Advances in flow cytometric serology for canine visceral leishmaniasis: diagnostic applications when distinct clinical forms, vaccination and other canine pathogens become a challenge.

We have previously reported the applicability of flow cytometry anti-fixed Leishmania infantum chagasi promastigotes IgG (FC-AFPA-IgG) as a novel serological device for laboratorial diagnosis of CVL. Herein, we validate throughout a blind study applied into a broader range of coded sera samples that FC-AFPA-IgG at serum dilution 1:8192 have an outstanding performance to discriminate the serological reactivity of canine visceral leishmaniasis (CVL, n=64) and Leishmune vaccines (VAC, n=62) and non-infected controls (NI, n=25). Moreover, we have evaluated the performance of indirect immunofluorescence antibody test (IFAT) and the crude-antigen enzyme-linked immunosorbent assay (ELISA) in parallel with FC-AFPA-IgG, to discriminate the seroreactivity of NI, CVL and VAC. Our data demonstrated that both ELISA and FC-AFPA-IgG showed similar performance to detect the seronegativity in 100% of NI, whereas FC-AFPA-IgG displayed better performance to exclude seropositivity in 100% of VAC. The high kappa agreement indexes observed suggested similar performance between these two serological testes when distinct clinical forms of CVL become a challenge. Furthermore, the FC-AFPA-IgG applied at sera dilution 1:8192 showed a remarkable performance to discriminate CVL from other co-endemic canine infections with high co-negativity in dogs infected with Trypanosoma cruzi and Leishmania braziliensis (86% and 84%, respectively). In conclusion, the data presented here re-emphasize the applicability of FC-AFPA-IgG as an innovative methodology able to discriminate post-infection imunomediated seroreactivity from that triggered by prophylactic immunization with minor cross-reactivity with other relevant canine pathogens, which may contribute as a supplementary assay for the CVL immunodiagnosis.

Clinical value of anti-Leishmania (Leishmania) chagasi IgG titers detected by flow cytometry to distinguish infected from vaccinated dogs.

Leishmune vaccination covers a broader number of endemic areas of canine visceral leishmaniasis (CVL) and therefore the development of new serological devices able to discriminate CVL from Leishmune vaccinees becomes an urgent need considering the post-vaccine seroconversion detected throughout conventional methodologies. Herein, we have described the establishment of a flow cytometry based methodology to detect anti-fixed L. (L.) chagasi promastigotes antibodies (FC-AFPA-IgG, FC-AFPA-IgG1 and FC-AFPA-IgG2) in sera samples from Leishmania (Leishmania) chagasi infected dogs and Leishmune vaccinees. The results of FC-AFPA were reported along the sera titration curve (1:128-1:524,288), as percentage-of-positive-fluorescent-parasite (PPFP). The use of PPFP=20% as a cut-off edge to segregate negative and positive results at sera dilution 1:2048 revealed outstanding performance indexes that elect FC-AFPA-IgG and IgG2 (both detected by polyclonal FITC-labeled second step reagent) applicable to the serological diagnosis of CVL, with 100% of specificity for both IgG and IgG2 and 97 and 93% of sensitivity, respectively. Moreover, FC-AFPA-IgG, applied at sera dilution 1:2048, also appeared as a useful tool to discriminate L. chagasi infected dogs from Leishmune vaccinees, with 76% of specificity. Outstanding likelihood indexes further support the performance of FC-AFPA-IgG for exclusion diagnosis of CVL in Leishmune vaccinees. Analysis of FC-AFPA-IgG at sera dilution 1:8192 revealed the most outstanding indexes, demonstrating that besides the ability of PPFP

Immunological changes in canine peripheral blood leukocytes triggered by immunization with first or second generation vaccines against canine visceral leishmaniasis.

