Tick microbial communities within enriched extracts of Amblyomma maculatum.

Our objective of this study was to explore the bacterial microbiome in fresh or fresh-frozen adult Amblyomma maculatum (Gulf Coast ticks) using extracts enriched for microbial DNA. We collected 100 questing adult A. maculatum, surface disinfected them, and extracted DNA from individual ticks collected the same day or after storage at -80 °C. Because only extracts with microbial DNA concentrations above 2 ng/µL were considered suitable for individual analysis, we expected fewer samples to meet these requirements. Of individual ticks extracted, 48 extracts met this minimum concentration. We pooled 20 additional extracts that had lower concentrations to obtain seven additional pools that met the minimum DNA concentration. Libraries created from these 55 samples were sequenced using an Illumina MiSeq platform, and data sets were analyzed using QIIME to identify relative abundance of microorganisms by phylum down to genus levels. Proteobacteria were in greatest abundance, followed by Actinobacteria, Firmicutes, and Bacteroidetes, at levels between 1.9% and 6.4% average relative abundance. Consistent with the Francisella-like endosymbiont known to be present in A. maculatum, the genus Francisella was detected at highest relative abundance (72.9%; SE 0.02%) for all samples. Among the top ten genera identified (relative abundance ≥ 0.5%) were potential extraction kit contaminants, Sphingomonas and Methylobacterium, the soil bacterium Actinomycetospora, and the known A. maculatum-associated genus, Rickettsia. Four samples had Rickettsia at greater than 1% relative abundance, while nine additional samples had Rickettsia at low (0.01-0.04%) relative abundance. In this study, we used the entire microbe-enriched DNA extract for whole ticks for microbiome analysis. A direct comparison of the microbiome in microbe-enriched DNA and total genomic DNA extracts from halves of the same tick would be useful to determine the utility of this extraction method in this system. We anticipate that future tick microbiome studies will be valuable to explore the influence of microbial diversity on pathogen maintenance and transmission, and to evaluate niche-specific microbiomes within individual tick tissues.

In vivo effects of aspirin and cyclosporine on regulatory T cells and T-cell cytokine production in healthy dogs.

Cyclosporine and aspirin are routinely used in combination to treat immune-mediated hemolytic anemia (IMHA) in dogs. Cyclosporine is a potent immunosuppressive agent that targets T cell production of the cytokines IL-2 and IFN-γ. Low-dose aspirin is often used to inhibit platelet function in dogs with IMHA, since these animals are prone to life-threatening thromboembolic disease. In rodents and humans, aspirin and cyclosporine have both been shown to variably affect T cell cytokine production, and also numbers of circulating regulatory T cells (Tregs). In dogs, it has not yet been determined if concurrent aspirin alters the effects of cyclosporine on T-cell cytokine expression, or if either drug influences Treg numbers. In a crossover study, seven healthy young adult dogs were given either oral high-dose cyclosporine (10 mg/kg Q12 h), oral low-dose aspirin (1 mg/kg Q24 h), oral high-dose aspirin (10 mg/kg Q12 h), or combined low-dose aspirin with cyclosporine, each for 8 days, with a washout of at least 2 weeks after each treatment. Activated T cell cytokine expression (IL-2 & IFN-γ) and percent CD4 + CD25 + FOXP3+ Tregs were evaluated using flow cytometry, both prior to and on the last day of treatment. The difference between pre- and post-treatment values for each group, as well as the difference between treatment groups, was evaluated. Cyclosporine significantly decreased IL-2 and IFN-γ expression when used alone or in combination with low-dose aspirin. High-dose aspirin, but not low-dose aspirin, also significantly decreased IL-2 expression, although the decrease was not as marked as that seen with cyclosporine alone or in combination with aspirin. Neither low-dose nor high-dose aspirin significantly affected IFN-γ expression. No drug or drug combination affected Treg numbers. Low-dose aspirin given with cyclosporine creates the same degree of T-cell cytokine suppression as does cyclosporine alone, suggesting that the two drugs can be used concurrently without significantly altering the immunosuppressive mechanism of action of cyclosporine.

