Screening of Antimicrobial Activity of Essential Oils against Bovine Respiratory Pathogens - Focusing on Pasteurella multocida.

Administration of essential oils as natural plant products with antimicrobial activity might be an alternative to antibiotic treatment of bovine respiratory disease. The aim of this study was to analyse the in vitro antimicrobial activity of 11 essential oils against Pasteurella multocida isolated from the respiratory tract of calves using microdilution with determination of minimum inhibitory and bactericidal concentration as well as agar disc diffusion. Additionally, antimicrobial activity against Mannheimia haemolytica and bacteria in the Mannheimia clade was assessed by agar disc diffusion. Seven essential oil mixtures were also tested against all bacterial isolates. P. multocida was strongly inhibited by cinnamon cassia and lemongrass oil followed by coriander, winter savory, thyme, clove, and peppermint oil in the microdilution assays. Eucalyptus, wintergreen, spruce, and star anise oil showed lower activity. Comparison of both methods revealed an underestimation of cinnamon cassia oil activity by agar disc diffusion and conflicting results for wintergreen oil in microdilution, which precipitated in broth. Cinnamon cassia, thyme, wintergreen, lemongrass, and winter savory oil all showed strong antimicrobial activity against M. haemolytica. Bacteria in the Mannheimia clade were mostly inhibited by cinnamon cassia and thyme oil. Pasteurella isolates were more susceptible to inhibition by essential oils than Mannheimia isolates. Essential oil mixtures did not show stronger antibacterial activity than single essential oils. In conclusion, cinnamon cassia and lemongrass as well as coriander, winter savory, and thyme oil are promising candidates for treatment of P. multocida-associated bovine respiratory infections.

Antifungal in vitro Activity of Essential Oils against Clinical Isolates of Malassezia pachydermatis from Canine Ears: A Report from a Practice Laboratory.

BACKGROUND: The yeast Malassezia pachydermatis is a commensal but also the most isolated pathogenic yeast in canine otitis externa. Essential oils (EOs) exhibit antimicrobial activity and thus could be an alternative for treatment of otitis. OBJECTIVE: The aim of this study was to analyse the in vitro efficacy of EOs against M. pachydermatis isolates from canine ears. MATERIALS AND METHODS: Twenty-two EOs were tested for their in vitro activity against 15 M. pachydermatis isolates by agar disc diffusion. In addition, diluted EOs were tested and volatile antifungal activity was analysed by vapour assay. RESULTS: Most EOs showed in vitro efficacy against M. pachydermatis. A different susceptibility pattern of tested isolates was found. Overall, the most potential EO was lemon grass. Also, cinnamon leaf, clove, manuka, Indian melissa, oregano, palmarosa, and winter savory oil exhibited a strong antifungal activity. Volatile activity was proven by the inhibiting effect in vapour assay. CONCLUSION: EOs show in vitro activity against M. pachydermatis. This study gives scientific background for the use of EOs in veterinary medicine. Diverse susceptibility patterns of different M. pachydermatis strains emphasise the helpfulness of an aromatogram as one lead for the best choice of an EO for therapy.

Distinct Features of Canine Non-conventional CD4-CD8α- Double-Negative TCRαß+ vs. TCRγδ+ T Cells.

