Testing the Efficacy of a Multi-Component DNA-Prime/DNA-Boost Vaccine against Trypanosoma cruzi Infection in Dogs

Immunization of dogs with DNA-prime/DNA-boost vaccine (TcVac1) enhanced the Trypanosoma cruzi-specific type 1 antibody and CD8+ T cell responses that resulted in an early control of acute parasitemia and a moderate decline in pathological symptoms during chronic phase. Further improvement of vaccine-induced immunity would be required to achieve clinical and epidemiological benefits and prevent transmission of parasites from vaccinated/infected dogs to triatomines

Growth and Yield of Purple Kculli Corn Plants under Different Fertilization Schemes

Globally, corn is the most economically important crop, surpassing other cereals of economic importance. However, the tillage methods, monoculture and the abuse of synthetic agrochemicals used in Mexico have led to the loss of fertility and soil yield. In this sense, the application of alternative fertilization methods based on chemical fertilizer, organic matter and biofertilizer, applied alone or in combination, can stimulate the defense systems of corn plants and increase their yield. Therefore, in this research, some fertilization schemes were tested on purple corn plants of the Kculli race through the evaluation of some growth and yield variables, as well as the subsequent evaluation of the chemical characteristics of the corn grain produced in each fertilization scheme. The results indicate highly significant differences (p ≤ 0.05) between treatments, for the different growth and yield variables studied. Of all the fertilization schemes evaluated, treatment T7 obtained the best grain yield of 6.19 ± 0.07 t ha−1, with respect to treatment T1 of 1.02 ± 0.01 t ha−1, as well as the highest protein content and starch quality. Being clear the positive effect of the adequate contribution of the macro and micronutrients used exerts on the corn crop in each of the fertilization schemes studied. On the other hand, the analysis carried out on the grains was found within the values reported by other authors

Caracterización molecular de aislados de Trypanosoma cruzi de triatominos recolectados en los municipios del Estado de Hidalgo, México

Trypanosoma cruzi (T. cruzi), has been classified into six lineages using molecular typing markers, which are easily amplified by the polymerase chain reaction (PCR) technique. The objective of this work was to identify and geographically locate the isolates of T. cruzi that circulate naturally in triatomines of the municipalities of the State of Hidalgo, Mexico, through the amplification from the conserved region of the mini-exon gene by end point PCR. Method: 170 specimens of hematophagous insects from 14 municipalities from the state of Hidalgo, Mexico, were collected. Optic microscopy and PCR from triatomine fecal and digestive tissue samples were used for laboratory diagnostic of T. cruzi infection and T. cruzi lineage classification. Results: Three triatomines taxas were found: Triatoma dimidiata (87/170), Triatoma mexicana (14/170) y Triatoma gerstaeckeri (7/170). For 36.47% (62/170) of the collected specimens, species could not be determined and were classified as T. spp. T. cruzi infection was determined in 1.76% of the collected specimens through optic microscopy and in 11.18% through PCR. All the classified parasites correspond to the TcI biotype of T. cruzi. Most abundant populations of triatomines (80.58%), as well as, the highest percentage (10.58%) of T. cruzi infected insects, were found in the peridomestic ecotope. Conclusion: The most important vector found in the región of study was Triatoma dimidiata, followed by T. mexicana and T. gerstaekeri and the only T. cruzi biotype found to be infecting triatomines was TcI. The vectors were mainly distributed in the peridomiciliary habitats of the studied municipalities. Results indicate a T. cruzi represents a risk of infection for the inhabitants of the studied regions of the state of Hidalgo, Mexico.Trypanosoma cruzi (T. cruzi), está clasificado en seis linajes mediante marcadores moleculares de tipificación, que son fácilmente amplificados por técnicas de biología molecular. El objetivo del trabajo fue identificar y ubicar geográficamente a los aislados de T. cruzi que infectan naturalmente a los triatominos de los municipios del Estado de Hidalgo a través de la técnica de PCR, que amplifica fragmentos de la región intergénica del gen mini-exón. Método: Se recolectaron 170 muestras de insectos hematófagos en 14 municipios del Estado de Hidalgo, México. El diagnóstico de laboratorio en las muestras de heces y de tejido digestivo de los triatominos, se realizó de manera convencional por microscopia óptica y por la técnica de PCR para determinar la presencia o ausencia de T. cruzi y para la identificación del linaje correspondiente del parásito. Resultados: Se identificaron tres taxones de triatominos: Triatoma dimidiata (87/170), Triatoma mexicana (14/170) y Triatoma gerstaeckeri (7/170). En el 36.47% (62/170) de los especímenes colectados la especie no pudo ser identificada y se clasificaron únicamente como Triatoma spp. Se determinó la presencia del parásito en el 1.76% de los vectores analizados por el método parasitoscópico y en el 11.17% por el método de biología molecular. El total de los parásitos analizados corresponde al biotipo TcI de T. cruzi. En el ecotopo peridoméstico, se encontró la mayor abundancia de triatominos (80.58%) y el mayor porcentaje (10.58%) de infección por T. cruzi. Conclusiones: El vector más importante encontrado en la región en estudio fue Triatoma dimidiata seguido de T. mexicana y T. gerstaekeri y el biotipo con el que están mayormente infectados es el TcI. Los triatominos encontrados se distribuían principalmente en hábitats peridomésticos en los municipios estudiados. Los resultados indican la existencia de riesgo de infección para los habitantes de esas regiones endémicas del Estado de Hidalgo, México

Immune protection against Trypanosoma cruzi induced by TcVac4 in a canine model.