Effects of Leukoreduction and Storage on Erythrocyte Phosphatidylserine Expression and Eicosanoid Concentrations in Units of Canine Packed Red Blood Cells.

BACKGROUND: Storage of canine packed red blood cells (pRBCs) can increase erythrocyte phosphatidylserine (PS) expression and eicosanoid concentrations. HYPOTHESIS/OBJECTIVES: To determine the effects of leukoreduction on erythrocyte PS expression and eicosanoid concentrations in stored units of canine pRBCs. Our hypothesis was that leukoreduction would decrease PS expression and eicosanoid concentrations. ANIMALS: Eight healthy dogs. METHODS: In a cross-over study, units of whole blood were leukoreduced (LR) or non-LR and stored (10 and 21 days) as pRBCs. Samples were collected at donation, and before and after a simulated transfusion. PS expression was measured by flow cytometry, and concentrations of arachidonic acid (AA), prostaglandin F2α (PGF2α ), prostaglandin E2 (PGE2 ), prostaglandin D2 (PGD2 ), thromboxane B2 (TXB2 ), 6-keto-prostaglandin F1α (6-keto-PGF1α ), and leukotriene B4 (LTB4 ) were quantified by liquid chromatography-mass spectrometry. RESULTS: There was no change in PS expression during leukoreduction, storage, and simulated transfusion for non-LR and LR units. Immediately after leukoreduction, there was a significant increase in TXB2 and PGF2α concentrations, but during storage, these eicosanoids decreased to non-LR concentrations. In both LR and non-LR units, 6-keto-PGF1α concentrations increased during storage and simulated transfusion, but there was no difference between unit type. There was no difference in AA, LTB4 , PGE2 , and PGD2 concentrations between unit types. CONCLUSIONS AND CLINICAL IMPORTANCE: Leukoreduction, storage, and simulated transfusion do not alter erythrocyte PS expression. Leukoreduction causes an immediate increase in concentrations of TXB2 and PGF2α , but concentrations decrease to non-LR concentrations with storage. Leukoreduction does not decrease the accumulation of 6-keto-PGF1α during storage.

Rickettsia parkeri and "Candidatus Rickettsia andeanae" in Questing Amblyomma maculatum (Acari: Ixodidae) From Mississippi.

Amblyomma maculatum Koch (Acari: Ixodidae), the primary vector for Rickettsia parkeri, may also be infected with a rickettsia of unknown pathogenicity, "Candidatus Rickettsia andeanae." Infection rates with these rickettsiae vary geographically, and coinfected ticks have been reported. In this study, infection rates of R. parkeri and "Ca. R. andeanae" were evaluated, and rickettsial DNA levels quantified, in 335 questing adult A. maculatum collected in 2013 (n = 95), 2014 (n = 139), and 2015 (n = 101) from Oktibbeha County, MS. Overall infection rates of R. parkeri and "Ca. R. andeanae" were 28.7% and 9.3%, respectively, with three additional A. maculatum (0.9%) coinfected. While R. parkeri-infected ticks were detected all three years (34.7% in 2013; 13.7% in 2014; 43.6% in 2015), "Ca. R. andeanae" was not detected in 2013, and was detected at rates of 10.8% in 2014, and 15.8% in 2015. Interestingly, rickettsial DNA levels in singly-infected ticks were significantly lower in "Ca. R. andeanae"-infected ticks compared to R. parkeri-infected ticks (P < 0.0001). Thus, both infection rates and rickettsial DNA levels were higher for R. parkeri than "Ca. R. andeanae." Infection rates of R. parkeri were also higher, and "Ca. R. andeanae" lower, here compared to A. maculatum reported previously in Kansas and Oklahoma. As we continue to monitor infection rates and levels, we anticipate that understanding temporal changes will improve our awareness of human risk for spotted fever rickettsioses. Further, these data may lead to additional studies to evaluate potential interactions among sympatric Rickettsia species in A. maculatum at the population level.

Effects of oral cyclosporine on canine T-cell expression of IL-2 and IFN-gamma across a 12-h dosing interval.