The role of conventional TCRαß+CD4+ or TCRαß+CD8α+ single-positive (sp) T lymphocytes in adaptive immunity is well-recognized. However, non-conventional T cells expressing TCRαß or TCRγδ but lacking CD4 and CD8α expression [i.e., CD4-CD8α- double-negative (dn) T cells] are thought to play a role at the interface between the innate and adaptive immune system. Dn T cells are frequent in swine, cattle or sheep and predominantly express TCRγδ. In contrast, TCRγδ+ T cells are rare in dogs. In this study, we identified a high proportion of canine dn T cells in the TCRαß+ T cell population of PBMC, lymphatic and non-lymphatic organs. In PBMC, the frequency of this T cell subpopulation made up one third of the frequency of TCRαß+CD4+ sp, and almost half of the frequency of TCRαß+CD8α+ sp T cells (i.e., ~15% of all TCRαß+ T cells). Among TCRαß+CD4-CD8α- dn T cells of PBMC and tissues, FoxP3+ cells were identified indicating regulatory potential of this T cell subset. 80% of peripheral blood FoxP3+TCRαß+CD4-CD8α- dn T cells co-expressed CD25, and, interestingly, also the FoxP3-negative TCRαß+CD4-CD8α- dn T cells comprised ~34% CD25+ cells. Some of the FoxP3-positive TCRαß+CD4-CD8α- dn T cells co-expressed GATA-3 suggesting stable function of regulatory T cells. The frequency of GATA-3 expression by FoxP3-TCRαß+CD4-CD8α- dn T cells was even higher as compared with TCRαß+CD4+ sp T cells (20.6% vs. 11.9%). Albeit lacking FoxP3 and CD25 expression, TCRγδ+CD4-CD8α- dn T cells also expressed substantial proportions of GATA-3. In addition, TCRαß+CD4-CD8α- dn T cells produced IFN-γ and IL-17A upon stimulation. T-bet and granzyme B were only weakly expressed by both dn T cell subsets. In conclusion, this study identifies two dn T cell subsets in the dog: (i) a large (~7.5% in Peyer's patches, ~15% in lung) population of TCRαß+CD4-CD8α- dn T cells with subpopulations thereof showing an activated phenotype, high expression of FoxP3 or GATA-3 as well as production of IFN-γ or IL-17A and (ii) a small TCRγδ+CD4-CD8α- dn T cell subset also expressing GATA-3 without production of IFN-γ or IL-17A. It will be exciting to unravel the function of each subset during immune homeostasis and diseases of dogs.

Canine tissue-associated CD4+CD8α+ double-positive T cells are an activated T cell subpopulation with heterogeneous functional potential.

Canine CD4+CD8α+ double-positive (dp) T cells of peripheral blood are a unique effector memory T cell subpopulation characterized by an increased expression of activation markers in comparison with conventional CD4+ or CD8α+ single-positive (sp) T cells. In this study, we investigated CD4+CD8α+ dp T cells in secondary lymphatic organs (i.e. mesenteric and tracheobronchial lymph nodes, spleen, Peyer's patches) and non-lymphatic tissues (i.e. lung and epithelium of the small intestine) within a homogeneous group of healthy Beagle dogs by multi-color flow cytometry. The aim of this systematic analysis was to identify the tissue-specific localization and characteristics of this distinct T cell subpopulation. Our results revealed a mature extrathymic CD1a-CD4+CD8α+ dp T cell population in all analyzed organs, with highest frequencies within Peyer's patches. Constitutive expression of the activation marker CD25 is a feature of many CD4+CD8α+ dp T cells independent of their localization and points to an effector phenotype. A proportion of lymph node CD4+CD8α+ dp T cells is FoxP3+ indicating regulatory potential. Within the intestinal environment, the cytotoxic marker granzyme B is expressed by CD4+CD8α+ dp intraepithelial lymphocytes. In addition, a fraction of CD4+CD8α+ dp intraepithelial lymphocytes and of mesenteric lymph node CD4+CD8α+ dp T cells is TCRγδ+. However, the main T cell receptor of all tissue-associated CD4+CD8α+ dp T cells could be identified as TCRαß. Interestingly, the majority of the CD4+CD8α+ dp T cell subpopulation expresses the unconventional CD8αα homodimer, in contrast to CD8α+ sp T cells, and CD4+CD8α+ dp thymocytes which are mainly CD8αß+. The presented data provide the basis for a functional analysis of tissue-specific CD4+CD8α+ dp T cells to elucidate their role in health and disease of dogs.

Antibakterielle In-vitro-Wirksamkeit ätherischer Öle gegen veterinärmedizinisch relevante Keime klinischer Isolate von Hunden, Katzen und Pferden.

