Abstract The current study proposes to investigate the diversity and phylogeny of trypanosomes parasitizing wild birds from the Brazilian Atlantic Forest. Cytological examination was carried out by light microscopy of blood smears and positive birds were selected for amplification of the 18S rDNA sequence through PCR. The resulting amplicons were subjected to purification, cloning, and sequencing analysis. Phylogenetic reconstruction was conducted, including all avian trypanosomes representative’s lineages. A total of ten bird samples from species of Turdus flavipes (N=1/12), T. albicollis (N=1/8), Tachyphonus coronatus (N=6/121), Thamnophilus caerulescens (N=1/22) and Synallaxis spixi (N=1/8) were positive for Trypanosoma spp. In the six specimens of T. coronatus, five distinct lineages of Trypanosoma spp. 18S-rRNA were observed in ninety sequences obtained, and using the strategy of cloning independent PCR, it was possible to observe that two of them were related to T. avium (JB01/JB02), and three were closed related to T. bennetti (JB03/ JB04/JB05). Addionaly, all fifteen sequences obtained from T. caerulescens/ S. spixi/T. flavipes/T. albicollis were identical. The present research is the first study to access molecular diversity and polyparasitism by avian trypanosomes in Brazil. The current research exhibits the wide genetic variability in avian trypanosomes and its non-specific relationship with its avian hosts. Forest S PCR purification analysis conducted representatives representative s N=1/12, N112 N N=1/12 , 1 12 (N=1/12) T N=1/8, N18 N=1/8 8 N=6/121, N6121 N=6/121 6 121 (N=6/121) N=1/22 N122 22 (N=1/22 (N=1/8 spp 18SrRNA SrRNA rRNA JB01/JB02, JB01JB02 JBJB JB01/JB02 JB01 JB02 JB (JB01/JB02) JB03/ JB03 (JB03 JB04/JB05. JB04JB05 JB04/JB05 . JB04 JB05 JB04/JB05) Addionaly spixiT spixi/T flavipesT flavipes/T identical Brazil nonspecific non specific hosts N11 N=1/1 (N=1/12 N1 N=1/ N612 N=6/12 (N=6/121 N=1/2 N12 2 (N=1/2 (N=1/ JB01JB0 JB01/JB0 JB0 (JB01/JB02 (JB0 JB04JB0 JB04/JB0 (N=1/1 N=1 N61 N=6/1 (N=6/12 (N=1 JB01JB JB01/JB (JB01/JB0 (JB JB04JB JB04/JB N= N6 N=6/ (N=6/1 (N= (JB01/JB N=6 (N=6/ (N (N=6

ABSTRACT: Cattle trypanosomiasis imposes significant economic burdens on the global livestock industry. The causative agents of this disease belong to the protozoan Trypanosoma genus. This study aims to perform detection (parasitological and molecular) and genetic characterization to analyze Trypanosoma spp. in cattle from 15 municipalities in the state of Rio de Janeiro, focusing on the 18S rDNA and Cathepsin-L (CatL) gene of Trypanosoma vivax and Trypanosoma theileri. A total of 389 blood samples from 15 dairy cattle farms in the state of Rio de Janeiro were collected, and DNA was extracted for subsequent PCR amplification of Trypanosoma spp. 18S rDNA and CatL genes. The resulting amplicons underwent sequencing and alignment for phylogenetic analysis, with comparisons made to GenBank isolates. Concerning parasitological analysis, blood smears presented 4.4% of positive cattle (n=17/389) for T. vivax and did not show any trypomastigote forms of T. theileri. The absolute frequency of Trypanosoma spp. through molecular detection targeting 18S rDNA was 11.6% (45/389). However, when performing species-specific PCRs, the T. vivax frequency, determined through CatL gene PCR, was 12.8%, and the T. theileri frequency was 3.6%. Phylogenetic analysis based on 18S rDNA revealed low diversity among T. vivax sequences, suggesting potential host