Elizabeth G. Davis

Cathelicidins: microbicidal activity, mechanisms of action, and roles in innate immunity.

Antimicrobial peptides are important host-defense molecules of innate immunity. Cathelicidins are a diverse family of potent, rapidly acting and broadly effective antimicrobial peptides, which are produced by a variety of cells. This review examines the classification, antimicrobial spectrum, mechanism of action, and regulation of cathelicidins.

Characterization of peripheral blood and pulmonary leukocyte function in healthy foals

Studies in infants and foals indicate an age-dependent maturation of peripheral lymphocyte subsets. The age-dependent relationship for maturation of cellular immune responses, such as phagocytosis and lymphocyte responses of the peripheral and pulmonary-derived leukocytes, has not been characterized in foals. Lymphocyte subpopulations, mitogen stimulation response of lymphocytes, lymphokine-activated killing cell activity, phagocytosis and oxidative burst activity, and serum immunoglobulin (Ig) classes G and M concentrations were determined in developing foals. This study illustrates age-dependent changes in immunoglobulin class concentrations, lymphocyte subsets, and EqMHC Class II expression in cells of the peripheral blood and lungs of developing neonatal-to-weanling foals. The increase in peripheral blood and BAL B-lymphocytes and serum immunoglobulins in developing foals suggests expansion of immune cell populations during a time in which environmental pathogen exposure is great. General immune function, mitogenic responses, LAK cell activity, opsonized phagocytosis, and oxidative burst activity of newborns was similar to the adult horse. Total immune-cell numbers, rather than function, seemed to be the limiting factor in the development of the equine neonatal immune system. There was an age-related percent increase in the appearance of pulmonary lymphocytes, but a percent decrease in macrophages. Although development of the respiratory immune system follows changes in the peripheral blood, cellular expansion, activation, and migration may occur at a slower pace, making the respiratory environment susceptible to pathogens prior to optimal immune system maturity.

Innate Immunity and Host Defense Peptides in Veterinary Medicine

Recent years have witnessed a surge in interest directed at innate immune mechanisms. Proper conceptualization of the key elements of innate immunity, however, is still a work in progress, because most research in immunology traditionally has been focused on components of the acquired immune response. The question of why an animal stays healthy in a world filled with many dangers is perhaps as interesting as why it sometimes surrenders to disease. Consequently, studies with an increased focus on inborn mechanisms of animal host defense may help further the development of appropriate preventative and therapeutic measures in veterinary medicine. Host defense peptides (HDPs) are central effector molecules of innate immunity, and are produced by virtually all living species throughout the plant and animal kingdoms. These gene-encoded peptides play a central role in multiple, clinically relevant disease processes. Imbalances in the expression of HDPs can lead to overt pathology in different organ systems and cell types in all species studied. In addition, HDPs are an ancient group of innate chemical protectors, which are now evaluated as model molecules for the development of novel natural antibiotics and immunoregulatory compounds. This review provides an overview of HDPs and is aimed at veterinary practitioners as well as basic researchers with an interest in comparative immunology involving small and large animal species.

Equine recurrent airway obstruction: pathogenesis, diagnosis, and patient management

Recurrent airway obstruction is a condition that affects some older horses maintained in confinement. Clinical signs range from exercise intolerance with occasional cough to dyspnea at rest. Bronchoalveolar lavage cytology is characterized by neutrophilic leukocytosis (15%-85%) and is recommended for making the diagnosis in horses with mild to moderate disease. Environmental management combined with periodic bronchodilator and antiinflammatory corticosteroid therapy yields the best prognosis for disease remission.

Neonatal enterocolitis associated with coronavirus infection in a foal: a case report.

3. Edwards S, Sands JJ: 1994, Evidence of circovirus infection in British pigs. Vet Rec 134:680–681. 4. Ellis JA, Hassard L, Clarke EG, et al.: 1998, Isolation of circovirus-like virus from lesions with post-weaning multisystemic wasting syndrome. Can Vet J 39:44–51. 5. Ellis J, Krakowka S, Lairmore M, et al.: 1999, Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets. J Vet Diagn Invest 11:3–14. 6. Hamel AL, Lin LL, Nayar GPS: 1998, Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. J Virol 72:5262–5267. 7. Kennedy S, Allan G, McNeilly F, et al.: 1998, Procine circovirus infection in Northern Ireland. Vet Rec 142:495–496. 8. Kiupel M, Stevenson GW, Mittal SK, et al.: 1998, Circoviruslike viral associated disease in weaned pigs in Indiana. Vet Pathol 35:303–307. 9. LeCann P, Albina E, Madec F, et al.: 1997, Piglet wasting disease. Vet Rec 141:600. 10. Meehan BM, McNeilly F, Todd D, et al.: 1998, Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. J Gen Virol 79:2171–2179. 11. Morozov I. Sirinarumitr T, Sorden SD, et al.: 1998, Detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome. J Clin Microbiol 36:2535– 2541. 12. Segales J, Sitjar M, Dorningo M, et al.: 1997, First report of post weaning multisystemic wasting syndrome in Spain. Vet Rec 141:600–601. 13. Studdert MJ: 1993, Circoviridae: new viruses of pigs, parrots and chickens. Aust Vet J 4:121–122. 14. Tischer I, Rasch R, Tochtermann G: 1974, Characterization of papovavirusand piconavirus-like particles in permanent pig kidney cell lines. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Abt 1 Orig 26:153–167. 15. Todd D, Niagro FD, Ritchies BW, et al.: 1991, Comparison of three animals viruses with circular single-stranded DNA genomes. Arch Virol 117:129–135.

