Strategies for phase matching control in a multidimensional Floquet state spectroscopy.

Floquet state spectroscopy is an optical analogue of multiple quantum coherence nuclear magnetic resonance (MQC-NMR). Tunable ultrafast excitation pulses resonantly excite multiple states in a sample to form the Floquet state. The Floquet state emits multiple coherent beams at frequencies and in directions that conserve energy and momenta. The different output beams differ in the time ordering and coherences created by the excitation beams. They correspond to the different methodologies in the NMR family. Isolating a specific beam and monitoring the output intensity as a function of excitation frequencies creates multidimensional spectra containing cross-peaks between coupled states. The frequency range of the multidimensional spectra is limited by phase matching constraints. This paper presents a new, to the best of our knowledge, active phase matching strategy that increases the versatility of multidimensional Floquet state spectroscopy through both longer sample path lengths and larger spectral ranges.

The PIKfyve complex regulates the early melanosome homeostasis required for physiological amyloid formation.

The metabolism of PI(3,5)P2 is regulated by the PIKfyve, VAC14 and FIG4 complex, mutations in which are associated with hypopigmentation in mice. These pigmentation defects indicate a key, but as yet unexplored, physiological relevance of this complex in the biogenesis of melanosomes. Here, we show that PIKfyve activity regulates formation of amyloid matrix composed of PMEL protein within the early endosomes in melanocytes, called stage I melanosomes. PIKfyve activity controls the membrane remodeling of stage I melanosomes, which regulates PMEL abundance, sorting and processing. PIKfyve activity also affects stage I melanosome kiss-and-run interactions with lysosomes, which are required for PMEL amyloidogenesis and the establishment of melanosome identity. Mechanistically, PIKfyve activity promotes both the formation of membrane tubules from stage I melanosomes and their release by modulating endosomal actin branching. Taken together, our data indicate that PIKfyve activity is a key regulator of the melanosomal import-export machinery that fine tunes the formation of functional amyloid fibrils in melanosomes and the maintenance of melanosome identity.This article has an associated First Person interview with the first author of the paper.

Three Dimensional Triply Resonant Sum Frequency Spectroscopy Revealing Vibronic Coupling in Cobalamins: Toward a Probe of Reaction Coordinates.

Triply resonant sum frequency (TRSF) spectroscopy is a fully coherent mixed vibrational-electronic spectroscopic technique that is ideally suited for probing the vibrational-electronic couplings that become important in driving reactions. We have used cyanocobalamin (CNCbl) and deuterated aquacobalamin (D2OCbl+) as model systems for demonstrating the feasibility of using the selectivity of coherent multidimensional spectroscopy to resolve electronic states within the broad absorption spectra of transition metal complexes and identify the nature of the vibrational and electronic state couplings. We resolve three short and long axis vibrational modes in the vibrationally congested 1400-1750 cm-1 region that are individually coupled to different electronic states in the 18 000-21 000 cm-1 region but have minimal coupling to each other. Double resonance with the individual vibrational fundamentals and their overtones selectively enhances the corresponding electronic resonances and resolves features within the broad absorption spectrum. This work demonstrates the feasibility of identifying coupling between different pairs of vibrational states with different electronic states that together form the reaction coordinate surface of transition metal enzymes.

Francisella tularensis subsp. tularensis group A.I, United States.

We used whole-genome analysis and subsequent characterization of geographically diverse strains using new genetic signatures to identify distinct subgroups within Francisella tularensis subsp. tularensis group A.I: A.I.3, A.I.8, and A.I.12. These subgroups exhibit complex phylogeographic patterns within North America. The widest distribution was observed for A.I.12, which suggests an adaptive advantage.

Genetic interaction between MTMR2 and FIG4 phospholipid phosphatases involved in Charcot-Marie-Tooth neuropathies.