Einleitung: Ätherische Öle sind die Grundlage der Aromatherapie. Unter anderem wird ihnen eine antibakterielle Wirkung zugeschrieben. In dieser Studie sollte die In-vitro-Wirksamkeit ätherischer Öle gegen ein breites Spektrum veterinärmedizinisch relevanter Erreger getestet werden. Methoden: Die antibakterielle Aktivität von 16 ätherischen Ölen wurde mittels Agardiffusionstest bestimmt. Getestet wurden grampositive und gramnegative Erreger, die aus klinischen Isolaten von Hunden, Katzen und Pferden aus der veterinärmedizinischen Routinediagnostik stammten. Die Einteilung der Wirksamkeit in nicht, gering-, mittel- und hochgradig wirksam erfolgte anhand der Größe der Hemmhofradien des Bakterienwachstums. Ergebnisse: Generell zeigten sich sowohl grampositive als auch gramnegative Erreger empfindlich gegen einige der getesteten ätherischen Öle. Nicht nur gegen Staphylokokken, sondern auch gegen Methicillin-resistente Stämme der Staphylokokken wiesen die ätherischen Öle in vitro eine nicht zu vernachlässigende Wirkung auf. Pasteurella multocida stellte sich als eher sensibler Keim heraus, während Pseudomonas aeruginosa als vollkommen resistenter Keim eine Ausnahme bildete. Teebaum-, Oregano-, und Bergbohnenkrautöl waren die potentesten Öle. Zusätzlich zeigten sich bei den grampositiven Erregern Lemongrasöl und bei den gramnegativen Erregern Thymianöl als gut wirksam. Schlussfolgerung: Ätherische Öle verfügen in vitro über eine antibakterielle Aktivität gegen klinische Isolate von Hunden, Katzen und Pferden. Diese Studie bietet eine Grundlage für die Anwendung ätherischer Öle in der Veterinärmedizin. Es zeichneten sich Tendenzen im Wirkspektrum einzelner ätherischer Öle bzw. im Grad der Wirksamkeit ätherischer Öle hinsichtlich einzelner Erregerspezies ab, allerdings lässt sich keine sichere Vorhersage über ihre Wirksamkeit gegen einen spezifischen Keim eines individuellen Patienten treffen. Deswegen sollte vor einer Therapie mit ätherischen Ölen deren individuelle Wirksamkeit mittels Aromatogramm getestet werden.

Canine peripheral blood CD4+CD8+ double-positive Tcell subpopulations exhibit distinct Tcell phenotypes and effector functions.

Canine peripheral blood CD4+CD8α+ double-positive (dp) Tcells represent a small population of T lymphocytes that are not only derived from single-positive (sp) CD4+ but also from CD8+ Tcells. Dp Tcells can be subdivided in three different subpopulations according to their expression levels of CD4 and CD8α, i.e. the CD4dimCD8αbright, CD4brightCD8αbright, and the CD4brightCD8αdim subsets. Previously we could show that total canine peripheral CD4+CD8α+ dp Tcells have features of activated Tcells. Here we demonstrate that the individual dp Tcell subsets distinctly differ in their activation status and phenotype. Thus, among the dp Tcell subsets the CD4dimCD8αbright subpopulation has the lowest and the CD4brightCD8αbright subset the highest frequency of CD25+ cells pointing to the CD4brightCD8αbright subset with the highest activation status. Consistent with that, analysis of CD44 vs. CD62L expression demonstrates that the CD4brightCD8αbright subset contains the highest frequencies of effector-memory Tcells (TEM). Upon in vitro stimulation, the CD4brightCD8αbright and CD4dimCD8αbright dp Tcell subsets show the highest frequencies of IFN-γ producing cells. Expression of granzyme B was found exclusively in the CD4dimCD8αbright dp Tcell subset indicating cytotoxic potential of this dp Tcell subset. Furthermore, T-bet expression was restricted to the CD4dimCD8αbright subset, while Foxp3+ regulatory Tcells were detected in the CD4brightCD8αdim dp Tcell subset. In conclusion, canine dp Tcells are phenotypically and functionally heterogeneous consistent with the diverse characteristics of their single-positive CD4+ and CD8+ Tcell progenitors. The data provide the basis for future in vivo studies to elucidate the role of individual dp Tcell subsets in host defense and/or immune pathological diseases of dogs.

Peripheral canine CD4(+)CD8(+) double-positive T cells - unique amongst others.

T lymphocytes co-expressing CD4 and CD8 ("double-positive T cells") are commonly associated with a thymic developmental stage of T cells. Their first description in humans and pigs as extrathymic T cells with a memory phenotype almost 30 years ago came as a surprise. Meanwhile peripheral double-positive T cells have been described in a growing number of different species. In this review we highlight novel data from our very recent studies on canine peripheral double-positive T cells which point to unique features of double-positive T cells in the dog. In contrast to porcine CD4(+)CD8(+) T cells forming a homogenous cellular population based on their expression of CD4 and CD8α, canine CD4(+)CD8(+) T cells can be divided into three different cellular subsets with distinct expression levels of CD4 and CD8α. Double-positive T cells expressing CD8ß are present in humans and dogs but absent in swine. Moreover, canine CD4(+)CD8(+) T cells can not only develop from CD4(+) single-positive T cells but also from CD8(+) single-positive T cells. Together, this places canine CD4(+)CD8(+) T cells closer to their human than porcine counterparts since human double-positive T cells also appear to be heterogeneous in their CD4 and CD8α expression and have both CD4(+) and CD8(+) T cells as progenitor cells. However, CD4(+) single-positive T cells are the more potent progenitors for canine double-positive T cells, whereas CD8(+) single-positive T cells are more potent progenitors for human double-positive T cells. Canine double-positive T cells have an activated phenotype and may have as yet unrecognized roles in vivo in immunity to infection or in inflammatory diseases such as chronic infection, autoimmunity, allergy, or cancer.

