in Ca2+-free medium. An amiloride-inhibitable Na+/H+ exchanger is present in the membrane of human endothelial cells, its apparent Km for extracellular Na+ is 25 mM, and its activity is greatly enhanced when the cytoplasm is acidified. These results suggest that Na+/H+ exchange activation by thrombin and the resulting intracellular alkalinization play a direct role in the induction of Ca2+ influx and PAF synthesis in human endothelial cells.     

Regulation of cytoplasmic pH of cultured bovine corneal endothelial cells in the absence and presence of bicarbonate

Summary Single sodium-channel currents were measured in neuroblastoma cells after inhibition of inactivation by chloramine-T (CHL-T), sea anemone toxin II (ATX-II) and scorpion toxin (SCT). The decaying phase of the averaged single-channel currents recorded with 90-msec pulses in cell-attached patches was clearly slower than that of the unmodified channels, suggesting inhibition of macroscopic inactivation. Each substance caused repetitive openings and a moderate increase in the channel open time. AtV _m =RP+20 mV andT=12°C, the mean channel open times were 1.4, 1.6 and 1.8 msec for CHL-T, ATX-II and SCT, respectively, as opposed to 1.07 msec for native channels. Open-time histograms could be best fitted by the sum of two exponentials. The time constants of the fits were similar for histograms constructed from single openings and from openings during bursts. This suggests that the population of channels is homogeneous and that in bursts the same open conformations of channels occur as in single openings. Mean burst durations for bursts consisting of more than one opening atV _m =RP+20 mV were 4.9, 5.8 and 6.1 msec for CHL-T, ATX-II and SCT, respectively. Burst open-time histograms constructed from two or three openings were fitted by the gamma function. The different time constants of the fits obtained for ATX-II and SCT suggested multiple open conformations of channels for openings of bursts. However, significantly different open-time histograms constructed from the first, second and third openings of bursts could not be obtained systematically. A positive correlation was found for the dwell time of the first and the second, as well as for the second and the third opening of bursts with each substance, but a negative one for the dwell time of an opening and the neighboring closing of bursts with ATX-II. The results suggest a model with multiple open and inactivated states. In this model the inactivated states are weakly absorbing.