Intracellular IFN-γ and IL-4 levels of CD4 + and CD8 + T cells in the peripheral blood of naturally infected (Leishmania infantum) symptomatic dogs before and following a 4-week treatment with miltefosine and allopurinol: a double-blinded, controlled and cross-sectional study.

BACKGROUND: Canine leishmaniosis (CanL) is a systemic disease caused by the protozoan parasite Leishmania infantum with a wide spectrum of clinical signs, with cutaneous, ocular, renal and lymphoreactive conditions prevailing in the clinical setting. The immune system plays a pivotal role in the evolution of Leishmania infection and its response to antileishmanial treatment. Cytokines are important immune response mediators that are released by activated lymphocytes and less so by other immunocytes. In dogs with leishmaniosis, IFN-γ and IL-4 have been recognized as the main activators of cellular and humoral immunity, respectively. The objective of this study was to investigate intracellular IL-4 and IFN-γ expression by CD4 + and CD8 + lymphocytes in the peripheral blood of symptomatic dogs before and after combined antileishmanial treatment with miltefosine and allopurinol. RESULTS: Postantileishmanial treatment CD4 + IL-4 + and CD8 + IL-4 + cell counts were significantly decreased, although no similar changes were observed in the comparisons made between the pre- and posttreatment CD4 + IFN-γ + and CD8 + IFN-γ + counts and ratios. CONCLUSION: The findings indicate that IL-4 production by T cells may facilitate the symptomatic phase of CanL, whereas IFN-γ production by CD4 + and CD8 + cells may indicate its negligible role in the evolution of natural CanL and perhaps the equivocal positive influence of antileishmanial treatment.

Cutaneous Disease as Sole Clinical Manifestation of Protothecosis in a Boxer Dog.

Prototheca wickerhamii is ubiquitous, saprophytic achlorophyllous algae that cause opportunistic infections in the dog and cat and disseminated disease usually in immunocompromised animals. In this report an uncommon case of canine cutaneous protothecosis is presented. A 6-year-old female boxer was brought in with skin lesions that consisted of nodules and generalized footpad hyperkeratosis, depigmentation, and erosion. Cytology and histopathology showed pyogranulomatous inflammation along with organisms containing round sporangia with spherical sporangiospores. PCR and sequencing identified the causal organism as Prototheca wickerhamii. Therapy applied in this patient with either fluconazole alone or combination of amphotericin B and itraconazole proved effective only for footpad lesions but not for skin nodules. Systemic therapy seems to be ineffective for skin nodules, at least in chronic cases of canine cutaneous protothecosis. Although canine protothecosis usually presents with the disseminated form, cutaneous disease as sole clinical manifestation of the infection may also be witnessed.

Claw histopathology and parasitic load in natural cases of canine leishmaniosis associated with Leishmania infantum.

Histological lesions and the presence of Leishmania spp. amastigotes in claw tissues were investigated in 40 dogs with leishmaniosis, with (16/40--group A) or without (24/40--group B) generalized onychogryphosis. Following euthanasia, the entire third phalanx with intact claw was amputated, formalin fixed, decalcified in a formic acid solution, embedded in paraffin, sectioned longitudinally and stained with haematoxylin and eosin, and acid orcein-Giemsa. Nested polymerase chain reaction (PCR) was used for the detection of Leishmania amastigotes. Lichenoid mononuclear infiltration (all dogs in group A, 21 of 24 dogs in group B), basal keratinocyte vacuolation (nine of 16 dogs in group A, 15 of 24 dogs in group B) and dermoepidermal clefting (13 of 16 dogs in group A, 18 of 24 dogs in group B) were the most prominent histopathological findings. There was no difference in the frequency and severity of these lesions between the two groups. Leishmania amastigotes could not be visualized in the dermis of any of the H&E sections, but their presence was demonstrated by nested PCR in three of 16 dogs in group A and two of 24 dogs in group B. However, the frequency of positive nested PCRs was not significantly different between the two groups. In conclusion, claw histopathology in symptomatic dogs with leishmaniosis, either with or without onychogryphosis is mainly characterized by mononuclear lichenoid dermatitis with or without interface dermatitis and dermoepidermal clefting, and is not accompanied by substantial local parasitism.

Determination of intracellular cytokines IFN-gamma and IL-4 in canine T lymphocytes by flow cytometry following whole-blood culture.

This report describes a whole-blood flow cytometric method for the determination of intracellular cytokines IFN-gamma and IL-4 in canine T lymphocyte subpopulations. Conjugated anti-cytokine antibodies and commercially available reagents for cell fixation and permeabilization were used. Canine peripheral blood was cultured with a combination of phorbol-12-myristate-13-acetate (PMA) and ionomycin to promote cytokine synthesis in each cell, along with monensin to increase the sensitivity of the method by retaining IFN-gamma and IL-4 within the cell to detectable levels. The optimum concentrations of PMA and ionomycin were determined. Maximum IFN-gamma expression from both CD4+ and CD8+ T lymphocytes was detected after 6 h of incubation of cell culture, while maximum IL-4 production took 6 h from CD4+ cells and 4 h from CD8+ cells. This method is a simple immunologic technique for measuring intracellular cytokines which could be of value in the investigation of canine immunological response mainly in various intracellular and extracellular infections, since IFN-gamma and IL-4 are considered key cytokines activating the cellular and humoral immunity, respectively.