segregation. This study emphasizes the high frequency of positive samples by PCR when compared to direct parasitological exams. Additionally, T. vivax phylogeny targeting 18S rDNA hints at sequence clustering related to host species. Importantly, this investigation unveils, for the first time in Rio de Janeiro’s cattle, the circulation of T. theileri lineage ThI, encompassing genotypes IIB and IF. This discovery expands our understanding of this parasite’s geographical distribution and genetic diversity. ABSTRACT industry genus spp 1 S CathepsinL Cathepsin L (CatL 38 collected genes isolates 44 4 4.4 n=17/389 n17389 n 17 (n=17/389 T 116 11 6 11.6 45/389. 45389 45/389 . 45 (45/389) However speciesspecific species specific PCRs 128 12 8 12.8% 36 3 3.6% sequences segregation exams Additionally Importantly unveils Janeiros s ThI IF parasites parasite 4. n=17/38 n1738 (n=17/38 11. 4538 45/38 (45/389 12.8 3.6 n=17/3 n173 (n=17/3 453 45/3 (45/38 12. 3. n=17/ n17 (n=17/ 45/ (45/3 n=17 n1 (n=17 (45/ n=1 (n=1 (45 n= (n= (4 (n (

RESUMO: A tripanossomíase bovina impõe significativos ônus econômicos à indústria pecuária global. Os agentes causadores dessa doença pertencem a protozoários do gênero Trypanosoma. Objetivou-se, com este estudo, realizar detecção (parasitológica e molecular) e caracterização genética de Trypanosoma spp. em bovinos de 15 municipalidades do estado do Rio de Janeiro, com foco na sequência 18S rDNA e no gene Cathepsin-L (CatL) de Trypanosoma vivax e Trypanosoma theileri. Um total de 389 amostras de sangue de 15 fazendas leiteiras no estado do Rio de Janeiro foram coletadas, e o DNA foi extraído para subsequente amplificação por PCR dos genes 18S rDNA e CatL de Trypanosoma spp. Os amplicons resultantes foram submetidos a sequenciamento e alinhamento para análise filogenética, com comparações realizadas com isolados do GenBank. No que se refere à análise parasitológica, os esfregaços de sangue apresentaram 4,4% de bovinos positivos (n=17/389) para T. vivax e não mostraram nenhuma forma tripomastigota de T. theileri. A frequência absoluta de Trypanosoma spp. através da detecção molecular visando 18S rDNA foi de 11,6% (45/389). No entanto, ao realizar PCRs específicos de espécies, a frequência de T. vivax, determinada por PCR do gene CatL foi de 12,8%, e a frequência de T. theileri foi de 3,6%. A análise filogenética com base no 18S rDNA revelou baixa diversidade entre as sequências de T. vivax, sugerindo uma possível segregação de hospedeiros. Este estudo enfatiza a alta frequência de amostra positiva pela PCR quando comparada com a parasitológica direta. Além disso, a filogenia de T. vivax direcionada ao 18S rDNA sugere agrupamento de sequências relacionado à espécie hospedeira. Importante destacar que esta investigação revela, pela primeira vez no gado do Rio de Janeiro, a circulação da linhagem ThI de T. theileri, abrangendo os genótipos IIB e IF. Esta descoberta amplia nosso entendimento sobre a distribuição geográfica e diversidade genética desse parasito. RESUMO global Objetivouse, Objetivouse Objetivou se, Objetivou-se spp 1 S CathepsinL Cathepsin L (CatL 38 coletadas GenBank 44 4 4,4 n=17/389 n17389 n 17 (n=17/389 T 116 11 6 11,6 45/389. 45389 45/389 . 45 (45/389) entanto espécies 128 12 8 12,8% 36 3 3,6% hospedeiros direta disso hospedeira revela IF parasito 4, n=17/38 n1738 (n=17/38 11, 4538 45/38 (45/389 12,8 3,6 n=17/3 n173 (n=17/3 453 45/3 (45/38 12, 3, n=17/ n17 (n=17/ 45/ (45/3 n=17 n1 (n=17 (45/ n=1 (n=1 (45 n= (n= (4 (n (