We previously reported that autosomal recessive demyelinating Charcot-Marie-Tooth (CMT) type 4B1 neuropathy with myelin outfoldings is caused by loss of MTMR2 (Myotubularin-related 2) in humans, and we created a faithful mouse model of the disease. MTMR2 dephosphorylates both PtdIns3P and PtdIns(3,5)P(2), thereby regulating membrane trafficking. However, the function of MTMR2 and the role of the MTMR2 phospholipid phosphatase activity in vivo in the nerve still remain to be assessed. Mutations in FIG4 are associated with CMT4J neuropathy characterized by both axonal and myelin damage in peripheral nerve. Loss of Fig4 function in the plt (pale tremor) mouse produces spongiform degeneration of the brain and peripheral neuropathy. Since FIG4 has a role in generation of PtdIns(3,5)P(2) and MTMR2 catalyzes its dephosphorylation, these two phosphatases might be expected to have opposite effects in the control of PtdIns(3,5)P(2) homeostasis and their mutations might have compensatory effects in vivo. To explore the role of the MTMR2 phospholipid phosphatase activity in vivo, we generated and characterized the Mtmr2/Fig4 double null mutant mice. Here we provide strong evidence that Mtmr2 and Fig4 functionally interact in both Schwann cells and neurons, and we reveal for the first time a role of Mtmr2 in neurons in vivo. Our results also suggest that imbalance of PtdIns(3,5)P(2) is at the basis of altered longitudinal myelin growth and of myelin outfolding formation. Reduction of Fig4 by null heterozygosity and downregulation of PIKfyve both rescue Mtmr2-null myelin outfoldings in vivo and in vitro.

Ultrafast reversible self-assembly of living tangled matter.

Tangled active filaments are ubiquitous in nature, from chromosomal DNA and cilia carpets to root networks and worm collectives. How activity and elasticity facilitate collective topological transformations in living tangled matter is not well understood. We studied California blackworms (Lumbriculus variegatus), which slowly form tangles in minutes but can untangle in milliseconds. Combining ultrasound imaging, theoretical analysis, and simulations, we developed and validated a mechanistic model that explains how the kinematics of individual active filaments determines their emergent collective topological dynamics. The model reveals that resonantly alternating helical waves enable both tangle formation and ultrafast untangling. By identifying generic dynamical principles of topological self-transformations, our results can provide guidance for designing classes of topologically tunable active materials.

Collecting-Gathering Biophysics of the Blackworm L. variegatus.

Many organisms exhibit collecting and gathering behaviors as a foraging and survival method. Certain benthic macroinvertebrates are classified as collector-gatherers due to their collection of particulate matter as a food source, such as the aquatic oligochaete Lumbriculus variegatus (California blackworms). Blackworms demonstrate the ability to ingest organic and inorganic materials, including microplastics, but previous work has only qualitatively described their possible collecting behaviors for such materials. The mechanism through which blackworms consolidate discrete particles into a larger clumps remains unexplored quantitatively. By analyzing a group of blackworms in a large arena with an aqueous algae solution, we discover that their relative collecting efficiency is proportional to population size. Examining individual blackworms under a microscope reveals that both algae and microplastics physically adhere to the worm's body due to external mucus secretions, which cause the materials to clump around the worm. We observe that this clumping reduces the worm's exploration of its environment, potentially due to thigmotaxis. To validate the observed biophysical mechanisms, we create an active polymer model of a worm moving in a field of particulate debris with a short-range attractive force on its body to simulate its adhesive nature. We find that the attractive force increases gathering efficiency. This study offers insights into the mechanisms of collecting-gathering behavior, informing the design of robotic systems, as well as advancing our understanding the ecological impacts of microplastics on benthic invertebrates.

Collecting-Gathering Biophysics of the Blackworm Lumbriculus variegatus.

Many organisms exhibit collecting and gathering behaviors as a foraging and survival method. Benthic macroinvertebrates are classified as collector-gatherers due to their collection of particulate matter. Among these, the aquatic oligochaete Lumbriculus variegatus (California blackworms) demonstrates the ability to ingest both organic and inorganic materials, including microplastics. However, earlier studies have only qualitatively described their collecting behaviors for such materials. The mechanism by which blackworms consolidate discrete particles into a larger clump remains unexplored quantitatively. In this study, we analyze a group of blackworms in a large arena with an aqueous algae solution (organic particles) and find that their relative collecting efficiency is proportional to population size. We found that doubling the population size (N = 25-N = 50) results in a decrease in time to reach consolidation by more than half. Microscopic examination of individual blackworms reveals that both algae and microplastics physically adhere to the worm's body and form clumps due to external mucus secretions by the worms. Our observations also indicate that this clumping behavior reduces the worm's exploration of its environment, possibly due to thigmotaxis. To validate these observed biophysical mechanisms, we create an active polymer model of a worm moving in a field of particulate debris. We simulate its adhesive nature by implementing a short-range attraction between the worm and the nearest surrounding particles. Our findings indicate an increase in gathering efficiency when we add an attractive force between particles, simulating the worm's mucosal secretions. Our work provides a detailed understanding of the complex mechanisms underlying the collecting-gathering behavior in L. variegatus, informing the design of bioinspired synthetic collector systems, and advances our understanding of the ecological impacts of microplastics on benthic invertebrates.

