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Background: This cross-sectional investigation aimed to evaluate the prevalence of IgM and IgG anti-Toxoplasma gondii antibodies and the associated risk factors among healthy blood donors in Khorasan Razavi Province, northeast of Iran from Nov 2014 to May 2015. Methods: Overall, 491 serum samples from apparently healthy blood donors referred the six biggest blood centers in Razavi Khorasan, Iran, were screened for IgG and IgM anti-T. gondii antibodies by enzyme-linked immunosorbent assay (ELISA). A structured questionnaire was used to obtain information on risk factors for T. gondii infection. Nested PCR was also used to detect DNA of T. gondii in the IgM-positive samples by using of B1 and RE (Repetitive Element) as marker for amplifying fragment size of 531 bp and 164 bp in PCR method. Results: Totally, 200 (40.7%) samples were seropositive for anti-T. gondii antibodies; 184 (37.5%) donors tested seropositive for only IgG antibody, 8 (1.6%) tested seropositive for both IgM and IgG and 8 (1.6%) were positive for IgM antibody alone. Several risk factors significantly related to T. gondii seropositivity in the univariate analysis at P

An attenuated line of Leishmania infantum (the H-line), developed through exposure to gentamicin, has been shown to protect dogs against canine visceral leishmaniasis. A specific diagnostic test to differentiate dogs vaccinated with the attenuated line from dogs infected with L. infantum wild-type (L. infantum WT) could be a valuable tool in evaluating the effectiveness of canine vaccination. In this study, 28 healthy dogs were allocated into four groups. In Group I and Group II (eight dogs per group), dogs were immunized subcutaneously (s.c.) with L. infantum H-line, and the dogs of Group II challenged s.c. with L. infantum WT, at 2 months post-immunization. In Group III, eight animals were challenged s.c. with L. infantum WT, and four dogs of Group IV were injected s.c. with PBS. We found that sera from vaccinated dogs recognize a 21 kDa antigen of promastigotes of L. infantum H-line but not of L. infantum WT, whereas sera from unvaccinated dogs challenged with L. infantum WT, recognized a 21 kDa antigen of promastigotes of L. infantum WT but not of L. infantum H-line. Sera from dogs challenged with L. infantum WT with prior vaccination with L. infantum H-line, recognized a 21 kDa antigen of both L. infantum WT and L. infantum H-line. These results suggest that the Western blot analysis of antibodies against 21 kDa antigens of L. infantum H-line and WT may be a useful technique for distinguishing between dogs vaccinated with L. infantum H-line and dogs naturally infected with L. infantum WT.

An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. Here, we show that L. infantum H-line induced significantly higher levels of IFN-γ and lower levels of IL-10 compared with those in dogs infected with L. infantum wild type (WT). Anti-Leishmania-specific total IgG, IgG1, and IgG2 antibodies were present in the serum of all infected dogs, with levels of IgG2 subclass highest in the sera of dogs inoculated with L. infantum H-line. Relatively high levels of IgG1 were found in the sera of dogs infected with L. infantum WT. Six of seven dogs immunized intradermally (i.d.) with the attenuated line later showed a positive skin test to leishmanin, whereas the dogs infected with L. infantum WT did not. No clinical abnormalities were observed, and no parasites found in the visceral organs of the dogs inoculated intravenously (i.v.) with L. infantum H-line over 24 months post-inoculation. Dogs which had been immunized with L. infantum H-line i.d. 12 months previously were protected against challenge with L. infantum WT. These data suggest that the L. infantum H-line was safe and induced a protection which is correlated with cellular immunity in dogs.

