Bovine mastitis and ecology of Streptococcus uberis

Bovine mastitis caused by Streptococcus uberis is a common problem in pasture-based dairying systems. This study examines both the ecology of S. uberis and infection of the bovine mammary gland on a New Zealand dairy farm. Initially, the REP-PCR strain typing method was developed and the potential of MALDI-TOF mass spectrometry evaluated as a strain typing method. While strain-specific mass spectra were obtained with MALDI-TOF mass spectrometry, the irreproducibility of spectra was its major downfall. With further work, this rapid method could be very useful for strain typing S. uberis on a large scale. Using optimised REP-PCR and anchored typing methods, multiple S. uberis strains were isolated and strain typed from the dairy environment, including farm races and paddocks, faeces, teat skin, the cow body and from intramammary infections. High strain diversity was observed in all sampled locations; however, some strains were found at more than one site, suggesting transmission may occur between the environment and cows. The most likely means of S. uberis distribution throughout the dairy farm was via excretion with faeces and, although not all cow faeces contained this pathogen, the gastrointestinal tract of some cows appeared to be colonised by specific strains, resulting in persistent shedding of this bacteria in the faeces. Infection of the mammary gland is likely to occur through contamination of the teat skin with highly diverse environmental strains of S. uberis. However, only one or two strains are generally found in milk from mastitis cases, suggesting that, although infection may arise from a random or opportunistic event, a strain selection process may take place. Intramammary challenge with multiple strains of S. uberis revealed that selection of a single infective strain can occur within the mammary gland. The predominance of one strain over others may be related to production of virulence factors allowing enhanced ability to establish in the gland and evade the immune response, or due to direct competition between strains through the production of antimicrobial factors such as bacteriocins. In addition to strain-specific factors, the individual cow and quarter response may play an important role in the development of infection and selection of the infective strain. Using results from this study, a model of S. uberis strain transmission has been proposed, which includes potential mechanisms of infection and persistence of S. uberis within the mammary gland

NGF Causes TrkA to Specifically Attract Microtubules to Lipid Rafts

Membrane protein sorting is mediated by interactions between proteins and lipids. One mechanism that contributes to sorting involves patches of lipids, termed lipid rafts, which are different from their surroundings in lipid and protein composition. Although the nerve growth factor (NGF) receptors, TrkA and p75NTR collaborate with each other at the plasma membrane to bind NGF, these two receptors are endocytosed separately and activate different cellular responses. We hypothesized that receptor localization in membrane rafts may play a role in endocytic sorting. TrkA and p75NTR both reside in detergent-resistant membranes (DRMs), yet they responded differently to a variety of conditions. The ganglioside, GM1, caused increased association of NGF, TrkA, and microtubules with DRMs, but a decrease in p75NTR. When microtubules were induced to polymerize and attach to DRMs by in vitro reactions, TrkA, but not p75NTR, was bound to microtubules in DRMs and in a detergent-resistant endosomal fraction. NGF enhanced the interaction between TrkA and microtubules in DRMs, yet tyrosine phosphorylated TrkA was entirely absent in DRMs under conditions where activated TrkA was detected in detergent-sensitive membranes and endosomes. These data indicate that TrkA and p75NTR partition into membrane rafts by different mechanisms, and that the fraction of TrkA that associates with DRMs is internalized but does not directly form signaling endosomes. Rather, by attracting microtubules to lipid rafts, TrkA may mediate other processes such as axon guidance

Experimentally induced intramammary infection with multiple strains of Streptococcus uberis

The effect of infusing a mixture of 5 Streptococcus uberis strains into mammary quarters of 10 lactating cows was investigated. All 5 strains, which included 2 originally isolated from the dairy environment and 3 from clinical cases of mastitis, were capable of establishing an intramammary infection when infused individually. However, when the 5 strains were infused together, a single strain predominated in 7 out of 10 quarters. One strain in particular prevailed in 4 mammary quarters and was also found to inhibit the growth of the other 4 strains with deferred antagonism on esculin blood agar. The genes required for the production of bacteriocins nisin U and uberolysin were identified in this strain, whereas the other 4 strains contained only uberolysin genes. Direct competition may have occurred between strains within the mammary gland but competition was not apparent when cultured together in UHT milk, where no strain predominated. Although the mechanism is unknown, these results imply that a selection process can occur within the mammary gland, leading to a single strain that is detected upon diagnosis of mastitis