Oxygenation-Controlled Collective Dynamics in Aquatic Worm Blobs.

Many organisms utilize group aggregation as a method for survival. The freshwater oligochaete, Lumbriculus variegatus (California blackworms) form tightly entangled structures, or worm "blobs", that have adapted to survive in extremely low levels of dissolved oxygen (DO). Individual blackworms adapt to hypoxic environments through respiration via their mucous body wall and posterior ciliated hindgut, which they wave above them. However, the change in collective behavior at different levels of DO is not known. Using a closed-loop respirometer with flow, we discover that the relative tail reaching activity flux in low DO is ∼75x higher than in the high-DO condition. Additionally, when flow rate is increased to suspend the worm blobs upward, we find that the average exposed surface area of a blob in low DO is ∼1.4x higher than in high DO. Furthermore, we observe emergent properties that arise when a worm blob is exposed to extreme DO levels. We demonstrate that internal mechanical stress is generated when worm blobs are exposed to high DO levels, allowing them to be physically lifted off from the bottom of a conical container using a serrated endpiece. Our results demonstrate how both collective behavior and the emergent generation of internal mechanical stress in worm blobs change to accommodate differing levels of oxygen. From an engineering perspective, this could be used to model and simulate swarm robots, self-assembly structures, or soft material entanglements.

The effect of aerosolized bacterial lysate on experimentally induced Mannheimia haemolytica pneumonia in calves.

Bovine respiratory disease (BRD) often occurs during specific periods of increased susceptibility when stress, viral infection, or reduced air quality are thought to suppress respiratory defences. The innate immune system is rapidly responsive and broadly protective and could be a target for preventing BRD during these periods of increased susceptibility. This study tested the hypothesis that stimulation of pulmonary innate immune responses by aerosol delivery of a lysate of killed Escherichia coli and Staphylococcus aureus bacteria would protect calves against Mannheimia haemolytica pneumonia. Ten clean-catch colostrum-deprived Holstein calves were randomly assigned to receive either aerosolized bacterial lysate or saline 24 hours before M. haemolytica challenge. Effects of this treatment on clinical, hematologic, microbiologic, and pathologic outcomes were assessed. Compared to controls, lysate-treated calves had lower serum haptoglobin and blood leukocyte and neutrophil concentrations following M. haemolytica challenge. There were no differences in temperature, heart and respiratory rates, clinical scores, ultrasound lesions, or number of M. haemolytica in the nasal cavity or lung. Thus, treatment with bacterial lysate prior to M. haemolytica challenge appeared to ameliorate early measures of inflammation but did not provide sufficient protection to substantially alter the course of disease.

La maladie respiratoire bovine (BRD) survient souvent pendant des périodes spécifiques de sensibilité accrue lorsque le stress, une infection virale ou une qualité de l'air réduite sont censés supprimer les défenses respiratoires. Le système immunitaire inné est rapidement réactif et largement protecteur et pourrait être une cible pour prévenir la BRD pendant ces périodes de sensibilité accrue. Cette étude a testé l'hypothèse selon laquelle la stimulation des réponses immunitaires innées pulmonaires par la délivrance d'aérosols d'un lysat de bactéries Escherichia coli et Staphylococcus aureus tuées protégerait les veaux contre la pneumonie à Mannheimia haemolytica. Dix veaux Holstein dont on a limité la contamination bactérienne et privés de colostrum ont été répartis au hasard pour recevoir soit un lysat bactérien en aérosol, soit une solution saline 24 heures avant une infection défi par M. haemolytica. Les effets de ce traitement sur les résultats cliniques, hématologiques, microbiologiques et pathologiques ont été évalués. Comparativement aux témoins, les veaux traités au lysat présentaient des concentrations sériques d'haptoglobine et de leucocytes et de neutrophiles sanguins plus faibles après la provocation par M. haemolytica. Il n'y avait aucune différence dans la température, les fréquences cardiaques et respiratoires, les scores cliniques, les lésions échographiques ou le nombre de M. haemolytica dans la cavité nasale ou les poumons. Ainsi, le traitement avec un lysat bactérien avant la provocation par M. haemolytica a semblé améliorer les réactions précoces de l'inflammation mais n'a pas fourni une protection suffisante pour modifier substantiellement l'évolution de la maladie.(Traduit par Docteur Serge Messier).

