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Background: Canine atopic dermatitis (cAD) is a chronic disease characterised by hypersensitivity to environmental allergens. Oclacitinib maleate selectively inhibits pro-inflammatory mediators associated with cAD. However, the impact of chronic oclacitinib use on immunocompetence requires further investigation., Objectives: Herein, we examined the potential immunomodulatory effects of prolonged oclacitinib treatment in dogs., Animals: Thirteen privately owned dogs with cAD, treated with 0.4-0.6 mg/kg oclacitinib for 12 months., Methods and Materials: Pruritus level was evaluated using a pruritus Visual Analog Scale (pVAS) and the canine atopic dermatitis extent and severity index, 4 th iteration (CADESI IV). Peripheral blood samples were collected for routine laboratory assays and lymphocyte subtypes were analysed using flow cytometry. Antigen-specific intracellular cytokine production from CD4+ and CD8+ T lymphocytes was analysed following in vitro stimulation by Dermatophagoides farinae antigens., Results: Oclacitinib treatment significantly reduced pVAS and CADESI-04 scores, by 51% and 86.7%, respectively. Flow cytometric analysis revealed increased CD4+ and CD14+ lymphocyte populations. The cytokine profile at 360 days after treatment initiation was similar to that before treatment and was not associated with clinical relapse., Conclusion: Oclacitinib, when administered at the currently labelled dose for one year, is associated with a significant increase in circulating CD4+ T cells, but does not alter cytokine production from antigen-stimulated T cells. The results reported do not support evidence for immunosuppression mediated by the mechanisms evaluated in this study., (© 2021 ESVD and ACVD.)

Visceral leishmaniasis (VL), caused by digenetic protozoa of the genus Leishmania , is the most severe form of leishmaniasis. Leishmania infantum is one of the species responsible for VL and the disease caused is considered a zoonosis whose main reservoir is the dog. Canine visceral leishmaniasis (CVL) can lead to the death of the animal if left untreated. Furthermore, the available pharmocologial treatment for CVL presents numerous disadvantages, such as relapses, toxicity, drug resistance, and the fact treated animals continue to be reservoirs when treatment fails to achieve parasitological cure. Moreover, the available VL control methods have not been adequate when it comes to controlling parasite transmission. Advances in immune response knowledge in recent years have led to a better understanding of VL pathogenesis, allowing new treatments to be developed based on immune system activation, often referred to as immunotherapy. In fact, well-defined protocols have been described, ranging from the use of immunomodulators to the use of vaccines. This treatment, which can also be associated with chemotherapy, has been shown to be effective in restoring or inducing an adequate immune response to reduce parasitic burden, leading to clinical improvement. This review focuses on immunotherapy directed at dogs infected by L. infantum , including a literature review of what has already been done in dogs. We also introduce a promising strategy to improve the efficacy of immunotherapy., (Copyright © 2019 Gonçalves, Leite, Resende, Mariano, Silveira, Melo-Júnior, Ribeiro, de Oliveira, Soares, Santos, Marques, Galdino, Martins-Filho, Dutra, da Silveira-Lemos and Giunchetti.)

Canine atopic dermatitis (CAD) is a chronic, pruritic, genetic, and inflammatory disease. Its pathogenesis is very complex and involves skin barrier defects and immune system dysfunction. This study aimed to investigate hematological, biochemical, clinical, and immunological parameters to contribute to the identification of biomarkers applied to CAD. The results of the analysis on hematologic and clinical parameters showed increased neutrophil numbers and decreased lymphocyte counts. The ex vivo immunophenotyping of leukocytes demonstrated increased counts of circulating neutrophils, in addition to a high frequency of CD4 + T-cells and elevated CD4 + /CD8 + T-cell ratio, which were the hallmark of atopic animals. Moreover, atopic dogs presented a mixed immune response, displaying both CD4 + and CD8 + T-cell subsets as relevant sources of IFN-γ and IL-4 cytokines. The morbidity analyzed by the CADESI index demonstrated that CAD severity is related to the low frequency of circulating CD14 + monocytes, CD21 + B-cells, and CD8 + T-cells. The reported biomarkers would be useful in CAD monitoring for treatment and prognosis analysis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Live attenuated Leishmania donovani parasites as LdCen(-/-) were shown to confer protective immunity against Leishmania infection in mice, hamsters, and dogs. Strong immunogenicity in dogs vaccinated with LdCen(-/-) has been previously reported, including increased antibody response favoring Th1 response lymphoproliferative responses, CD4(+) and CD8(+) T-cells activation, increased levels of Th1 and reduction of Th2 cytokines, in addition to a significant reduction in parasite burden after 18 and 24 months post virulent parasite challenge., Methods: Aimed at validating a new method using in vitro co-culture systems with macrophages and purified CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells of immunized dogs with both LdCen(-/-) and Leishmune® to assess microbicide capacity of macrophages and the immune response profile as the production of IFN-γ, TNF-α, IL-12, IL-4 and IL-10 cytokines., Results and Discussion: Our data showed co-cultures of macrophages and purified T-cells from dogs immunized with LdCen(-/-) and challenged with L. infantum were able to identify high microbicidal activity, especially in the co-culture using CD4(+) T-cells, as compared to the Leishmune® group. Similarly, co-cultures with CD8(+) T-cells or CD4(+):CD8(+) T-cells in both experimental groups were able to detect a reduction in the parasite burden in L. infantum infected macrophages. Moreover, co-cultures using CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells from immunized dogs with both LdCen(-/-) and Leishmune® were able to identify higher levels of IFN-γ and IL-12 cytokines, reduced levels of IL-4 and IL-10, and a higher IFN-γ/IL-10 ratio. While the highest IFN-γ levels and IFN-γ/IL-10 ratio were the hallmarks of LdCen(-/-) group in the co-culture using CD4(+) T-cells, resulting in strong reduction of parasitism, the Leishmune® immunization presented a differential production of TNF-α in the co-culture using CD4(+):CD8(+) T-cells., Conclusion: The distinct conditions of co-culture systems were validated and able to detect the induction of immune protection. The method described in this study applied a new, more accurate approach and was able to yield laboratory parameters useful to test and monitor the immunogenicity and efficacy of Leishmania vaccines in dogs.