The duration of immunosuppressive effects following oral cyclosporine in dogs is unknown. This study used flow cytometry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to evaluate the effects of high-dose oral cyclosporine across a 12-h dosing interval. Expression of interleukin-2 (IL-2) and interferon-gamma (IFN-γ) was compared before and after 8 days of cyclosporine at 10 mg/kg every 12 h in six healthy dogs. Samples were collected at 0, 2, 4, and 8 h postdosing for analysis of unactivated and activated T-cell and whole blood cytokine expression using flow cytometry and qRT-PCR, respectively, and at 0, 2, 4, 6, 8, and 10 h postdosing for measurement of cyclosporine concentrations. Flow cytometry and qRT-PCR both demonstrated significant marked reductions in IL-2 and IFN-γ levels at 0, 2, 4, and 8 h after dosing compared to pretreatment levels (P < 0.05) for activated samples, with less consistent effects observed for unactivated samples. Both flow cytometry and qRT-PCR are viable techniques for measuring cyclosporine pharmacodynamics in dogs, yielding comparable results with activated samples. Two hours postdrug administration is the preferred time for concurrent assessment of peak drug concentration and cytokine expression, and T-cell activation is needed for optimal results.

Pharmacodynamic monitoring of canine T-cell cytokine responses to oral cyclosporine.

BACKGROUND: Pharmacodynamic assays measure the immunosuppressive effects of cyclosporine on T-cells and offer an alternative assessment of efficacy in individual patients. OBJECTIVE: To assess the immunosuppressive effects of high and low dosage cyclosporine on canine T-cells and to develop a novel testing system for individualized dose adjustment. ANIMALS: Seven healthy female Walker hounds. METHODS: Experimental study using a paired comparison design. Flow cytometry was used to measure T-cell expression of IL-2, IL-4, and IFN-γ. Cytokine expression 8 days after oral administration of high and low dosages of cyclosporine was compared to baseline and washout values, respectively. The high dosage was initially 10 mg/kg q12h and was then adjusted to attain established immunosuppressive trough blood drug concentrations (>600 ng/mL). The low dosage was 5 mg/kg q24h. RESULTS: High dosage cyclosporine resulted in significant decreases in IL-2 and IFN-γ expression (P = .0156, P = .0156), but not IL-4 expression (P = .2188). Low dosage cyclosporine was associated with a significant decrease in IFN-γ expression (P = .0156), while IL-2 expression was not affected (P = .1094). CONCLUSIONS AND CLINICAL IMPORTANCE: T-cell function is suppressed at trough blood drug concentrations exceeding 600 ng/mL, and is at least partially suppressed in some dogs at low dosages. Direct evaluation of T-cell function could be an effective, more sensitive alternative to measuring blood drug concentrations for monitoring immunosuppressive therapy.

Cyclosporine A affects the in vitro expression of T cell activation-related molecules and cytokines in dogs.

Cyclosporine is a powerful immunosuppressive drug that is being used with increasing frequency to treat a wide range of immune-mediated diseases in the dog. To date, ideal dosing protocols that will achieve immunosuppression with cyclosporine in dogs remain unclear, and standard methods that can measure effectiveness of immunosuppression have not been established. The aim of our study was to evaluate the effects of in vitro cyclosporine exposure on a panel of molecules expressed by activated T cells to ascertain their potential as biomarkers of immunosuppression in dogs. Blood was drawn from six healthy dogs, and peripheral blood mononuclear cells (PBMC) were isolated and activated. Half of the cells were incubated with 200 ng/mL cyclosporine prior to activation, and the other half were not exposed to cyclosporine. Samples were analyzed using flow cytometry, and the expression of intracellular cytokines IL-2, IL-4, and IFN-γ was evaluated after 6, 12, and 24h of drug exposure. Each cytokine exhibited a time-dependent suppression profile, and all but two samples activated in the presence of cyclosporine showed lower cytokine expression than untreated controls. We also evaluated the expression of the surface T cell activation molecules CD25 and CD95 by flow cytometry after 36 h of drug exposure. Expression of these surface molecules decreased significantly when activated in the presence of cyclosporine. Our results suggest that suppressed expression of the markers related to T cell activation could potentially be utilized as an indicator of the efficacy of cyclosporine therapy in dogs.