Canine CD4(+)CD8(+) double-positive T cells can develop from CD4(+) and CD8(+) T cells.

For a long time the expression of the CD4 and CD8 receptor on peripheral blood T cells was thought to be mutually exclusive. However, in canine peripheral blood, similar to other species as swine or human for example, mature CD4(+)CD8(+) double-positive (dp) T cells exist which simultaneously express both surface receptors and have features of activated T cells. Canine CD4(+)CD8(+)dp T cells are heterogeneous and can be divided into three subpopulations by their intensity of CD4 and CD8α expression: CD4(bright)CD8α(bright), CD4(dim)CD8α(bright) and CD4(dim)CD8α(dim). The number of CD4(+)CD8α(+)dp T cells increases after in vitro stimulation of canine peripheral blood mononuclear cells (PBMC) raising the question of their progenitor(s). Thus, the aim of our study was to characterize the progenitor(s) of canine CD4(+)CD8α(+)dp T cells. By cell tracing experiments we identified both CD4(+) single-positive (sp) and also CD8α(+)sp T cells as progenitors of canine CD4(+)CD8α(+)dp T cells after in vitro stimulation. CD4(+)sp T cells almost exclusively upregulate a CD8αα homodimer, whereas CD8α(+)sp T cells can become CD4(+)CD8αß(+) or CD4(+)CD8αα(+). Even in the absence of other cells, highly purified CD4(+)sp T cells can become double-positive upon in vitro stimulation, whereas highly purified CD8α(+)sp T cells fail to do so. However, CD8α(+)sp T cells can additionally express CD4 when stimulated in the presence of CD4(-)CD8α(-) double-negative (dn) cells or more efficiently when stimulated in the presence of CD4(+)sp T cells. Soluble factors secreted by CD4(+)sp T cells are sufficient for the upregulation of CD4 on CD8α(+)sp T cells, but direct cell-cell contact between CD4(+)sp and CD8α(+)sp T cells is more efficient. mRNA analysis shows that additional CD4 expression on CD8α(+)sp T cells results from de novo synthesis. Thus, uptake of soluble CD4 or trogocytosis is less likely as mechanism for generation of canine double-positive T cells. CD4(+)CD8α(+)dp T cells are highly activated independent of their origin except when generated in coculture of CD8α(+)sp T cells with CD4(-)CD8α(-)dn cells. Overall, in dog, CD4(+)sp T cells are the more potent progenitors of CD4(+)CD8α(+)dp T cells compared to CD8α(+)sp T cells.

Canine CD4+CD8+ double positive T cells in peripheral blood have features of activated T cells.

In dogs a CD4(+)CD8(+) double positive T cell subpopulation exists that has not been phenotypically defined yet. We demonstrate that canine CD4(+)CD8(+) T cells are mature CD1a(-) and TCRαß(+) T cells. To analyse the activation potential of CD4(+)CD8(+) T cells, PBMC from dogs vaccinated against canine distemper virus (CDV) were re-stimulated with CDV. Upon antigen-specific stimulation, the CD4(+)CD8(+) T cell fraction increases and consists nearly exclusively of proliferated cells. Similarly, other features of activated effector/memory T cells such as up-regulation of CD25 and MHC-II as well as down-regulation of CD62L (L-selectin) were observed in CD4(+)CD8(+) T cells after stimulation. Canine CD4(+)CD8(+) T cells are less abundant, but more heterogeneous than porcine ones, comprising a small proportion expressing the ß chain of CD8 in addition to the CD8α chain, like human CD4(+)CD8(+) T cells. In summary, this analysis provides the basis for functional characterisation of the in vivo relevance of CD4(+)CD8(+) T cells in T-cell mediated immunity.