Chagas disease, caused by Trypanosoma cruzi, is endemic in southern parts of the American continent. Herein, we have tested the protective efficacy of a DNA-prime/T. rangeli-boost (TcVac4) vaccine in a dog (Canis familiaris) model. Dogs were immunized with two-doses of DNA vaccine (pcDNA3.1 encoding TcG1, TcG2, and TcG4 antigens plus IL-12- and GM-CSF-encoding plasmids) followed by two doses of glutaraldehyde-inactivated T. rangeli epimastigotes (TrIE); and challenged with highly pathogenic T. cruzi (SylvioX10/4) isolate. Dogs given TrIE or empty pcDNA3.1 were used as controls. We monitored post-vaccination and post-challenge infection antibody response by an ELISA, parasitemia by blood analysis and xenodiagnosis, and heart function by electrocardiography. Post-mortem anatomic and pathologic evaluation of the heart was conducted. TcVac4 induced a strong IgG response (IgG2>IgG1) that was significantly expanded post-infection, and moved to a nearly balanced IgG2/IgG1 response in chronic phase. In comparison, dogs given TrIE or empty plasmid DNA only developed high IgG titers with IgG2 predominance in response to T. cruzi infection. Blood parasitemia, tissue parasite foci, parasite transmission to triatomines, electrocardiographic abnormalities were significantly lower in TcVac4-vaccinated dogs than was observed in dogs given TrIE or empty plasmid DNA only. Macroscopic and microscopic alterations, the hallmarks of chronic Chagas disease, were significantly decreased in the myocardium of TcVac4-vaccinated dogs. We conclude that TcVac4 induced immunity was beneficial in providing resistance to T. cruzi infection, evidenced by control of chronic pathology of the heart and preservation of cardiac function in dogs. Additionally, TcVac4 vaccination decreased the transmission of parasites from vaccinated/infected animals to triatomines

T cell and cytokine response in vaccinated dogs.

<p>(<b>A</b>) Spleen cells were obtained from vaccinated and non-vaccinated dogs at one year after challenge infection with <i>T. cruzi</i>. Spleen cells were incubated for 30 min with FITC-conjugated anti-CD8 and PE-conjugated anti-CD4 antibodies and CD4⁺ and CD8⁺ T cell subsets monitored by flow cytometry. (<b>B</b>) The circulatory IFN-γ level was measured by an ELISA. Data are presented as mean ± SD (*<i>p</i><0.05).</p

Morphological alterations in the heart.

<p>Dogs were vaccinated and infected with <i>T. cruzi</i> as above. Shown are representative morphologic alterations of the heart during the acute infection phase (60 dpi) in dogs injected with vector only (<b>B&B1</b>) or immunized with TcVac1 (<b>C&C1</b>). Images of normal heart (<b>A&A1</b>) are shown for comparison. Horizontal arrows show right ventricle wall thinning characteristic of ventricle dilation. Vertical arrows show pale striated epicardium and myocardium, characteristic of necrosis produced after inflammatory response to infection.</p

The antibody response to <i>T. cruzi</i> infection was polarized to type 1 in vaccinated dogs.

<p>Dogs were vaccinated as above, and infected with <i>T. cruzi</i>. An ELISA was performed to evaluate sera levels (1∶100-dilution) of <i>T. cruzi</i>-specific IgM (<b>A</b>), IgG (<b>B</b>), IgG1 (<b>C</b>), and IgG2 (<b>D</b>) antibodies at 0, 15, 30, 45, and 60 days post-infection.</p

TcVac1-induced antibody response in dogs.

<p>Dogs were vaccinated with TcVac1 or injected with vector only, and sera were collected two weeks after each immunization dose. An ELISA was performed to monitor the sera levels (1∶50 dilution) of parasite-specific IgM (<b>A</b>), IgG (<b>B</b>), IgG1 (<b>C</b>) and IgG2 (<b>D</b>). The vaccine-induced antigen-specific (TcG1, TcG2 and TcG4) antibody response in sera collected 14 days after last dose of vaccine was determined using recombinant antigens. Data are presented as mean ± SD (*<i>p</i><0.05, vaccinated versus non-vaccinated, n = 6 per experiment).</p

Histological analysis of hearts.

<p>Dogs were vaccinated, and challenged with <i>T. cruzi</i>. Heart tissue sections (5-ìM) from left ventricle, septum, and right ventricle were obtained at 60 days post-infection (acute phase), and stained with hematoxylin-eosin. Shown are representative micrographs of dogs injected with vector only (<b>B,B1,B2</b>) or immunized with TcVac1 (<b>C,C1,C2</b>). Micrographs from normal/uninfected dogs (<b>A,A1,A2</b>) are shown for comparison. Vertical arrows show amastigote nests and horizontal arrows show lymphocyte infiltration, and cardiomyocytes destruction.</p

Shown are plasma levels of myeloperoxidase activity (A) and nitrite content (B) in dogs immunized with vector only or TcVac1 vaccine during the course of 0–60 days post-infection.

<p>Data are presented as mean ± SD (*<i>p</i><0.05).</p