Phylogeography of Francisella tularensis subspecies holarctica from the country of Georgia.

BACKGROUND: Francisella tularensis, the causative agent of tularemia, displays subspecies-specific differences in virulence, geographic distribution, and genetic diversity. F. tularensis subsp. holarctica is widely distributed throughout the Northern Hemisphere. In Europe, F. tularensis subsp. holarctica isolates have largely been assigned to two phylogenetic groups that have specific geographic distributions. Most isolates from Western Europe are assigned to the B.Br.FTNF002-00 group, whereas most isolates from Eastern Europe are assigned to numerous lineages within the B.Br.013 group. The eastern geographic extent of the B.Br.013 group is currently unknown due to a lack of phylogenetic knowledge about populations at the European/Asian juncture and in Asia. In this study, we address this knowledge gap by describing the phylogenetic structure of F. tularensis subsp. holarctica isolates from the country of Georgia, and by placing these isolates into a global phylogeographic context. RESULTS: We identified a new genetic lineage of F. tularensis subsp. holarctica from Georgia that belongs to the B.Br.013 group. This new lineage is genetically and geographically distinct from lineages previously described from the B.Br.013 group from Central-Eastern Europe. Importantly, this new lineage is basal within the B.Br.013 group, indicating the Georgian lineage diverged before the diversification of the other known B.Br.013 lineages. Although two isolates from the Georgian lineage were collected nearby in the Ukrainian region of Crimea, all other global isolates assigned to this lineage were collected in Georgia. This restricted geographic distribution, as well as the high levels of genetic diversity within the lineage, is consistent with a relatively older origin and localized differentiation. CONCLUSIONS: We identified a new lineage of F. tularensis subsp. holarctica from Georgia that appears to have an older origin than any other diversified lineages previously described from the B.Br.013 group. This finding suggests that additional phylogenetic studies of F. tularensis subsp. holarctica populations in Eastern Europe and Asia have the potential to yield important new insights into the evolutionary history and phylogeography of this broadly dispersed F. tularensis subspecies.

Zinc Source and Concentration Altered Physiological Responses of Beef Heifers during a Combined Viral-Bacterial Respiratory Challenge.

To determine the effects of zinc supplementation on the immune response to a combined viral-bacterial respiratory disease challenge, thirty-two beef heifers (255 ± 15 kg) were subjected to a 30-d period of Zn depletion, then randomly assigned to one of three treatment diets fed for 30 d before the challenge: (1) supplementation with 100 mg of Zn from Zn sulfate/kg of DM (Zn100), (2) supplementation with 200 mg of Zn from Zn sulfate/kg of DM (Zn200), and (3) supplementation with 80 mg of Zn/kg of DM from zinc methionine and 20 mg of Zn from Zn sulfate/kg of DM (ZinMet). After the 30-d supplementation period, all heifers were fitted with indwelling vaginal temperature (VT) devices and intra-nasally challenged with 1 × 108 PFU bovine herpesvirus-1 on d -3, and then allowed to rest in outdoor pens for 3 d. On d 0, each heifer was challenged intra-tracheally with an average dose of 2.38 × 107 CFU Mannheimia haemolytica (MH), fitted with an indwelling jugular catheter, and then moved into individual stalls in an environmentally-controlled enclosed barn. Whole blood samples were collected at 1-h (serum) and 2-h (complete blood counts) intervals from 0 to 8 h, and at 12, 24, 36, 48, 60, 72, 168, and 360 h relative to MH challenge. Data were analyzed using the MIXED procedure of SAS specific for repeated measures with fixed effects of treatment, time, and their interaction. There was a treatment effect (p < 0.01) for VT such that Zn200 heifers had greater VT than Zn100 and ZinMet heifers. There was a trend (p = 0.10) for a serum cortisol treatment effect with Zn100 heifers having greater cortisol than ZinMet heifers. Total leukocytes and lymphocytes were greater (p ≤ 0.01) in Zn100 heifers than Zn200 and ZinMet heifers, whereas monocytes were less (p = 0.05) in ZinMet heifers than Zn100 and Zn200 heifers. Concentrations of IL-6 were greater (p = 0.02) in ZinMet heifers than Zn100 and Zn200 heifers. Concentrations of IFN-γ were greater in Zn200 heifers than ZinMet heifers at 0 h, and Zn100 heifers from 0 to 12 h post-MH challenge (treatment x time p = 0.02). Serum haptoglobin was not affected by treatment or treatment x time (p ≥ 0.36) but increased over time (p < 0.01) in all groups. There was a trend (p = 0.11) for ZinMet heifers to have less severe nasal lesion scores than Zn100 heifers. The observed differential physiological responses in this study indicate that zinc source and concentration may alter the response to a bovine respiratory challenge in heifers.