TrkA is bound to microtubules in floating DRMs. Floating DRMs were isolated after 10

<p> <b>min NGF treatment and in vitro reactions.</b> A) Microtubules were immunoprecipitated from the floating peak with anti-β-tubulin and western blotted for TrkA (upper panel) and tubulin (lower panel). B) In vitro reactions without (ATP only) or with biotinylated tubulin added during the last 5 min of the reaction (+biotin-tubulin) were performed. The floating DRM peak was immunoprecipitated with streptavidin or anti-TrkA (indicated) and western blotted for anti-TrkA (upper panel) and anti-biotin (lower panel). p75^NTR was not reproducibly detected in microtubule immunoprecipitations from DRMs under these conditions. C, D) Images of permeabilized cells after in vitro reactions with biotinylated tubulin added during the last 5 min of the reaction. Texas red streptavidin stained discreet foci at or near the plasma membrane, shown in a group of permeabilized cells in C and an individual cell in D.</p

The ganglioside, GM1 affects the partitioning of NGF, its receptors, and microtubules in floating DRMs.

<p>GM1 was pre-incubated with cells at 65 µM. Control samples in the same experiment were treated identically except no GM1 was added. Cells were bound to ¹²⁵I-NGF and sonicated DRMs were floated on iodixanol equilibrium gradients as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone-0035163-g001" target="_blank">Figure 1</a> (0 min). A) Western blots showing that TrkA and tubulin were increased in floating DRMs with GM1 treatment. B) ¹²⁵I-NGF in floating DRMs (left, plotted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone-0035163-g001" target="_blank">Figure 1</a>) increased with GM1 treatment (filled circles) compared to control (open triangles). The amount of ¹²⁵I-NGF in floating DRMs, plotted as a fraction of that in the whole cell as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone-0035163-g002" target="_blank">Figure 2</a>, increased in GM1-treated cells (p<0.1). C) Data from western blots with anti-TrkA, -p75^NTR, -flotillin and -tubulin (indicated) were quantified and the amount in the floating DRM peak coincident with ¹²⁵I-NGF are plotted as the percent of the whole cell for control and GM1-treated cells. The amount of p75^NTR in the floating DRMs decreased by half (from 19% to 8%) after GM1 treatment (p<0.1). Flotillin was also decreased (p<0.1), while TrkA (p<0.1) and tubulin (p<0.01) were increased by GM1.</p

Amount of Radioactive Ligands Associated With Cell Fractions.

<p>Cells were bound to radiolabelled ligand, washed, and subjected to internalization 10 min at 37°C. Mechanical permeabilization, fractionation, and detergent extraction was performed exactly as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone.0035163-Grimes1" target="_blank">[35]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone.0035163-Grimes2" target="_blank">[36]</a>.</p

TrkA and p75^NTRin endosomes and endosomal DRMs.

<p>A) Endosomes from cells treated 10 min with NGF. Organelles that emerged from cells mechanically permeabilized by a single pass through a Balch homogenizer were subjected to velocity sedimentation followed by equilibrium flotation as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone.0035163-Mccaffrey1" target="_blank">[7]</a>. Shown is the flotation equilibrium gradient from velocity gradient fraction 3, which contains TrkA and p75^NTR endosomes (indicated) that floated to their equilibrium density. Blots were probed with anti-pTrkA, -p75^NTR, and -SHP-1 (indicated). B) The detergent-resistant fraction containing endosomes from untreated or NGF-treated cells before (no reaction) or after in vitro reactions (+ATP) was fractionated on iodixanol velocity gradients as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone.0035163-Maccormick1" target="_blank">[34]</a>. Pools from the bottom of the gradient containing microtubules (MT) and control samples from the top of the gradient (C) were collected for immunoprecipitations with anti-tubulin (MTIP). One-ninth of each sample was TCA precipitated before immunoprecipitation (input). Western blots were probed with anti-TrkA and anti-p75^NTR (indicated). pTrkA was not detected in endosomal DRMs (not shown).</p

Association of NGF receptors and cytoskeletal elements with DRMs under different experimental conditions.