Diagnosing canine visceral leishmaniasis (CVL) is a critical challenge since conventional immunoserological tests still present some deficiencies. The current study evaluated a prototype flow cytometry serology test, using antigens and fluorescent antibodies that had been stored for 1 year at 4°C, on a broad range of serum samples. Noninfected control dogs and Leishmania infantum-infected dogs were tested, and the prototype test showed excellent performance in differentiating these groups with high sensitivity, specificity, positive and negative predictive values, and accuracy (100% in all analyses). When the CVL group was evaluated according to the dogs' clinical status, the prototype test showed outstanding accuracy in all groups with positive serology (asymptomatic II, oligosymptomatic, and symptomatic). However, in dogs which had positive results by PCR-restriction fragment length polymorphism (RFLP) but negative results by conventional serology (asymptomatic I), serological reactivity was not observed. Additionally, sera from 40 dogs immunized with different vaccines (Leishmune, Leish-Tec, or LBSap) did not present serological reactivity in the prototype test. Eighty-eight dogs infected with other pathogens (Trypanosoma cruzi, Leishmania braziliensis, Ehrlichia canis, and Babesia canis) were used to determine cross-reactivity and specificity, and the prototype test performed well, particularly in dogs infected with B. canis and E. canis (100% and 93.3% specificities, respectively). In conclusion, our data reinforce the potential of the prototype test for use as a commercial kit and highlight its outstanding performance even after storage for 1 year at 4°C. Moreover, the prototype test efficiently provided accurate CVL serodiagnosis with an absence of false-positive results in vaccinated dogs and minor cross-reactivity against other canine pathogens.

The control of canine visceral leishmaniasis (CVL) is imperative, but euthanasia of seropositive dogs has been highly criticized. Commonly used, immunodiagnostic tests, including Dual-Path Platform®, enzyme-linked immunosorbent assay, and immunofluorescent antibody test, have failed at detecting asymptomatic dogs in endemic areas. In this context, new serological methods are needed. Flow cytometry serology has demonstrated potential as a test with excellent performance for CVL. In this study, we proposed to establish the best conditions for preserving Leishmania infantum promastigote antigens employed in this serology test. During 12 months of follow-up, promastigotes were maintained in different preservatives (phosphate-buffered saline with 3% fetal bovine serum, phenol 0.35%, thimerosal 0.01%, and formaldehyde 0.5%) and stored at 3 distinct temperatures (25 °C, 4 °C, and -20 °C). During the study period, the morphological characteristics of the promastigotes were assessed by flow cytometry according to the forward and side scatter parameters and also under optical microscopic analysis. Reactivity performance was evaluated as the percentage of positive fluorescent parasites in the sera of naturally infected and noninfected dogs. Microbiological analysis was performed at 2 time points, the first and sixth months, to rule out contamination of stored promastigotes. Taken together, our results indicated that the best conditions to preserve fixed L. infantum antigens were storage in formaldehyde at 4 °C. Promastigotes presented the best morphological profile, with appropriate antigenic stability even at 4 °C, in an inexpensive preservative for a long period of conservation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Human visceral leishmaniasis, one of the most important zoonoses, is caused by the protozoa Leishmania chagasi (syn. L. infantum) and is present as a fatal disease common in South America and Europe where dogs and wild canids are the main reservoirs. A vaccine against visceral leishmaniasis would be an important tool in the control of this disease in dogs. Although the current strategies for vaccination against leishmaniasis are based on the use of recombinant antigens, killed vaccines are still attractive in terms of stability of their biochemical composition and antigenicity, cost, and safety. Here we evaluate the immunogenicity of a whole parasite vaccine as a promising candidate against canine leishmaniasis, demonstrated by cellular reactivity, changes in the cellular profile of the peripheral blood and by the differential production of immunoglobulins. Our results showed that immunization elicited mainly a strong cellular reactivity and increase in T-lymphocytes, particularly the subpopulation CD8(+) that would be related to the control of tissue parasitism. In addition, a higher production of anti-Leishmania total IgG, characterized by mixed isotypes profile (IgG1 and IgG2), was demonstrated.