Effect of aerosolized bacterial lysate on development of naturally occurring respiratory disease in beef calves.

BACKGROUND: Bovine respiratory disease (BRD) is a major problem affecting beef cattle after arrival to feedlots. Alternatives to antibiotics are needed for prevention. HYPOTHESIS: Stimulation of pulmonary innate immune responses at the time of arrival to a feedlot reduces the occurrence and severity of BRD. ANIMALS: Sixty beef steers at high risk of BRD. METHODS: Randomized, double-blinded, placebo-controlled study. Calves received saline or a lysate of Staphylococcus aureus and Escherichia coli by aerosol, at 16 hours after feedlot arrival. Calves were monitored for 28 days for disease outcomes and levels of Mycoplasma bovis and Mannheimia haemolytica in nasal swabs. RESULTS: Death from M bovis pneumonia was significantly greater in lysate-treated animals (6/29, 24%) compared to controls (1/29, 3%; odds ratio = 10.2; 95% confidence interval [CI] = 1.1-96.0; P = .04). By 28 days after arrival, 29/29 lysate-treated calves had ultrasonographic pulmonary consolidation compared to 24/29 control calves (P = .05). Lysate-treated calves had lower weight gain compared to control calves (-8.8 kg, 95% CI = -17.1 to -0.5; P = .04), and higher body temperatures on days 4, 7, and 21 (0.19°C; 95% CI = 0.01-0.37; P = .04). Nasal M bovis numbers increased over time and were higher in lysate-treated calves (0.76 log CFU, 95% CI = 0.3-1.2; P = .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Aerosol administration of a bacterial lysate exacerbated BRD in healthy high-risk beef calves, suggesting that respiratory tract inflammation adversely affects how calves respond to subsequent natural infection with M bovis and other respiratory pathogens.

Immune Responses and Performance Are Influenced by Respiratory Vaccine Antigen Type and Stress in Beef Calves.

The study objective was to determine if a combined weaning and transportation stress model affected performance, antibody, endocrine, or hematological responses to modified-live virus (MLV) or killed virus (KV) respiratory vaccination in beef steers. In total, 48 calves (Day 0 BW = 226 ± 6.2 kg) from a single origin were used in a 2 × 2 factorial to evaluate main effects of stress model, vaccine type, and their interaction, resulting in four treatments (n = 12/treatment) including non-stress control (C) with KV (CKV), C with MLV (CMLV), stress model implementation (S) with KV (SKV), and S with MLV (SMLV). The C calves were weaned at the origin ranch on Day -37 and transported 472 km to the study site on Day -21 to allow acclimation. The S calves were weaned on Day -3, transported 460 km to a research facility on Day -2, held overnight, and transported 164 km to the study site on Day -1 to mimic the beef cattle marketing process. Vaccines were administered on Day 0 and KV was revaccinated on Day 14. The animal was the experimental unit and dependent variables were analyzed using PROC MIXED with repeated measures (day). A stress model effect (p = 0.01) existed for DMI from Day 0 to Day 7 with greater DMI for C (6.19 vs. 4.64 kg/day) when compared to S. The MLV groups had reduced (p = 0.05) ADG from Day 0 to Day 56, compared to KV. There was a vaccine type × day (p < 0.01) interaction with increased (p ≤ 0.01) PI3V- and IBRV-specific antibody titers for KV on Day 21; conversely, MLV had increased (p ≤ 0.01) BVDV titers on Days 14, 28, 35, 42, 49, and 56. Increased (p ≤ 0.05) BRSV titers were observed in a stress model × day (p < 0.01) interaction for S on Days 21, 28, 36, and 42; however, C exceeded S in BVDV-specific antibody concentration on Days 21, 28, and 49. A day effect (p < 0.01) was observed for serum haptoglobin with the greatest (p < 0.01) concentration on Day 3. Serum cortisol concentration was greater (p ≤ 0.04) for C vs. S on Days -2, 0, 1, 3, and 5. Total leukocytes were decreased for C vs. S on Days 0, 1, 3, 5, 7, 14, and 21 (p ≤ 0.02). A reduction (p ≤ 0.04) in total leukocytes was observed for MLV on Days 5, 7, and 14 vs. KV. Neutrophils and neutrophil:lymphocyte were markedly increased (p ≤ 0.01) for S on Day -2, whereas neutrophils were decreased (p ≤ 0.01) on Days 1 and 21 for S. Monocytes were decreased on Days 1, 5 and 7 for MLV (p ≤ 0.04) and Days -2 to 14 for S (p ≤ 0.03). Eosinophils were reduced (p = 0.007) for S vs. C on Day -2, yet a distinct rebound response (p = 0.03) was noted for S on Day 0. The results indicate that S and MLV vaccination more profoundly induced immunomodulation in beef calves.