<p>A) Western blots showing TrkA and p75^NTR in floating DRMs prepared after 10 min NGF treatment and using nuclease (Benzonase) rather than sonication to break up nucleic acids prior to equilibrium density gradients. Western blots of flotation equilibrium gradients of detergent-resistant fraction were probed with anti-TrkA, -pTrkA, -SHP-1, -p75^NTR, and -tubulin (indicated). Blots include the detergent-sensitive (P1M) fraction and size standards (S) to the left of DRM gradient fractions. B) Left: ¹²⁵I-NGF in DRM without (open squares) and with (closed circles) in vitro reactions with ATP. Right: Quantification of chemiluminescent signals from western blots is compared to ¹²⁵I-NGF for Benzonase-treated samples as in A. Fraction number is plotted on the x-axis of plot on the left; fraction 1 has the highest density. Signals from western blots were quantified and plotted vs. density together with ¹²⁵I-NGF (closed circles) on the right. The y-axis for TrkA (closed squares), p75^NTR (open circles), SHP-1 (open squares), and tubulin (closed triangles) is arbitrary units (chemiluminescent pixel volume). C) Western blots showing actin in floating DRMs prepared as in A, with and without in vitro reactions with ATP (–, +ATP). Data obtained under these conditions for TrkA, tubulin, and actin were quantified and plotted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone-0035163-g002" target="_blank">Figure 2</a>. After 10 min internalization, in vitro reactions with ATP caused a significant increase of TrkA (p<0.01) and NGF and tubulin (p<0.001) in floating DRMs. A decrease in actin (+ATP) was noted but was not statistically significant.</p

NGF differently affects association of TrkA and p75^NTRwith floating DRMs.

<p>A) Floating DRMs were isolated using Benzonase treatment after 10 min without (open diamonds) and with (closed squares) NGF treatment without (no rxn, left) and with (+in vitro rxn, right) subsequent in vitro reactions with ATP. TrkA (upper panels) and p75^NTR (lower panels) were quantified from western blots probed simultaneously in the same antibody solution, exposed for the same amount of time, for all conditions. Data are plotted using the same y-axis (chemiluminescence for TrkA and p75^NTR) for all conditions. In vitro reactions had little influence on the amounts of TrkA in floating DRMs in the absence of NGF, but increased TrkA in DRMs in the presence of NGF. In vitro reactions caused p75^NTR to increase in the floating peak in the absence of NGF, but decrease in the presence of NGF. B) Amounts of TrkA, p75^NTR, tubulin, and flotillin in the floating DRM peak prepared using sonication as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#pone-0035163-g002" target="_blank">Figure 2</a>, plotted as the percent of the whole cell for control (–NGF) and NGF-treated (+NGF) for cells subjected to in vitro reactions. Under these conditions, NGF caused an increase in TrkA (p<0.1) and tubulin (p<0.05) in floating DRMs, yet caused a significant decrease (p<0.05) in p75^NTR in floating DRMs.</p

NGF associates with lipid rafts before and after initiation of membrane traffic and signal transduction.

<p>¹²⁵I-NGF was bound to PC12 cells in the cold, the cells were washed and warmed for the indicated periods of time. 0 min represents cells bound to NGF but not warmed. Flotation equilibrium iodixanol gradients were performed using sonication to resuspend the detergent-resistant fraction. A) Plots of DRM gradients after pulse-stimulation with ¹²⁵I-NGF for 0, 2, 10, and 30 min. Refractive index measurements were taken on each fraction and converted into density using a formula derived empirically (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035163#s4" target="_blank">Methods</a>); density is plotted on the x-axis. There also was non-floating NGF in the detergent-resistant pellet, whose distribution in fractions of higher density is consistent with diffusion in a bottom-loaded sample. B, C) Amount of NGF and density of floating DRMs. The amounts of ¹²⁵I-NGF in the floating DRM peak containing ¹²⁵I-NGF were quantified and compared to amounts in detergent-soluble membranes and other fractions (B, %WC=percent of whole cell). Amounts in the floating DRM peak are plotted as the percent in the whole cell. A transient increase in the density of the floating ¹²⁵I-NGF DRM peak was noted after 2 and 10 min (C). A higher density suggests a higher protein:lipid ratio. Error bars are SEM. D) DRM fraction from rat dorsal root ganglia neurons bound to ¹²⁵I-NGF and warmed for 10 min as above. The floating peak had a slightly higher density (1.18 g/ml) than that in PC12 cells (1.16 g/ml).</p