In this study, we summarized the major phenotypic/functional aspects of circulating leukocytes following canine immunization with Leishvaccine and Leishmune®. Our findings showed that Leishvaccine triggered early changes in the innate immunity (neutrophils and eosinophils) with late alterations on monocytes. Conversely, Leishmune(®) induced early phenotypic changes in both, neutrophils and monocytes. Moreover, Leishvaccine triggered mixed activation-related phenotypic changes on T-cells (CD4+ and CD8+ and B-lymphocytes, whereas Leishmune(®) promoted a selective response, mainly associated with CD8+ T-cell activation. Mixed cytokine profile (IFN-γ/IL-4) was observed in Leishvaccine immunized dogs whereas a selective pro-inflammatory pattern (IFN-γ/NO) was induced by Leishmune® vaccination. The distinct immunological profile triggered by Leishvaccine and Leishmune® may be a direct consequence of the distinct biochemical composition of these immunobiological, i.e. complex versus purified Leishmania antigen along with Bacillus Calmette-Guérin (BCG) versus saponin adjuvant. Both immunobiologicals are able to activate phagocytes and CD8+ T-cells and therefore could be considered as a putative vaccines against canine visceral leishmaniasis (CVL).

T-cell-derived cytokines, nitric oxide production by peripheral blood monocytes and seric anti-Leishmania (Leishmania) chagasi IgG subclass patterns following immunization against canine visceral leishmaniasis using Leishvaccine and Leishmune.

It is generally accepted that distinct cytokine expression by the cellular immune response plays a critical role during the outcome of experimental as well as natural canine visceral Leishmaniasis (CVL). Despite the fact that immunoprophylaxis of CVL has become an important control strategy and protective immunity has been reported upon immunization with whole as well as purified Leishmania antigens, the cytokine profile of T-cells triggered by anti-CVL vaccines still remain to be determined. Herein, we have developed a cross-sectional analysis of German Shepherd dogs submitted to vaccination protocols with Leishvaccine (n=6) and Leishmune (n=6). Our data identified distinct immunological profiles elicited by Leishvaccine and Leishmune, with the Leishvaccine triggering a mixed, IFN-gamma and IL-4, cytokine pattern in addition to high levels of anti-Leishmania IgG1, whereas the Leishmune induced an immunological pattern characterized by enhanced levels of IFN-gamma, NO and anti-Leishmania chagasi IgG2. It was important to notice that despite the distinct immunological patterns triggered by Leishvaccine and Leishmune, the ability of both immunobiologicals to activate T-cell-derived IFN-gamma synthesis further suggesting their immunogenic potential against CVL. These findings added support to our hypothesis that both antigenic composition (whole antigen in Leishvaccine versus purified antigen in Leishmune) as well as the adjuvant nature (BGC and saponin) used for the vaccine formulation may count for the distinct activation pattern observed.

Despite Leishvaccine and Leishmune trigger distinct immune profiles, their ability to activate phagocytes and CD8+ T-cells support their high-quality immunogenic potential against canine visceral leishmaniasis.

Phenotypic features of peripheral blood leukocytes have been investigated as a pre-requisite to characterize the protective immunity attributed to both Leishvaccine and Leishmune. Our results showed that either those vaccine were accompanied by distinct profiles on innate immune compartment. While Leishvaccine promoted early changes in phenotypic features of neutrophils and eosinophils with late involvement of monocytes, Leishmune induced early and persistent activation of neutrophils and monocytes, without changes on eosinophil activation status. Regarding the adaptive immunity, Leishvaccine sponsored a mixed profile, associated with phenotypic changes of T and B-lymphocytes. Major phenotypic changes in CD4+ T-cells with transient activation of CD8+ T-cell, besides decreased frequency of B-cell expressing CD32 were the hallmark of Leishvaccine. In contrast, Leishmune was associated with phenotypic changes in T-lymphocytes, particularly in CD8+ T-cells, and selective up-regulation of CD3+CD5+LowCD8+ cells. We hypothesized that this dissimilar alteration in immunological events would represent phenomenon directly related with the molecular nature of these vaccines besides the distinct adjuvants employed. However, it is important to emphasize that both immunobiologicals are able to activate phagocytes and CD8+ T-cells and therefore could be considered priority vaccines with a high-quality immunogenic potential against CVL.

