Novel genotypes of Hepatozoon spp. in small mammals, Brazil

BACKGROUND: Small mammals (rodents and marsupials) have been poorly explored for the occurrence of apicomplexan (genus Hepatozoon and genera of the order Piroplasmorida) and Anaplasmataceae agents in Brazil. Thus, this study investigated the occurrence of Hepatozoon spp., Piroplasmorida, and Anaplasmataceae agents in small mammals in seven forest fragments in Brazil. METHODS: During 2015–2018, small mammals were captured in six forest fragments in the State of São Paulo (Cerrado and Atlantic Forest biomes) and one fragment in the State of Mato Grosso do Sul (Pantanal biome). Mammal blood, liver, spleen, and lung samples were tested molecularly for the presence of DNA of Hepatozoon, Piroplasmorida, and Anaplasmataceae agents. RESULTS: A total of 524 mammals were captured, comprising seven species of marsupials, 14 rodents, two carnivores, and one Cingulata. Four novel haplotypes (1, 2, 3, 4) of Hepatozoon spp. were detected in small mammals from different biomes. In São Paulo state, haplotype 1 was detected in rodents from Cerrado and a transition area of Cerrado and Atlantic Forest biomes, whereas haplotype 2 was detected in rodents from the Atlantic Forest biome. On the other hand, haplotypes 3 and 4 were restricted to rodents and marsupials, respectively, from the Pantanal biome of Mato Grosso do Sul. No host species shared more than one haplotype. Despite these distinct geographical and host associations, our phylogenetic analyses indicated that the four Hepatozoon haplotypes belonged to the same clade that contained nearly all haplotypes previously reported on rodents and marsupials, in addition to several reptile-associated haplotypes from different parts of the world. No mammal samples yielded detectable DNA of Piroplasmorida agents. On the other hand, the Anaplasmataceae-targeted polymerase chain reaction (PCR) assay amplified a sequence 100% identical to the Wolbachia pipientis endosymbiont of the rodent filarid Litomosoides galizai. CONCLUSIONS: We report a variety of Hepatozoon haplotypes associated with small mammals in three Brazilian biomes: Cerrado, Atlantic Forest, and Pantanal. Through phylogenetic analyses, the Hepatozoon agents grouped in the rodent-marsupial-reptile large clade of Hepatozoon spp. from the world. The detection of a W. pipientis associated with the rodent filarid L. galizai indicates that the rodent was infected by filarial nematodes. GRAPHICAL ABSTRACT: [Image: see text]