The results obtained in this study show classic clinical signs of CD described by others[6, 15, 25, 26]. The sample size was appropriate for the DNA vaccination canine model in CD and was similar to what has been reported by other research groups in Mexico and other countries of America[6, 8, 26–29].
The DNA-immunized/infected dogs did not have body temperature increases during the acute phase of the disease. The hypothesis of glandular hypertrophy in the murine model during the acute stage of CD has been reported, mainly because of numerous parasite nests in the gland parenchyma with atrophy of secretory structures. This event could have taken place in our model; however, we were unable to confirm this since no histological analysis during this phase of the present study was performed. The dogs immunized with the recombinant plasmids had clinically evident immune responses as evidenced by lymph node enlargement, however, during the post-infection period, the lymphadenopathy was less noticeable. To mount an immune response, lymphocytes must re-circulate between the blood and lymph nodes, recognize antigens upon contact with specialized presenting cells, proliferate to expand a small number of clonally-relevant lymphocytes, differentiate to antibody-producing plasma cells or effector T cells, exit from lymph nodes, migrate to tissues, and engage in host-protective activities such as lymphadenomegaly or fever. Alternatively, fever has not been established as a constant sign of CD in dogs, but other physical signs such as lymph node enlargement always occur[13–15].
In dogs, the tachyarrhythmias have been documented in a continuous or paroxistic (intermittent) manner and are associated with several etiologies, including dilated myocardiopathy, which can be caused by CD, hypertrophic myocardiopathy, endocarditis, valvular disease, congenital defects, cardiac tumors, etc. These may worsen and progress to heart failure, which may result in death. In our study, immunization with the recombinant plasmids was able to halt the heart rate increase during the acute and/or chronic stages of CD, which diminished tachyarrhythmia presentation.
Electrocardiographic tracings were analyzed according to the published data in canine and feline cardiology[23, 24] and our results can be compared to human patients with CD[33–36]. Some electrocardiographic findings observed in this study are consistent with the most frequent electrocardiographic features reported when the canine model has been used in CD studies[6, 8, 15, 25, 26], such as myocardial infarction and/or pericarditis, second-degree AV block, left ventricle enlargement (dilation or hypertrophy), left and right BBB, and VPC.
In disagreement with findings reported by others[8, 9, 26, 28], we did not observe any severe life-threatening arrhythmias, which is likely due to the strain used. Any dog infected with the T. cruzi Ninoa strain exhibited clinical signs that were severe enough to result in death. We chose the Ninoa strain as a reference Mexican strain, whose characterization fulfilled the criteria for being classified as biodeme 3 because of its biological behavior, tissue tropism, and virulence in a susceptible model. Here, we demonstrate that the Ninoa strain of T. cruzi is able to establish an infection with the development of some characteristic clinical signs of acute and chronic stages of CD as described in dogs[13–15].
In a previous study with mice immunized with the pBCSP and pBCSSP4 plasmids, the T. cruzi TcSP and TcSSP4 genes were also effective at inducing antigen-specific antibodies that recognized the surface of T. cruzi and produced a Th1 immune response, which conferred significant protection against T. cruzi experimental infection in BALB/c mice[17, 18]. Our results indicate that the protective effect provided by the TcSP and TcSSP4 genes was similar; however, immunization with the pBCSP plasmid avoided heart rate increases in both stages of the infection and prevented severe heart conduction abnormalities such as myocardial infarction, second-degree AV block or left BBB. This plasmid encodes the trans-sialidase superfamily protein that is present in all three forms of T. cruzi and is recognized by sera from chagasic patients, unlike the TcSSP4 gene that codes for a glycoprotein associated with the transformation of trypomastigotes into amastigotes. Thus, the slight difference in the protective effect is likely because the TcSP gene induces immunity against all forms of the parasite, whereas the TcSSP4 gene only does so against a stage-specific surface antigen present on the amastigote form. Further studies of immunizations with the two plasmids in combination are required to determine if there is a possible synergistic effect.
We conclude that plasmid DNA vaccination with T. cruzi genes induces a moderate level of protection in immunized dogs because this strategy 1) avoids acute phase fever, 2) induces an immune response that manifests as lymph node enlargement as part of host-protective activity, 3) avoids heart rate increases during the acute and/or chronic stages, and 4) most interestingly, halts the symptomatic progression to severe heart conduction abnormalities.