Changes in antimicrobial susceptibility profiles of Mycoplasma bovis over time.

Mycoplasma bovis is a major cause of pneumonia, arthritis, and mastitis in cattle and can lead to significant economic losses. Antimicrobial resistance is a concern and further limits the already short list of drugs effective against mycoplasmas. The objective of this study was to examine changes in in vitro minimum inhibitory concentrations (MICs) of antimicrobials of aminoglycoside, fluoroquinolone, lincosamide, macrolide, pleuromutilin, phenicol, and tetracycline classes for 210 M. bovis isolates collected from 1978 to 2009. The MIC50 values of the various antimicrobials were also compared. The MIC50 levels for enrofloxacin and danofloxacin remained low (0.25 µg/mL) across all 3 decades. MIC50 levels for tetracyclines, tilmicosin, and tylosin tartrate were low in the 1980s, then increased in the 1990s and remained high. In the 1980s, MIC50 levels were low for clindamycin, spectinomycin, and tulathromycin, increased in the 1990s to 8 µg/mL (clindamycin) and 32 µg/mL (spectinomycin and tulathromycin), then decreased again in the 2000s. Members of the fluoroquinolone class of antimicrobials had the lowest MIC50 levels across all 3 decades, which suggests in vitro susceptibility of M. bovis to this class of antimicrobials. Statistically significant associations were observed between MIC values for chlortetracycline, oxytetracycline, tylosin tartrate, and tilmicosin; between clindamycin, tulathromycin, spectinomycin, and tiamulin; and between tylosin tartrate and clindamycin. Changes in MIC levels of various antimicrobials over time show the importance of monitoring the susceptibility of mycoplasmas to antimicrobials. The number of antimicrobials that showed elevated MIC50 levels, and therefore possibly reduced in vitro effectiveness against M. bovis, supports initiatives that promote prudent use of antimicrobials in agriculture.

Mycoplasma bovis est une cause majeure de pneumonie, d'arthrite, et mammite chez les bovins et peut entrainer des pertes économiques significatives. La résistance antimicrobienne est une préoccupation et réduit encore plus la courte liste déjà existante de médicaments efficaces contre les mycoplasmes. L'objectif de la présente étude était d'examiner in vitro les changements des concentrations minimales inhibitrices (CMI) des antimicrobiens des classes des aminoglycosides, des fluoroquinolones, des lincosamides, des macrolides, des pleuromutilines, des phénicoles, et des tétracyclines envers 210 isolats de M. bovis collectionnés entre 1978 et 2009. Les valeurs de CMI50 des différents antimicrobiens ont également été comparées. Les valeurs de CMI50 de l'enrofloxacine et de la danofloxacine sont demeurées faibles (0,25 µg/mL) au cours des trois décennies. Les valeurs de CMI50 pour les tétracyclines, le tilmicosin et le tartrate de tylosine étaient basses dans les années 1980s, puis augmentèrent dans les années 1990s et sont demeurées élevées. Durant les années 1980s, les valeurs de CMI50 étaient basses pour la clindamycine, la spectinomycine, et la tulathromycine, augmentèrent dans les années 1990s jusqu'à 8 µg/mL (clindamycine) et 32 µg/mL (spectinomycine et tulathromycine), puis ont diminué encore dans les années 2000s. Les membres de la classe des fluoroquinolones avaient les valeurs de CMI50 les plus faibles au cours des trois décennies examinées, ce qui suggère une sensibilité in vitro de M. bovis à cette classe d'antibiotiques. Des associations statistiquement significatives furent notées entre les valeurs de CMI de la chlortétracycline, l'oxytétracycline, le tartrate de tylosine, et le tilmicosin; entre la clindamycine, la tulathromycine, la spectinomycine, et la tiamulin; et entre le tartrate de tyloosine et la clindamycine. Les changements dans les valeurs de CMI de différents antibiotiques dans le temps démontrent l'importance de suivre la sensibilité des mycoplasmes aux antimicrobiens. Le nombre d'antimicrobiens qui a démontré des valeurs élevées de CMI50, et ainsi une efficacité in vitro réduite envers M. bovis, encourage les initiatives qui font la promotion de l'usage prudent des antimicrobiens en agriculture.(Traduit par Docteur Serge Messier).

