Trypanosoma cruzi in the chicken model: Chagas-like heart disease in the absence of parasitism

Abstract

Background: The administration of anti-trypanosome nitroderivatives curtails Trypanosoma cruzi infection in Chagas disease patients, but does not prevent destructive lesions in the heart. This observation suggests that an effective treatment for the disease requires understanding its pathogenesis.

Methodology/principal findings: To understand the origin of clinical manifestations of the heart disease we used a chicken model system in which infection can be initiated in the egg, but parasite persistence is precluded. T. cruzi inoculation into the air chamber of embryonated chicken eggs generated chicks that retained only the parasite mitochondrial kinetoplast DNA minicircle in their genome after eight days of gestation. Crossbreeding showed that minicircles were transferred vertically via the germ line to chicken progeny. Minicircle integration in coding regions was shown by targeted-primer thermal asymmetric interlaced PCR, and detected by direct genomic analysis. The kDNA-mutated chickens died with arrhythmias, shortness of breath, cyanosis and heart failure. These chickens with cardiomyopathy had rupture of the dystrophin and other genes that regulate cell growth and differentiation. Tissue pathology revealed inflammatory dilated cardiomegaly whereby immune system mononuclear cells lyse parasite-free target heart fibers. The heart cell destruction implicated a thymus-dependent, autoimmune; self-tissue rejection carried out by CD45(+), CD8γδ(+), and CD8α lymphocytes.

Conclusions/significance: These results suggest that genetic alterations resulting from kDNA integration in the host genome lead to autoimmune-mediated destruction of heart tissue in the absence of T. cruzi parasites.

Figure 1. Elimination of Trypanosoma cruzi infection early in Gallus gallus embryonic development.

A) Top panel shows 330 bp bands formed by PCR amplified minicircles kDNA templates harvested at several stages of the chicken embryonic development, after hybridization with a specific probe; Bottom panel shows bands formed by PCR amplified from same embryos after separation in 1% agarose gel and hybridization with a specific nDNA probe; the 188 bp nDNA band was diagnostic of the parasite persistence in the host tissue. B) Sensitivity of the PCR with nDNA primers Tcz1/2. Lanes 1 and 2, control DNA from kDNA negative and from kDNA-mutated chickens; Lanes 3 to 7, mix of 200 ng of control chicken DNA with increasing amounts of T. cruzi DNA, respectively: 1 fg, 10 fg, 1 pg, and 100 pg, and 1 ng. The hybridization with the radiolabeled 188-bp probe improved the technique sensitivity (10 fg), which reached 24-fold below the diploid T. cruzi total DNA.

Figure 2. Trypanosoma cruzi-free inflammatory myocardiopathy in one week-old chick.

A) Cardiomegaly in a chick hatched from T. cruzi inoculated egg. B) Histopathology showing severe inflammatory infiltrates and non-parasitized heart cell lyses by cytotoxic lymphocytes. C) Normal heart size of a mock control chick. D) Normal heart histology of control chick. H-E staining, magnification 200 X.

Figure 3. Retention of the kDNA minicircle from Trypanosoma cruzi in the Gallus gallus genome.

A) PCR amplification of kDNA from 12 adult chickens hatched from T. cruzi-infected eggs with primer set s35/s36 and hybridization with NsiI-digested, whole kDNA labeled as probe. The lower panel shows the absence of nDNA by the Tcz1/2 primer set, hybridized with a 188 bp probe. B) G. gallus somatic cell's DNA templates from F3 progeny, which were separated in 1% agarose gels, revealed the minicircle 330-bp band in the absence of nDNA by using specific primer sets and hybridization with the 188 bp probe. C) G. gallus germ line DNA templates from parental 1 and 2, and from mated progeny in the F1 (8 and 9) and F2 (19 and 20) amplified with primer sets s35/s36 or Tcz1/2 and probed as described.