An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. A vaccine trial was conducted using 103 naive dogs from a leishmaniosis non-endemic area (55 vaccinated and 48 unvaccinated) brought into an endemic area of southeast Iran. No local and/or general indications of disease were observed in the vaccinated dogs immediately after vaccination. The efficacy of the vaccine was evaluated after 24 months (4 sandfly transmission seasons) by serological, parasitological analyses and clinical examination. In western blot analysis of antibodies to L. infantum antigens, sera from 10 out of 31 (32.2%) unvaccinated dogs, but none of the sera from vaccinated dogs which were seropositive at100, recognized the 21 kDa antigen of L. infantum wild-type (WT). Nine out of 31 (29%) unvaccinated dogs, but none of vaccinated dogs, were positive for the presence of Leishmania DNA. One out of 46 (2.2%) vaccinated dogs and 9 out of 31 (29%) unvaccinated dogs developed clinical signs of disease. These results suggest that gentamicin-attenuated L. infantum induced a significant and strong protective effect against canine visceral leishmaniosis in the endemic area.

An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. A vaccine trial was conducted using 103 naive dogs from a leishmaniosis non-endemic area (55 vaccinated and 48 unvaccinated) brought into an endemic area of southeast Iran. No local and/or general indications of disease were observed in the vaccinated dogs immediately after vaccination. The efficacy of the vaccine was evaluated after 24 months (4 sandfly transmission seasons) by serological, parasitological analyses and clinical examination. In western blot analysis of antibodies to L. infantum antigens, sera from 10 out of 31 (32.2%) unvaccinated dogs, but none of the sera from vaccinated dogs which were seropositive at >100, recognized the 21 kDa antigen of L. infantum wild-type (WT). Nine out of 31 (29%) unvaccinated dogs, but none of vaccinated dogs, were positive for the presence of Leishmania DNA. One out of 46 (2.2%) vaccinated dogs and 9 out of 31 (29%) unvaccinated dogs developed clinical signs of disease. These results suggest that gentamicin-attenuated L. infantum induced a significant and strong protective effect against canine visceral leishmaniosis in the endemic area., Author Summary A 24 month vaccine trial was conducted using 103 leishmania free dogs in an area of southeast Iran endemic for visceral leishmaniosis. The dogs were vaccinated with gentamicin-attenuated line of Leishmania infantum. No local and/or general indications of disease were observed in the vaccinated dogs immediately after vaccination with an attenuated line of Leishmania infantum. Nine out of 31 (29%) unvaccinated dogs, but none of those vaccinated, were positive for presence of Leishmania DNA by PCR. In western blots, sera from 10 out of 31 (32.2%) unvaccinated dogs, but none of the sera from vaccinated dogs, recognized the 21 kDa antigen of Leishmania infantum wild-type. One out of 46 (2.2%) vaccinated dogs and 9 out of 31 (29%) unvaccinated dogs developed clinical signs of disease. The attenuated Leishmania infantum induced a significant and strong protective effect against Leishmania infantum infection in the field.

SUMMARYThe impact of immunization with gentamicin-attenuatedLeishmania infantum(H-line) on the immunophenotypic profile of popliteal lymph node (PLN) and peripheral blood mononuclear cells (PBMCs) of dogs was assessed by flow cytometry and immunohistochemistry. Compared with the dogs infected withL. infantumwild-type (Group WT), there was a significantly higher percentage of CD4+, CD44+T cells and CD14+, MHC-II+cells and a lower percentage of CD4+CD25+regulatory T cells in PLN of the immunized dogs withL. infantumH-line (Group H). The percentage of CD4+and CD8+T cells in PBMCs of immunized dogs was higher than that in dogs of Group WT. The CD4:CD8 ratio in PLN of dogs of Group H was significantly higher than that in dogs of Group WT. A significantly higher percentage of CD21+B cells and a lower percentage of CD79b+cells were found in PLN of the immunized dogs compared with dogs of Group WT. Immunohistochemical investigation showed no parasites in the PLN of immunized dogs whereas there were parasites in the PLN of 60% of dogs infected withL. infantumWT. In this study, the immunophenotypic profile of mononuclear cells of the immunized dogs correlates with cellular immunity.