Effect of tracheal antimicrobial peptide on the development of Mannheimia haemolytica pneumonia in cattle.

Bacterial pneumonia causes significant economic loss to the beef industry and occurs at times of stress and viral infection. Administering antibiotics to at-risk calves is often used to prevent the disease, but alternatives to mass treatment with antibiotics are needed. Tracheal antimicrobial peptide (TAP), a ß-defensin naturally produced by bovine airways, has bactericidal activity against the pathogens that cause pneumonia in cattle. However, TAP expression is suppressed by glucocorticoid (stress) and viral infection. We hypothesized that delivering TAP to the respiratory tract would prevent development of pneumonia in calves infected with Mannheimia haemolytica. Clean-catch calves (i.e. obtained prior to contact with the dam) were challenged by aerosol with M. haemolytica, and TAP or water was delivered to the respiratory tract at 0.3, 2 and 6 hours post-infection. TAP treatment did not protect against development of disease. Calves treated with TAP had similar bacterial loads in the nasal cavity and lung compared to calves treated with water. Similarly, TAP treatment did not affect the development of clinical signs, elevated rectal temperatures, or increased levels of blood neutrophils, haptoglobin and fibrinogen that occurred after bacterial challenge. Postmortem gross and histologic lung lesions were also similar in the two groups. To determine why there was a lack of protective effect, we tested the effect of substances in respiratory lining fluid on the bactericidal activity of TAP. Physiologic concentrations of sodium chloride inhibited TAP bactericidal activity in vitro, as did serum at concentrations of 0.62 to 2.5%, but concentrated bronchoalveolar lavage fluid had no consistent effect. These findings suggest that TAP does not have in vivo bactericidal activity against M. haemolytica because of interference by physiological sodium chloride levels and by serum. Thus, administration of TAP may not be effective for prevention of M. haemolytica pneumonia.

Development of an aerosolized Mannheimia haemolytica experimental pneumonia model in clean-catch colostrum-deprived calves.

Mannheimia haemolytica is an important cause of bovine respiratory disease (BRD). BRD is usually a multifactorial disease with host factors and viral infections influencing pathogenesis. Previous studies that have attempted to experimentally induce pneumonia using aerosolized M. haemolytica alone have produced inconsistent results, yet an aerosol model would be useful to study the details of early infection and to investigate the role of innate defences in pathogenesis. The objective of these studies was to develop and characterize an aerosolized M. haemolytica disease model. In an initial study, conventionally raised calves with higher levels of antibody against M. haemolytica leukotoxin developed acute respiratory distress and diffuse alveolar damage, but did not develop bronchopneumonia, following challenge with M. haemolytica serotype 1. Clean-catch colostrum-deprived calves challenged with 1 × 1010 colony forming units of M. haemolytica serotype 1 consistently developed bronchopneumonia, with elevations in rectal temperature, serum haptoglobin, plasma fibrinogen, and blood neutrophils. Mannheimia haemolytica serotype 1 was consistently isolated from the nasal cavities and lungs of challenged calves. Despite distribution of aerosol and isolation of M. haemolytica in all lung lobes, gross lesions were mainly observed in the cranioventral area of lung. Gross and histologic lesions included neutrophilic bronchopneumonia and fibrinous pleuritis, with oat cells (necrotic neutrophils with streaming nuclei), and areas of coagulative necrosis, which are similar to lesions in naturally occurring BRD. Thus, challenge with M. haemolytica serotype 1 and use of clean-catch colostrum-deprived calves with low or absent antibody titres allowed development of an effective aerosol challenge model that induced typical clinical disease and lesions.