Efeito do mês dentro da estação da monta sobre a fertilidade de éguas inseminadas com sêmen fresco diluído / Effect of month within the breeding season upon fertility of mares inseminated with diluted fresh semen / Efecto del mes de la estación del monta sobre la fertilidad de yeguas inseminadas con semen fresco diluido

Noventa e quatro ciclos, de cinquentas e nova éguas foram analisados com objetivos de estudas o efeito do mês de ovulação sobre a fertilidade de éguas inseminadas com sêmen fresco diluído. As éguas foram rufiadas e inseminadas às segundas, quartas e sextas-feiras, a partir de um folículo de 3,0 a 3,5 cm de diâmetro, com sêmen de apenas um garanhão de fertilidade comprovada, diluído para um volume inseminante de 10 ml com diluidor de mínima contaminação. Os ciclos foram agrupados de acordo com o mês de ovulação (novembro, dezembro, janeiro, fevereiro/março). as taxas de concepção, ao primeiro ciclho, para os grupos novembro, dezembro, janeiro, fevereiro/março foram de 61,11% (11/18); 84,21%(16/19); 55,56%(5/9); 53,85%(7/13), respectivamente, sem que houvesse diferenças entre estas (P>0,05). Após quatro ciclos, as taxas de concepção foram de 61,11% (11/18); 70,37% (19/27); 50,00% (8/16); 42,42% (14/33), respectivamen te, na mesma ordem anterior (P>0,05). Entretanto, foi observada uma menor eficiência de prenhez no mês fevereiro/março. Conclui-se então que o mês de março foi o responsável pela menor fertilidade das éguas.(AU)

Ninety four estral cuycles of fifty nine mares were analyzed to study the effect of ovulation month of mares inseminated with fresh diluted fresh semen. The mares were teased and inseminate on Mondays, Wednesdays and Fridays, from one follicle of 3.0 to 3.5 cm of niameter, with semen from one stallion of proven fertility, extended with minimum contamination extender. The cycles were grouped according the ovulation month (November, December, January, February/Mach). The conception rates for the first cycle for groups November, December, January, Frebuary/March were 61,11% (11/18) 84,21% (16/19); 55,56% (5/9); 53,85% (7/13), respectively, without differences among then (P>0,05). The conception/cicle rates after four cicles were 61,11% (11/18); 70,37% (19/27); 50,00% (8/16); respectively, without differences among then (P>0,05). Homewer, a smaller pregnancy efficiency was observed in the month February/March. It was concluded that the month of March was responsible for the smallest fertility of the mares.(AU)

noventa y cuatro ciclos de cincuenta y nove hembras equinas fueron analizados para estudas el efecto del mes sobre la fertilidad de estas yeaguas inseminadas con semen fresco diluido. Las yeguas fueron inseminadas los lunes, miércoles y viernes, a partir de un folíulo de 3,0 a 3,5 centímetros de diámetro, con semen de un solo garañón de fertilidad comprobada, diluido para un volume inseminante de 10 mL con diluidor de mínima contaminación. Los resultados fuerón agrouped, en del acuerdo con el mes de la ovulación de las yeguas (noviembre, deciembre, enero, febrero/marzo). Las tazas de concepción, al primer ciclo para los grupos fueron del 61,11% (11/18); del 84,21% (16/19); del 55,56% (5/9) y del 53,85% (7/13), respectivamente, sin que hubiera diferencias entre estas (P>0,05). Después de 4 ciclos, las tazas de concepción fuero del 61,11%(11/18); del 70,37%(19/27); del 50,00%(8/16); del 42,42%(14/33), respectivamente, en el mismo orden anterior (P>0,05). Sin embardo, fue una menor eficiencia de la preñez en el mes febrero/marzo. Con base en los resultados obtenidos, el mes de marzo fue responsable por una menor fertilidad de las yeguas.(AU)