Molecular detection and genetic diversity of arthropod and trematode-borne agents in non-hematophagous bats from midwestern Brazil

Author(s)

Priscila Ikeda

• Document type: Doctoral Thesis
• Press: Jaboticabal. 2021-10-15.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Agrárias e Veterinárias. Jaboticabal
• Defense date: 2021-08-05
• Advisor: Marcos Rogério André; Rosangela Zacarias Machado

Abstract

It is estimated that 75% of emerging diseases comprise zoonoses, most of which have wild animals as sources of infection; of these, about 22.8% are carried by arthropod vectors. Thus, monitoring the presence of pathogens in wild animals in niches shared with humans becomes an important preventive method for zoonotic infections. Bartonellaceae and Anaplasmataceae families comprise Gram-negative, facultative and obligate intracellular Alphaproteobacteria, respectively, that have been identified in a wide variety of mammals, including humans. On the other hand, hemotrophic mycoplasmas are epierythrocytic Gram-negative bacteria lacking cell wall, which can cause from asymptomatic infections to severe hemolytic anemia, both in animals and in humans. Coxiella burnetii is the main representative of the Coxiellaceae family (Gammaproteobacteria) and is known to cause Q fever in humans. Despite being transmitted by aerosols, this agent has been detected in several ectoparasites that may act in its life cycle. Piroplasmids, on the other hand, are protozoa of the order Piroplasmida, which can cause hemolytic anemia in farm and domestic animals and, occasionally, in humans. Although the abovementioned agents have been increasingly studied in bats, little is known about their occurrence and genetic diversity in bats from Brazil. The main ectoparasites found in these animals are Streblidae and Nycteribiidae flies, soft ticks, and Spinturnicidae and Macronyssidae mites. Considering the abovementioned pathogens of interest in Public Health and the expressive representation of animals of the order Chiroptera in Brazil, this study aimed to investigate the occurrence and genetic diversity of Anaplasmataceae, Bartonellaceae, Mycoplasmataceae, Coxiellaceae agents and protozoa of the order Piroplasmida in chiropterans and their respective ectoparasites collected in a periurban area of the city of Campo Grande, state of Mato Grosso do Sul, in central-western Brazil. A total of 418 samples (135 spleen, 133 blood and 150 ectoparasites) were collected from 135 animals of the Phyllostomidae, Vespertillionidae and Mollossidae families. Based on molecular assays, positivity of 18.13% (34/418) for Bartonella spp. was found among all collected samples. Out of these, 17 samples had their amplified Bartonella gltA amplicons cloned in order to obtain 3 clones of each one aiming at assessing the genotypic diversity within the same sample. Among the 51 Bartonella gltA-clones obtained, 13 different genotypes were found, with at least two occurring within individual samples. Phylogenetic analyses based on the ftsZ, rpoB and nuoG genes confirmed the high genotype diversity of Bartonella spp. in bats and their ectoparasites. For Anaplasmataceae agents, positivity of 1.67% (7/418), 11.96% (50/418) and 13.63% (57/418) was observed for Anaplasma spp. (16S rRNA), Ehrlichia spp. (dsb) and Neorickettsia spp. (16S rRNA), respectively. The sequences obtained were closely related to Anaplasma phagocytophilum, Ehrlichia minasensis, Neorickettsia risticii, and Neorickettsia findlayensis by BLASTn analyses, and phylogenetically related to Ehrlichia ruminantium based on gltA gene. This is the first molecular evidence of Anaplasmataceae agents in bats and associated ectoparasites from Brazil. Positivity of 13.6% (57/418) for hemoplasmas was found. Among the 24 sequences obtained by the 16S rRNA gene, it was possible to find 12 different hemoplasma genotypes that were distributed into “Haemofelis group”. The two obtained 23S rRNA hemoplasma sequences were closely related to “Candidatus Mycoplasma haematohydrochaerus”, M. haemofelis and M. haemocanis. All bats’ blood and spleen samples were negative in the qPCR for C. burnetii based on the IS1111 gene. Seventeen of the 135 (12.6%) bats were positive for Piroplasmida (18S rRNA gene). This is the first molecular report of piroplasmids in bats from Brazil. Phylogenetic analysis showed the possible occurrence of two new species: Piroplasmid n. sp. Artibeus spp., which was closely related to Babesia canis and B. vogeli, and Piroplasmid n. sp. P. discolor, which formed a monophyletic clade. Unfortunately, the ectoparasite samples could not be used in the PCR assays for piroplasmids and C. burnetii. While Streblidae flies showed positivity in molecular assays for Bartonella spp. (11/64), Ehrlichia spp. (7/64) and Mycoplasma spp. (1/64), Ornithodoros hasei ticks were positive for Ehrlichia spp. (3/19), Anaplasma spp. (1/19) and Neorickettsia spp. (1/19). Spinturnicidae and Macronyssidae mites were positive for Bartonella spp. (4/21 Macronyssidae) and Ehrlichia spp. (18/21 Macronyssidae and 6/46 Spinturnicidae). Also, co-positivity for the different agents studied was observed. In conclusion, non-hematophagous bats from a periurban area of central-western Brazil act as hosts for several genotypes of Bartonella spp. and hemoplasms, Ehrlichia spp. closely related to E. ruminantium, and for two possible new species of piroplasmids. (AU)

• FAPESP's process: 17/14124-4 - Detection and molecular characterization of Anaplasmataceae, Bartonellaceae and Mycoplasmaceae agents in bats sampled in a peri-urban region of Central-Western Brazil
• Grantee: Priscila Ikeda
• Support Opportunities: Scholarships in Brazil - Doctorate