Range-wide genetic analysis of Dermacentor variabilis and its Francisella-like endosymbionts demonstrates phylogeographic concordance between both taxa.

BACKGROUND: The American dog tick, Dermacentor variabilis, is an important vector of pathogens to humans, wildlife and domestic animals in North America. Although this tick species is widely distributed in the USA and Canada, knowledge of its range-wide phylogeographic patterns remains incomplete. METHODS: We carried out a phylogenetic analysis of D. variabilis using samples collected from 26 USA states and five Canadian provinces. Tick samples (n = 1053 in total) originated from two main sources: existing archives (2000-2011), and new collections made from 2012 to 2013. We sequenced a 691 bp fragment of the cox1 gene from a subset (n = 332) of geographically diverse D. variabilis. DNA extracted from individual ticks (n = 1053) was also screened for a Francisella-like endosymbiont, using a targeted 16S rRNA sequencing approach, and important pathogens (Rickettsia spp. and Coxiella burnetii), using species-specific quantitative PCR assays. RESULTS: Maximum parsimony analysis of cox1 sequences revealed two major groups within D. variabilis with distinct geographical distributions: one from the eastern USA/Canada (Group 1) and one from the west coast states of the USA (California and Washington; Group 2). However, genetic subdivisions within both of these two major groups were weak to moderate and not tightly correlated with geography. We found molecular signatures consistent with Francisella-like endosymbionts in 257 of the DNA extracts from the 1053 individual ticks, as well as Rickettsia spp. and Coxiella burnetii in a small number of ticks (n = 29 and 2, respectively). Phylogenetic patterns for Francisella-like endosymbionts, constructed using sequence data from the bacterial 16S rRNA locus, were similar to those for D. variabilis, with two major groups that had a nearly perfect one-to-one correlation with the two major groups within D. variabilis. CONCLUSIONS: Our findings reveal a distinct phylogenetic split between the two major D. variabilis populations. However, high levels of genetic mixture among widely separated geographical localities occur within each of these two major groups. Furthermore, our phylogenetic analyses provide evidence of long-term tick-symbiont co-evolution. This work has implications for understanding the dispersal and evolutionary ecology of D. variabilis and associated vector-borne diseases.

Effects of binge alcohol exposure on Burkholderia thailandensis-alveolar macrophage interaction.

Alcohol consumption has diverse and well-documented effects on the human immune system and its ability to defend against infective agents. One example is melioidosis, a disease caused by infection with Burkholderia pseudomallei, which is of public health importance in Southeast Asia and Northern Australia, with an expanding global distribution. While B. pseudomallei infections can occur in healthy humans, binge alcohol use is progressively being recognized as a major risk factor. Although binge alcohol consumption has been considered as a risk factor for the development of melioidosis, no experimental studies have investigated the outcomes of alcohol exposure on Burkholderia spp. infection. Therefore, we proposed the use of non-pathogenic B. thailandensis E264 as a useful BSL-1 model system to study the effects of binge alcohol exposure on bacteria and alveolar macrophage interactions. The MH-S alveolar macrophage (AMs) cell line was used to characterize innate immune responses to infection in vitro. Our results showed that alcohol exposure significantly suppressed the uptake and killing of B. thailandensis by AMs. Alveolar macrophages incubated in alcohol (0.08%) for 3 h prior to infection showed significantly lower bacterial uptake at 2 and 8 h post infection. Activated AMs with IFN-γ and pre and post-incubation in alcohol when exposed to B. thailandensis released lower nitric oxide (NO) concentrations, compared to activated AMs with IFN-γ from non-alcoholic controls. As a result, B. thailandensis survival and replication increased ∼2.5-fold compared to controls. The presence of alcohol (1%) also increased bacterial survival within AMs. Alcohol significantly decreased bacterial motility compared to non-alcoholic controls. Increased biofilm formation was observed at 3 and 6 h when bacteria were pre-incubated in (0.08%) alcohol. These results provide insights into binge alcohol consumption, a culturally prevalent risk factor, as a predisposing factor for melioidosis.