Experimental model of a Trypanosoma cruzi strain via transplacental and breastfeeding transmission

It is estimated that at least two million women of childbearing age in Latin America are chronically infected with Trypanosoma (T.) cruzi, representing a persistent risk of congenital transmission1. Vertical transmission has been associated with maternal immune dysfunction, which may compromise foetal development and increase the risk of adverse outcomes2. According to the Brazilian consensus on Chagas disease (CD), breastfeeding represents a potential route of transmission when the parasite is present in milk, especially during the acute phase or in cases of nipple lesions with bleeding3,4. However, experimental evidence in lactating females during the acute phase is scarce, and reports of transmission via breastfeeding remain inconclusive. Despite global efforts to control CD, the disease continues to pose a public health challenge, particularly due to the asymptomatic nature of chronic infections and the lack of definitive evidence regarding transmission dynamics during pregnancy and lactation. In this context, we conducted an experimental study using BALB/c mice to investigate the potential for T. cruzi transmission via the placenta and breastfeeding. Eighteen neonates were evaluated 21 days after birth and divided into two groups: Group 1, comprising neonates born to infected females and nursed by uninfected females, to assess vertical transmission; and Group 2, comprising neonates born to uninfected females and nursed by infected females, to evaluate transmission through breastfeeding. The infected females received an intraperitoneal inoculation of 104 trypomastigotes/mL of the ZMC ZMC refers exclusively to the initials of the patient from whom the isolate was obtained, serving only as a clinical identifier. The strain was kindly provided by Prof. Dr. Vicente Amato Neto, from the Institute of Tropical Medicine at USP (University of São Paulo). strain of T. cruzi three weeks before delivery. After mating, parturition was induced with oxytocin (5 μL per animal). Neonates were immediately separated from their dams and allocated to groups as described above. The animals were housed in plastic cages (28 cm × 19 cm × 12 cm) containing autoclaved pine shavings and environmental enrichment with cardboard cones. Conditions were controlled: temperature (25 ± 2) °C, standard diet, and water ad libitum. After three weeks, neonates were euthanised with Thiopental® (Cristália, Itapira, SP, Brazil) at a dose of 0.03 g per 100 g body weight, diluted in saline and administered intraperitoneally. Following euthanasia, cardiac puncture was performed to obtain approximately 1 mL of blood per animal. Blood samples were pooled from every four animals within the same group for analysis. Additionally, the heart, liver, spleen, brain, intestine, and tongue were collected for imprint preparations and subsequent staining. Blood samples were processed using the Strout technique. Samples were centrifuged at ~250 × g for 5 min to remove red blood cells, followed by centrifugation at ~1 800 × g for 10 min. The sediment was examined on histological slides under light microscopy (400×), with 50 microscopic fields analysed per sample. Tissue fragments (heart, liver, spleen, brain, intestine, and tongue) were pressed onto glass slides, fixed in methanol for 5 min, and stained with Giemsa for 30 min. The slides were examined under light microscopy with immersion oil (1000×) to detect intracellular amastigote forms. Data were analysed using the Chi-squared test, with statistical significance set at P < 0.05. All procedures were performed in accordance with ethical principles for animal experimentation and were approved by the Animal Ethics Committee of São Paulo State University (CEUA- UNESP), protocol No. 1417/2022 on August 30, 2022. A total of 27 animals were included in the study, comprising 10 females infected with the low-virulence ZMC strain and 10 uninfected controls. Near parturition, litters were cross-fostered so that neonates were nursed by females from the opposite experimental condition. After three weeks, blood was collected by cardiac puncture, and organs were processed for imprint analysis. All infected dams were confirmed positive for T. cruzi by imprint, with cardiac tissue showing the highest positivity rate (3/5, 60.0%). Although the Strout technique yielded negative results for circulating trypomastigotes, the presence of intracellular amastigotes in tissues indicated a transition from the acute to the chronic phase of infection. A total of 138 slides were prepared for imprint analysis. The following animals were evaluated: five experimentally infected dams (to confirm infection status), nine neonates born to infected dams and nursed by uninfected dams, and nine neonates born to uninfected dams and nursed by infected dams (Figure 1).Figure 1: Imprint analysis of liver sections from BALB/c mice stained with Giemsa. The red circle indicates a cluster of amastigotes. Magnification: 100×. Source: author.All neonates born to infected dams and nursed by uninfected females exhibited at least one infected tissue, confirming vertical transplacental transmission. The most frequently affected organs were the heart and liver(5/9, 55.6%). Similarly, all neonates born to uninfected dams and nursed by infected females also presented at least one infected tissue, suggesting transmission via breastfeeding, with the heart being the most frequently infected organ (100%). Table 1 presents the results of the tissues evaluated by imprint analysis across the different groups.Table 1: Number and percentage of mice positive for the presence of amastigotes in tissues n (%).Historically, transmission of T. cruzi through breastfeeding has been controversial. Early studies by Nattan-Larrier and subsequent works by Disko, Krampitz, and Miles detected the parasite or antibodies in milk but failed to demonstrate successful transmission to offspring5-7. Later experiments by Campos et al. and Martins et al. reinforced the rarity of this route, suggesting that maternal antibodies transferred via milk may provide protection8,9. However, our findings demonstrate infection consistent with the chronic phase of CD in neonates nursed by infected dams, indicating that transmission through breastfeeding is possible under these conditions. In conclusion, our experimental data confirm effective infection of dams by the T. cruzi ZMC strain and demonstrate both transplacental and breastfeeding transmission routes, each resulting in 100% infection of neonates. The heart was consistently the most affected organ, followed by the liver and intestine. These results provide novel experimental evidence supporting the hypothesis that congenital and lactational transmission of T. cruzi may occur even during the chronic phase of infection, underscoring the need for further investigation into maternal-foetal and postnatal dynamics of parasite transmission to inform public health policies and preventive strategies. Conflict of interest statement The authors declare that there are no conflicts of interest. Funding This work was supported by a specialization grant from the SUS/ SP Human Resources Training Center, CEFORSUS/SP. Authors’ contributions B.C.C., R.M.S., and S.B.L.: Conceptualization, experiments, and manuscript drafting; R.M.S. and C.S.K.: Data analysis; All authors approved the final version of the manuscript. Publisher’s note The Publisher of the Journal remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Edited by Zhang Q, Lei Y, Pan Y