Figure 4. The tpTAIL-PCR strategy used to detect Trypanosoma cruzi kDNA integration into the Gallus gallus genome.

A) A chimera sequence with a fragment of kDNA minicircle conserved (dark blue) and variable (light blue) regions integrated in the locus NW_001471687.1 at chromosome 4 (AY237306) of the chicken genome (green) was used to obtain the host specific primer sets (Gg1 to Gg6). B) The tpTAIL-PCR amplifications were initiated (primary cycle) by annealing of the kDNA-specific S34 or S67 primers in combination with chicken-specific Gg1 to Gg 6 primers. Diluted products provided template for the secondary cycle with the S35 (sense/antisense) primers and the combinations of Gg primers. In the tertiary cycle a dilution of the secondary products was subjected to amplification with kDNA S36 or S67 antisense primers in combination with the Gg primers. C) These amplification products were separated in 1% agarose gels and transferred to nylon membrane, hybridized with the specific kDNA probe, then cloned and sequenced. The combinations of kDNA and targeted Gg1 to Gg6 are shown on top of the gel. The sequential PCR reactions amplified target kDNA-host DNA sequences with kDNA minicircles (blue) and the avian sequence (green).

Figure 5. Heredity of the integrations of Trypanosoma cruzi kDNA minicircles into several loci of the chicken genome.

Rows A, B, and C show the integrations, respectively, in the macrochromosomes, in the intermediate, and in the microchromosomes. The numeral(s) in brackets indicates the total times an insertion (red bar) was present at a chromosomal locus from animal source shown in Table S1.

Figure 6. Representation of the gRNA in the Trypanosoma cruzi kDNA minicircle integrated into Gallus gallus genome.

The kDNA conserved (dark blue) and variable (light blue) fragment (nts 1 to 286) is inserted in the PI-3K serine-threonine related kinase SMG1 (Supressor Morphogenetic Genitalia) at the locus NW_ 001471454.1. A CA-rich sequence block (CArsbI) microhomology intermediates the kDNA integration into the PI-3K exon. A gRNA cognate to Tbnd7ed ( Table 2 ) is present in the kDNA variable region (dotted line), which is formed by 55 nts in antisense direction (arrow). Short arrows indicate positions of kDNA primers.

Figure 7. Clinical and pathological findings in Gallus gallus with Trypanosoma cruzi kDNA mutations.

A) Nine month-old F1 hen displaying heart insufficiency by cyanosis of the comb (bottom left), and a control hen of the same age showing a bright red comb (top right). B) Deviation of cardiac axis from a-to-c over a six-month period. C) Increased cardiac heart/body indexes in chickens with kDNA integrations. The heart/body size indexes showed statistically significant differences (p≤0.05) in control and in kDNA-mutated chickens. D) Cardiomegaly (30 g) in a nine month-old hen that died of heart failure. E) Control heart (8 g) from a nine month-old hen. F to I, H-E, magnification 100X: F) Diffuse myocarditis showing immune system mononuclear cell infiltrates and lysis of target heart cells. The red circle depicts a minimal rejection unit whereby effectors lymphocytes destroy a target heart cell. G) Histology of control chicken heart. H) Intracardiac parasympathetic ganglion showing mononuclear cell infiltrates and neuronal cell lysis. I) Control plate showing normal histology of an intracardiac parasympathetic ganglion. J to N, series of histological analyses with kDNA-mutated chicken heart; control uninfected chicken heart tissue shown in the inserts: J) Lack of B cells in a destructive heart lesion treated with anti-Bu-1 monoclonal antibody. K) CD45⁺ lymphocytes identified (arrows) in heart lesions by a phycoerythrin-labeled specific monoclonal antibody. L) CD8⁺γδ immune lymphocytes (arrows) involved in severe destruction of the heart. M) Abundant CD8α⁺ T cells present in severe lesions with heart cell lysis. N) Mononuclear peripheral cells, monocytes and macrophages in the heart lesions.