Trypanosoma cruzi is mainly transmitted to mammals by vectors, but other transmission routes exist. Opossums can harbor the infectious form of the parasite in their anal glands, underscoring their potential role in non-vectorial transmission. In Mexico, Didelphis virginiana and D. marsupialis are the main opossum species. While T. cruzi has been detected in the anal gland secretions of various opossum species, their infectivity has only been confirmed in D. marsupialis and D. albiventris. Vertical transmission has also been proposed in D. virginiana. However, if this occurs in opossums, it remains unclear whether it happens during pregnancy or lactation. Our objective was to investigate the possible contribution of urban opossums to non-vectorial transmission of T. cruzi in the metropolitan area of Merida, Yucatan, in southeastern Mexico. Blood, anal gland secretions, and milk were collected from opossums captured in Merida, Mexico, all identified as D. virginiana using taxonomic keys and Cytb sequencing. By PCR, T. cruzi was detected in 16/102 opossums (15.69%) in at least one type of sample. The prevalence was 14.71% (15/102) in blood and 0.98% (1/102) in anal gland secretions. 1/22 milk samples (4.55%) tested positive. Blood of 37 offspring from T. cruzi-positive mothers was collected and tested negative. qPCR revealed that females with offspring tended to have lower parasite load in blood compared to females without offspring and males. Genotyping of the parasite through multiplex PCR revealed only the DTU TcI. This study agrees with previous works where D. virginiana was the most abundant opossum species in urban areas in southeastern Mexico and confirms that it is associated with TcI. Detection of T. cruzi in a sample of anal gland secretions underscores the potential risk represented by D. virginiana in non-vectorial transmission in urban areas of southeastern Mexico. Detection in the milk of a lactating female, along with the observed tendency towards a lower parasite load in females with offspring, highlight the importance of further investigating vertical transmission in D. virginiana.
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