Validation Of Complete Blood Count Methodology, And Determination Of The Relationship Between Endoparasite Load And Erythrocyte Values In New World Camelids

Background: Accurate measurement of RBCs (red blood cells) by automated hematology analyzers such as the ADVIA 120/2120 requires isovolumetric cell sphering; however, camelid RBC membranes are resistant to shape change. There are no published reports of method validation for hematologic analysis of camelid blood. Mycoplasma haemolamae and gastrointestinal nematodes can cause anemia in camelids. Parasite control programs aim to suppress parasite loads without promoting resistance, but there are few evidence-based guidelines for acceptable parasite loads in camelids.Objectives: 1) Demonstrate whether camelid RBCs sphere in the ADVIA sphering reagent, and determine the optimal ADVIA setting for CBC (complete blood count) analysis, 2) Compare M. haemolamae PCR status with RBC values, and 3) Determine the fecal egg count (FEC) threshold above which RBC values are consistently below the median of the reference interval.Methods: Camelid and canine blood were each added to ADVIA sphering reagent or saline, and evaluated by light microscopy for erythrocyte sphering. Camelid blood was analyzed on an ADVIA 120 hematology analyzer using one of three species settings, and values compared to manual measurements, including packed cell volume (PCV), Z2 Coulter counter RBC count, and calculations of other RBC values. Mycoplasma haemolamae was detected by real-time PCR. The number of trichostrongyle eggs per gram (epg) of feces was determined using the Modified McMaster’s test.Results and Conclusions: Camelid erythrocytes do not sphere when mixed with ADVIA sphering reagent. The ADVIA 120 equine setting provides the closest approximations to Z2 counter RBC count estimates, but ADVIA results for most other RBC values appear inaccurate. PCV, hemoglobin, and RBC count are not significantly different between M. haemolamae positive and negative animals, but are significantly lower in animals with FEC\u3e [greater than] 600epg. For all animals with FEC\u3e600epg, RBC values are below the medians of the reference intervals. Positive M. haemolamae PCR is not associated with lower RBC values in healthy camelids, consistent with previous reports that most infections are subclinical. Maintaining FEC below 600 epg is recommended in camelids

Dynamic Microtubules Promote Synaptic NMDA Receptor-Dependent Spine Enlargement

Most excitatory synaptic terminals in the brain impinge on dendritic spines. We and others have recently shown that dynamic microtubules (MTs) enter spines from the dendritic shaft. However, a direct role for MTs in long-lasting spine plasticity has yet to be demonstrated and it remains unclear whether MT-spine invasions are directly influenced by synaptic activity. Lasting changes in spine morphology and synaptic strength can be triggered by activation of synaptic NMDA receptors (NMDARs) and are associated with learning and memory processes. To determine whether MTs are involved in NMDAR-dependent spine plasticity, we imaged MT dynamics and spine morphology in live mouse hippocampal pyramidal neurons before and after acute activation of synaptic NMDARs. Synaptic NMDAR activation promoted MT-spine invasions and lasting increases in spine size, with invaded spines exhibiting significantly faster and more growth than non-invaded spines. Even individual MT invasions triggered rapid increases in spine size that persisted longer following NMDAR activation. Inhibition of either NMDARs or dynamic MTs blocked NMDAR-dependent spine growth. Together these results demonstrate for the first time that MT-spine invasions are positively regulated by signaling through synaptic NMDARs, and contribute to long-lasting structural changes in targeted spines

Enlargement of MT-invaded spines following acute activation of synaptic NMDARs.

<p>(A - C) Representative invaded and non-invaded spines from cells treated with Gly-0Mg²⁺ alone (A), or with Gly-0Mg²⁺ in the presence of APV (B) or nocodazole (C). <i>Top</i>, pseudocolored images of the DsRed2 signal intensity averaged over 10 minute time intervals spanning the time-lapse (i.e. before, during and after treatment with Gly-0Mg²⁺). In the left-most panel the dendrite and spines are outlined in white and are labeled (1 = invaded spine, 2 = non-invaded spine, D = dendrite shaft). Scale bars, 2 µm. <i>Middle</i>, kymographs for the invaded spines from the panels above show the timing of MT invasions during the time-lapse (EGFP-α-tubulin in green, DsRed2 in red). <i>Bottom</i>, normalized DsRed2 fluorescence intensities of the spines shown above at each frame in the time-lapse (10sec intervals; grey circles  =  invaded spines, filled black circles  =  non-invaded spines). Experimental paradigms are shown above the plot, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027688#pone-0027688-g001" target="_blank">Figure 1</a>. Light grey region from 0–10 minutes indicates the timing of the Gly-0Mg²⁺ treatment. Green-filled circles indicate the frames in which the spine was invaded by a MT (also shown in the kymographs above).</p

MTs are important for lasting NMDAR-dependent spine enlargement.

<p>(A) Normalized DsRed2 fluorescence intensities at each frame, averaged across all invaded (grey) and non-invaded control (black) spines from cells treated with Gly-0Mg²⁺ (mean ± SEM). Boxes represent 10-minute time averages of the respective traces (mean ±95%CI). Effects of time and spine-type (invaded vs. non-invaded) were assessed with a two-way ANOVA with repeated measures and a Bonferonni post-test to compare time columns. Experimental paradigms are shown above the plot, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027688#pone-0027688-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027688#pone-0027688-g003" target="_blank">3</a>. (B) Comparison of invaded (grey) and non-invaded (black) spine intensities in each time column after treatment with Gly-0Mg²⁺ (two-way ANOVA from (A) with Bonferonni post-test to compare invaded and non-invaded spines at each time-point) (mean ± SEM). (C - D) Normalized DsRed2 fluorescence intensities averaged across all invaded and non-invaded spines from cells treated with Gly-0Mg²⁺ in the presence of APV (C) or nocodazole (D). No significant differences were detected (two-way ANOVA with repeated measures). (E - F) Between-groups comparison of changes in DsRed2 intensity observed in invaded (E) and non-invaded (F) spines. Two-way ANOVA with repeated measures and Bonferroni post-test to compare cells treated with Gly-0Mg²⁺ alone, Gly-0Mg²⁺+APV, and Gly-0Mg²⁺+nocodazole at each time column (mean ± SEM). For all graphs, *p<0.05, **p<0.01, and *** p<0.001.</p

MT invasions trigger rapid spine enlargement that is more persistent following NMDAR activation.

<p>(A) MT-triggered average of normalized DsRed2 fluorescence intensities across all invaded spines (grey lines and symbols, n = 566 invasions) and non-invaded control spines (black lines and symbols, n = 566 invasions) from cells treated with Gly-0Mg²⁺. MT invasion onsets are aligned at t = 0. *** p<0.001; t-test comparing spine size one frame before (t =  −10 sec) with one frame after (t = 10 sec) invasion onset. Return to basal size was fit with a mono-exponential decay function from t = 10 sec to t = 10 min. (B) MT-triggered average of normalized DsRed2 fluorescence intensities across all invaded (n = 355) and non-invaded (n = 355) spines treated with Gly-0Mg²⁺ in the presence of APV. Symbols and analysis as in (A). (C) Population distribution of all MT lifetimes following treatment with Gly-0Mg²⁺ alone (grey open circles) or Gly-0Mg²⁺+APV (black filled circles). Best-fitting mono-exponential decay functions of invasion lifetimes for the two conditions are overlaid (grey and black lines).</p