Zoonotic visceral leishmaniasis (VL) is a widespread disease, and dogs are the main reservoirs for human parasite transmission. Hence, development of an effective vaccine that prevents disease and reduces the transmission of VL is required. As euthanasia of seropositive dogs is recommended in Brazil for VL epidemiological control, to include anti-VL canine vaccines as a mass control measure it is necessary to characterize the humoral responses induced by vaccination and if they interfere with the reactivity of vaccinated dogs in serological diagnostic tests. Leish-Tec(®) is an amastigote-specific A2 recombinant protein vaccine against canine visceral leishmaniasis (CVL) that is commercially available in Brazil. Here, we tested the immunogenicity of Leish-Tec(®) in a heterogeneous dog population by measuring A2-specific antibody responses. Healthy dogs (n=140) of various breeds were allocated to two groups: one group received Leish-Tec(®) (n=70), and the other group received a placebo (n=70). Anti-A2 or anti-Leishmania promastigote antigen (LPA) antibody levels were measured by ELISA in serum samples collected before and after vaccination. An immunochromatographic test (DPP) based on the recombinant K28 antigen was also used for serodiagnosis of CVL. Vaccinated animals, except one, remained seronegative for anti-LPA total IgG and anti-K28 antibodies. Conversely, seropositivity for anti-A2 total IgG antibodies was found in 98% of animals after vaccination. This value decreased to 81.13% at 6 months before rising again (98%), after the vaccination boost. Anti-A2 IgG2 and IgG1 titers were also increased in vaccinated animals relative to control animals. These data indicate that Leish-Tec(®) is immunogenic for dogs of different genetic backgrounds and that humoral responses induced by vaccination can be detected by A2-ELISA, but do not interfere with the LPA-ELISA and DPP diagnostic tests for CVL.

BACKGROUND Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy.OBJECTIVE This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL).METHODS A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection.FINDINGS Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis.MAIN CONCLUSIONS The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

The zoonotic potential of giardiasis, as proposed by WHO since the late 70's, has been largely confirmed in this century. The genetic assemblages A and B of Giardia duodenalis are frequently isolated from human and canine hosts. Most of the assemblage A strains are not infective to adult mice, which can limit the range of studies regarding to biology of G. duodenalis, including virulence factors and the interaction with host immune system. This study aimed to determine the infectivity in mice of an assemblage A Giardia duodenalis strain (BHFC1) isolated from a dog and to classify the strain in sub-assemblages (AI, AII, AIII) through the phylogenetic analysis of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. In addition, the proteomic profile of soluble and insoluble protein fractions of trophozoites was analyzed by 2D-electrophoresis. Accordingly, trophozoites of BHFC1 were highly infective to Swiss mice. The phylogenetic analysis of tpi and gdh revealed that BHFC1 clustered to sub-assemblage AI. The proteomic map of soluble and insoluble protein fractions led to the identification of 187 proteins of G. duodenalis, 27 of them corresponding to hypothetical proteins. Considering both soluble and soluble fractions, the vast majority of the identified proteins (n = 82) were classified as metabolic proteins, mainly associated with carbon and lipid metabolism, including 53 proteins with catalytic activity. Some of the identified proteins correspond to antigens while others can be correlated with virulence. Besides a significant complementation to the proteomic data of G. duodenalis, these data provide an important source of information for future studies on various aspects of the biology of this parasite, such as virulence factors and host and pathogen interactions.

Background Cell responses to different stress inducers are efficient mechanisms that prevent and fight the accumulation of harmful macromolecules in the cells and also reinforce the defenses of the host against pathogens. Vaccinia virus (VACV) is an enveloped, DNA virus, belonging to the Poxviridae family. Members of this family have evolved numerous strategies to manipulate host responses to stress controlling cell survival and enhancing their replicative success. In this study, we investigated the activation of the response signaling to malformed proteins (UPR) by the VACV virulent strain—Western Reserve (WR)—or the non-virulent strain—Modified Vaccinia Ankara (MVA). Methods Through RT-PCR RFLP and qPCR assays, we detected negative regulation of XBP1 mRNA processing in VACV-infected cells. On the other hand, through assays of reporter genes for the ATF6 component, we observed its translocation to the nucleus of infected cells and a robust increase in its transcriptional activity, which seems to be important for virus replication. WR strain single-cycle viral multiplication curves in ATF6α-knockout MEFs showed reduced viral yield. Results We observed that VACV WR and MVA strains modulate the UPR pathway, triggering the expression of endoplasmic reticulum chaperones through ATF6α signaling while preventing IRE1α-XBP1 activation. Conclusions The ATF6α sensor is robustly activated during infection while the IRE1α-XBP1 branch is down-regulated.

Leishmaniases are widespread neglected diseases with an incidence of 1.6 million new cases and 40 thousand deaths per year. Leishmania parasites may show distinct, species-specific patterns of virulence that lead to different clinical manifestations. It is well known that successive in vitro passages (SIVP) lead to the attenuation of virulence, but neither the metabolism nor the pathways involved in these processes are well understood. Herein, promastigotes of a virulent L. amazonensis strain recently isolated from mice was compared to SIVP derived and attenuated promastigotes, submitted to 10, 40, and 60 axenic passages and named R10, R40, and R60, respectively. In vitro assays and in vivo tests were performed to characterize and confirmed the attenuation profiles. A metabolomic fingerprint comparison of R0, R10, and R60 was performed by means of capillary electrophoresis, liquid and gas chromatography coupled to mass spectrometry. To validate the metabolomic data, qPCR for selected loci, flow cytometry to measure aPS exposure, sensitivity to antimony tartrate and ROS production assays were conducted. The 65 identified metabolites were clustered in biochemical categories and mapped in eight metabolic pathways: ABC transporters; fatty acid biosynthesis; glycine, serine and threonine metabolism; β-alanine metabolism; glutathione metabolism; oxidative phosphorylation; glycerophospholipid metabolism and lysine degradation. The obtained metabolomic data correlated with previous proteomic findings of the SVIP parasites and the gene expression of 13 selected targets. Late SIVP cultures were more sensitive to SbIII produced more ROS and exposed less phosphatidylserine in their surface. The correspondent pathways were connected to build a biochemical map of the most significant alterations involved with the process of attenuation of L. amazonensis. Overall, the reported data pointed out to a very dynamic and continuous metabolic reprogramming process, accompanied by changes in energetic, lipid and redox metabolisms, membrane remodeling and reshaping of parasite-host cells interactions, causing impacts in chemotaxis, host inflammatory responses and infectivity at the early stages of infection.