Intramuscular Administration of a Synthetic CpG-Oligodeoxynucleotide Modulates Functional Responses of Neutrophils of Neonatal Foals

Neutrophils play an important role in protecting against infection. Foals have age-dependent deficiencies in neutrophil function that may contribute to their predisposition to infection. Thus, we investigated the ability of a CpG-ODN formulated with Emulsigen to modulate functional responses of neutrophils in neonatal foals. Eighteen foals were randomly assigned to receive either a CpG-ODN with Emulsigen (N = 9) or saline intramuscularly at ages 1 and 7 days. At ages 1, 3, 9, 14, and 28, blood was collected and neutrophils were isolated from each foal. Neutrophils were assessed for basal and Rhodococcus equi-stimulated mRNA expression of the cytokines interferon-γ (IFN-γ), interleukin (IL)-4, IL-6, and IL-8 using real-time PCR, degranulation by quantifying the amount of β-D glucuronidase activity, and reactive oxygen species (ROS) generation using flow cytometry. In vivo administration of the CpG-ODN formulation on days 1 and 7 resulted in significantly (P<0.05) increased IFN-γ mRNA expression by foal neutrophils on days 3, 9, and 14. Degranulation was significantly (P<0.05) lower for foals in the CpG-ODN-treated group than the control group at days 3 and 14, but not at other days. No effect of treatment on ROS generation was detected. These results indicate that CpG-ODN administration to foals might improve innate and adaptive immune responses that could protect foals against infectious diseases and possibly improve responses to vaccination.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

High-throughput microscope counting of cyanobacteria using “cellcount”, a newly developed analysis package in the R programming language

Molecular approaches and novel method validations require the precise enumeration of cyanobacteria to validate cyanobacteria density, typically done via microscopic counts which are considered time consuming and technically challenging. Cell counting software tools, such as ImageJ, can help decrease enumeration time, but may offer little flexibility in software modifications and may incorrectly quantify different morphotypes. Here we provide an overview of the development and uses of the draft package cellcount, from the programming language R. We used previously published code described in Pokrzywinski et al. 2019 as a blueprint for the development of new functions and overall organization. The result is an open-source package capable of being expanded and modified by novice and experienced R users. Here, we analyzed concentrations of several species to demonstrate cellcount versatility and potential limitations. In addition, we compared cellcount against standard enumeration practices and in vivo pigment fluorescence to demonstrate ease of use and rapid analysis while maintaining the same accuracy. With the formation of this high-throughput approach, researchers can utilize cellcount for many applications, such as qPCR standard curve development, the development of biomass standard curves, and validation of emerging quantification techniques

Immunogenicity of an Electron Beam Inactivated <i>Rhodococcus equi</i> Vaccine in Neonatal Foals

<div><p><i>Rhodococcus equi</i> is an important pathogen of foals that causes severe pneumonia. To date, there is no licensed vaccine effective against <i>R. equi</i> pneumonia of foals. The objectives of our study were to develop an electron beam (eBeam) inactivated vaccine against <i>R. equi</i> and evaluate its immunogenicity. A dose of eBeam irradiation that inactivated replication of <i>R. equi</i> while maintaining outer cell wall integrity was identified. Enteral administration of eBeam inactivated <i>R. equi</i> increased interferon-γ production by peripheral blood mononuclear cells in response to stimulation with virulent <i>R. equi</i> and generated naso-pharyngeal <i>R. equi</i>-specific IgA in newborn foals. Our results indicate that eBeam irradiated <i>R. equi</i> administered enterally produce cell-mediated and upper respiratory mucosal immune responses, in the face of passively transferred maternal antibodies, similar to those produced in response to enteral administration of live organisms (a strategy which previously has been documented to protect foals against intrabronchial infection with virulent <i>R. equi</i>). No evidence of adverse effects was noted among vaccinated foals.</p></div