Simultaneous flow cytometric analysis of phagocytosis and oxidative burst activity in equine leukocytes.

This paper describes a method for simultaneously measuring phagocytosis and oxidative burst activity in equine peripheral blood leukocytes by flow cytometry. Opsonized propidium iodide-labelled Staphylococcus aureus (PI-Sa) was used to measure the uptake of bacteria by equine phacocytes and the oxidative burst activity by oxidation of dihydrorhodamine 123. The requirements to achieve optimal activity of phagocytosis and oxidative burst are described. The advantage of the simultaneous technique is that it provides both independent and comparative values for phagocytosis and the oxidative burst, for the detection of impaired mechanisms of microbial destruction. Furthermore, the technique allows evaluation of opsonization activity in this context.

Bayesian Geostatistical Analysis and Ecoclimatic Determinants of Corynebacterium pseudotuberculosis Infection among Horses

Kansas witnessed an unprecedented outbreak in Corynebacterium pseudotuberculosis infection among horses, a disease commonly referred to as pigeon fever during fall 2012. Bayesian geostatistical models were developed to identify key environmental and climatic risk factors associated with C. pseudotuberculosis infection in horses. Positive infection status among horses (cases) was determined by positive test results for characteristic abscess formation, positive bacterial culture on purulent material obtained from a lanced abscess (n = 82), or positive serologic evidence of exposure to organism (≥1:512)(n = 11). Horses negative for these tests (n = 172)(controls) were considered free of infection. Information pertaining to horse demographics and stabled location were obtained through review of medical records and/or contact with horse owners via telephone. Covariate information for environmental and climatic determinants were obtained from USDA (soil attributes), USGS (land use/land cover), and NASA MODIS and NASA Prediction of Worldwide Renewable Resources (climate). Candidate covariates were screened using univariate regression models followed by Bayesian geostatistical models with and without covariates. The best performing model indicated a protective effect for higher soil moisture content (OR = 0.53, 95% CrI = 0.25, 0.71), and detrimental effects for higher land surface temperature (≥35°C) (OR = 2.81, 95% CrI = 2.21, 3.85) and habitat fragmentation (OR = 1.31, 95% CrI = 1.27, 2.22) for C. pseudotuberculosis infection status in horses, while age, gender and breed had no effect. Preventative and ecoclimatic significance of these findings are discussed.

Pharmacokinetics of oral terbinafine in horses and Greyhound dogs

The objective of the study was to assess the pharmacokinetics of terbinafine administered orally to horses and Greyhound dogs. A secondary objective was to assess terbinafine metabolites. Six healthy horses and six healthy Greyhound dogs were included in the pharmacokinetic data. The targeted dose of terbinafine was 20 and 30 mg/kg for horses and dogs, respectively. Blood was collected at predetermined intervals for the quantification of terbinafine concentrations with liquid chromatography and mass spectrometry. The half-life (geometric mean) was 8.1 and 8.6 h for horses and Greyhounds, respectively. The mean maximum plasma concentration was 0.31 and 4.01 μg/mL for horses and Greyhounds, respectively. The area under the curve (to infinity) was 1.793 h·μg/mL for horses and 17.253 h·μg/mL for Greyhounds. Adverse effects observed in one study horse included pawing at the ground, curling lips, head shaking, anxiety and circling, but these resolved spontaneously within 30 min of onset. No adverse effects were noted in the dogs. Ions consistent with carboxyterbinafine, n-desmethylterbinafine, hydroxyterbinafine and desmethylhydroxyterbinafine were identified in horse and Greyhound plasma after terbinafine administration. Further studies are needed assessing the safety and efficacy of terbinafine in horses and dogs.

Determination of internal control for gene expression studies in equine tissues and cell culture using quantitative RT-PCR.

Quantitative reverse transcription polymerase chain reaction (RT-PCR) has become a basic, reliable and sensitive modern technique, in both biological research and clinical diagnosis, for investigation of gene expression and validation of cDNA microarray analysis. Accurate mRNA quantification using quantitative RT-PCR commonly requires data normalization through stable housekeeping genes (HKGs). Selection of HKGs for data normalization is critical for accurate mRNA quantification. Our objective was to evaluate a set of candidate HKGs as internal controls for gene expression studies using quantitative RT-PCR in equine tissues and cell culture. One-step quantitative RT-PCR for 6 HKGs was performed using total RNA from equine tissue samples and cultured peripheral blood mononuclear cells (PBMCs). The stability of HKGs was mainly evaluated by analysis of variance, analyses of the standard deviation and coefficient of variation of Ct, and change of Ct of HKGs between control and treated samples. 18S rRNA consistently showed the smallest standard deviation and coefficient of variation, and the least change of Ct between control and treated samples, thus was identified as the most stable HKG for mRNA data normalization in quantitative RT-PCR for studying gene expression in equine tissues and cultured PBMCs.

Molecular cloning and characterization of equine Toll-like receptor 9

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three leucine-rich repeat (LRR) motifs, with greater than 82% identity to human, monkey, bovine, canine, feline, porcine and ovine orthologs. Equine TLR9 mRNA expression was characterized for spleen, lymph node, and peripheral blood leukocyte samples. Flow cytometric analysis of equine TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in PMNs, CD4(+) and CD8(+) T-lymphocytes as well as other leukocytes; similar to human TLR9 expression. The conservation of equine TLR9 and high expression profile in leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity.