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Article

From Intact to Highly Degraded Mitochondrial Genes in Trypanosoma vivax: New Insights into Introduction from Africa and Adaptation to Exclusive Mechanical Transmission in South America

by
Herakles A. Garcia
1,*,
Adriana C. Rodrigues
1,
Carla M. F. Rodrigues
1,
Andrea S. Florentin
2,
Angélica M. Bethencourt
3,
Armando Reyna-Bello
4,
María Augusta Chávez-Larrea
4,5,
Carlos L. Pereira
6,
Zacharia Bengaly
7,
Desie Sheferaw
8,
Agapitus B. Kato
9,
Fernando Paiva
10,
Erney F. P. Camargo
1,† and
Marta M. G. Teixeira
1
1
Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 1374, São Paulo 05508-000, Brazil
2
Centro de Investigaciones y Transferencia de Formosa, Formosa 3600, Argentina
3
Faculty of Veterinary Science, Central University of Venezuela, Maracay 1050, Venezuela
4
Grupo de Investigación en Sanidad Animal y Humana (GISAH), Departamento de Ciencias de la Vida y la Agricultura, Universidad de las Fuerzas Armadas ESPE, P.O. Box 171-5-231, Sangolquí 171103, Ecuador
5
Research Unit of Epidemiology and Risk Analysis Applied to Veterinary Sciences (UREAR-ULiège), Fundamental and Applied Research for Animal and Health (FARAH) Center, Department of Infections and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, 4000 Liège, Belgium
6
National Administration of Conservation Areas (ANAC), Ministry of Land, Environment and Rural Development, Maputo 1196, Mozambique
7
Centre International of Recherche-Développement sur l’Élevage en zone Subhumide (CIRDES), Bobo-Dioulasso 01 BP 454, Burkina Faso
8
Faculty of Veterinary Medicine, Hawassa University, Hawassa P.O. Box 05, Ethiopia
9
Uganda Virus Research Institute, Plot 51-59 Nakiwogo Road, Entebbe P.O. Box 49, Uganda
10
Department of Veterinary Parasitology, Federal University of Mato Grosso do Sul, Campo Grande 79070-900, Brazil
*
Author to whom correspondence should be addressed.
†
In memoriam.
Parasitologia 2024, 4(4), 390-404; https://doi.org/10.3390/parasitologia4040035
Submission received: 23 September 2024 / Revised: 13 November 2024 / Accepted: 8 December 2024 / Published: 13 December 2024
(This article belongs to the Special Issue The Molecular Epidemiology of Parasites)
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Abstract

:
Trypanosoma vivax displays distinct mitochondrial genomes: intact in Africa and degraded in South America. This finding, formerly reported from four isolates, suggested an association between mitochondrial mutations and adaptation to exclusive mechanical transmission outside Africa, circumventing mitochondrial activity essential for cyclical transmission by tsetse flies. Here, we characterized the mitochondrial ND7 and COIII genes in 25 South American isolates of T. vivax from livestock from Argentina, Brazil, Colombia, Ecuador, and Venezuela, as well as in 11 African isolates from Mozambique, Ethiopia, Uganda, Nigeria, and Burkina Faso from tsetse flies, antelopes, and cattle. Phylogenetic inference supported the lineages TVV (Trypanosoma vivax vivax) and TVL (Trypanosoma vivax-like), predominant in South America/West Africa and East Africa, respectively. Both ND7 and COIII genes were intact in all African TVV and TVL isolates and, for the first time, in 10 South American isolates from Brazil. Remaining South American isolates exhibited some degree of gene erosion, including ~142 bp deletion at the 3′ end of ND7 disclosed in Venezuela and Colombia, ~163 bp deletion at the 3′ end of ND7 in Brazil and Argentina, and ~750 bp deletion from the 5′ end of ND7 to the 3′ end of COIII in Venezuela. Comprehensive analyses of whole mitochondrial genomes from additional South American and African isolates are crucial for formulating reliable hypotheses regarding the origin, absence, and rapid mitochondrial degradation observed in South American T. vivax.

1. Introduction

Trypanosoma vivax is a prominent pathogen affecting livestock in sub-Saharan Africa and Central and South America. Its introduction to the Americas most likely accompanied the importation of livestock from Spanish and Portuguese colonies in Africa by European colonizers over 500 years ago, imported directly from Africa or from Europe using intermediate ports in the Atlantic Islands of Cape Verde and the Canary Islands [1,2,3]. The movement of live livestock from African colonies to different regions of Central and South America persisted for centuries, facilitating the spread of many parasites, including African Trypanosoma evansi and T. vivax. In South America, T. vivax has been reported in Brazil, Venezuela, Colombia, French Guiana, Ecuador, and Argentina, with great negative impact on livestock production. T. vivax is the only trypanosome recognized as an important agent of disease in livestock in Central and South America, where infections manifest with weight loss or reduced weight gain, reduced milk yields, abortions, stillbirths, alterations in sperm parameters among other reproductive disorders in males, and high mortality due to hematological changes and, eventually, neurological disturbances [4,5,6,7,8,9]. The spectrum of T. vivax infection ranges from asymptomatic, mainly in beef cattle and water buffaloes in endemic areas, to severe and fatal acute infection, mostly in dairy cattle from nonendemic settings. Following fulminating outbreaks of acute disease in naïve herds, T. vivax trypanosomiasis tends to go into the enzootic state; however, asymptomatic animals act as sources of parasites to new outbreaks [4,6,10,11,12,13]. Recent confirmations of T. vivax in Asia, reported in cattle, buffalo, sheep, and goats from Iraq and camels from Iran, have been attributed to the importation of live animals from South America [14,15].
Trypanosoma vivax causes Nagana, a disease limiting livestock production with major socioeconomic impacts in sub-Saharan Africa [16,17], where it is transmitted cyclically by tsetse flies and mechanically by stable, horn, and tabanid flies [18,19,20,21,22]. In the American continent, T. vivax is exclusively found in its blood forms due to the absence of tsetse flies. Its transmission occurs exclusively mechanically through diverse biting flies and frequently through the iatrogenic route [23]. Phylogenetic studies demonstrated that T. vivax comprises two main phylogenetic lineages: TVV, prevalent in West Africa and South America, and TVL, predominant in East Africa. Recent studies showed increased complexity of TVV and TVL in Central and Eastern Africa, indicating greater genetic diversity in T. vivax than previously recognized [17,24,25,26,27,28,29,30]. The mechanical transmission of T. vivax has enabled its spread beyond the tsetse belt to Central and South America, unlike other African trypanosome species that rely exclusively on tsetse transmission and remain confined to sub-Saharan Africa. In tsetse flies, T. vivax development is restricted to the mouthparts, without stages in the midgut and salivary glands [31,32,33].
Cyclical transmission of African trypanosomes relies on mitochondrial protein-coding genes, which are arranged in a disk-shaped network composed by thousands of maxicircle and minicircle molecules of kinetoplast DNA. Maxicircles (20–35 kb) correspond to the mitochondrial genome, which encodes respiratory chain proteins, while minicircles, present in thousands of variable copies, encode guide rRNAs (gRNAs). The number and diversity of minicircles and gRNAs are directly associated with the extent of RNA editing, a mechanism necessary for decoding encrypted maxicircle transcripts ranging from pan-edited to non-edited genes. This process involves the acquisition of correct reading frames through a variable number of uridine insertions/deletions [34,35]. Maxicircle transcripts are properly edited in African isolates, contrasting with the reduced number and variability of minicircles and gRNAs limiting South American T. vivax to the host bloodstream [36,37,38]. The transition between insect and bloodstream forms requires rapid and extensive adaptations in trypanosome metabolism. In the bloodstream, trypanosomes rely solely on glycolysis within glycosomes to generate ATP from host plasma glucose, whereas in tsetse flies, amino acids like proline enable ATP production through mitochondrial oxidative phosphorylation [33,39,40].
Until recently, the only known trypanosomes exhibiting either partial or total absence of maxicircles were T. equiperdum, which has a large deletion, and T. evansi, which is characterized by a complete loss of maxicircles [34,41,42,43]. Recently, however, a substantial degradation of mitochondrial genes leading to the functional loss of mitochondria was observed in three T. vivax South American isolates. In contrast, the mitochondrial integrity remains preserved in the single isolate of African T. vivax so far characterized [37,38]. Mitochondrial erosion limited to South American T. vivax suggests a correlation with exclusive mechanical transmission [37,38]. Nevertheless, considering the relatively recent introduction of T. vivax in South America, the existence of T. vivax in tsetse-free regions in Africa, and the small number, underrepresented genetic diversity, and limited geographical origin of characterized isolates, many questions arise: (1) Did mitochondrial genes remain intact in all African T. vivax isolates? (2) Did mitochondrial gene sequences diverge according to the phylogenetic relationship and overall geographical structure of T. vivax? (3) Did a complex degree of mitochondrial degradation characterize South American T. vivax isolates, considering they were introduced over centuries from different regions in Africa? (4) Were mitochondrial mutations triggered upon the introduction of T. vivax to South America, and are they ongoing, following a determined pathway and timing toward the loss of the entire mitochondrial genome?
Addressing all the above questions is a challenging endeavor and would necessitate extensive research focusing on mitochondrial genomes in comprehensive T. vivax populations from both tsetse-infested and tsetse-free regions in Africa, as well as from Central and South America. In the present study, our aim was to contribute to the initial steps toward unraveling questions concerning variability in T. vivax mitochondrial genes. With this purpose, we compared the ND7 and COIII genes from isolates on both continents.

2. Materials and Methods

2.1. Trypanosoma vivax Isolates and DNA Preparation

The 36 T. vivax isolates included in our study are listed in Table 1. DNA was extracted as previously described in studies by Rodrigues et al. [25] and Garcia et al. [44]. Isolates from South America (SA, n = 25) were from this and our previous surveys as following: Six isolates from endemic settings in Venezuela, including asymptomatic cattle (n = 2) and water buffalo (n = 1), and symptomatic sheep (n = 1) and water buffaloes (n = 2) [11,45]. Isolates from Brazil were obtained from sick (n = 2) and asymptomatic (n = 1) cattle from non-endemic and endemic areas, respectively [10,24,45]; from sick (n = 4) and asymptomatic sheep (n = 2) from northeaster Brazilian [5,45]; and from asymptomatic water buffaloes (n = 4) from Brazilian Amazonia (Northern region) [45]. Additionally, four T. vivax isolates were from dairy cows from severe outbreaks in Argentina [7], one isolate was from healthy cattle from Colombia, and one isolate was obtained from sick cattle in Ecuador [12] (Table 1; Figure 1). African T. vivax isolates (n = 11) were from cattle from West countries: T. vivax Y486 from Nigeria [46] and one isolate from Burkina Faso (n = 1) [45]. The isolate of T. vivax Y484 was obtained from Nigerian cattle and is infective to calves, sheep, and goats, as well as mice, rats, and rabbits; cultivable in vitro; and transmissible by a wide range of tsetse species, and it was the isolate chosen for the genome project of T. vivax [46]. East African isolates were from an antelope Nyala [24] and from tsetse flies captured in Mozambique (n = 3) [25] and Ethiopia (n = 3) [47]. Two isolates were from tsetse flies captured in Uganda (n = 2), Central Africa [47] (Table 1; Figure 1).

2.2. PCR-Amplification of DNA Sequences Encoding ND7 and COIII Mitochondrial Genes

ND7 and COIII are genes that are contiguous and in the same orientation; thus, the amplification of both can be carried out using primers targeting the 5′ end of ND7 and the 3′ end of COIII, producing an amplicon of ~1.1 kb for the reference T. vivax Y486 [37]. Here, the PCR-amplification of the kDNA region encompassing the ND7 and COIII genes was carried out using oligonucleotides primers and PCR conditions previously defined [37] with some modifications. Briefly, we used the forward primer ContigB_F deletion (5′-GAGTGATTGAGTGGGAAAG-3′) as previously described by Greif et al. [37] and a new reverse primer (Reverse-new 5′-CGTGTTCCTCTCTCCG-3′) dislocated ~20 nucleotides downstream compared to the primer ContigB_R_deletion designed by Greif et al. [37]. Aiming to obtain a single DNA band spanning ND7/COIII genes, the annealing temperature was 61 °C, established using DNA from T. vivax Y486 in a gradient PCR assay. The estimated PCR-amplified length of ND7/COIII contiguous genes in T. vivax genomes was ~1042 bp for T. vivax Y486, while for the two South American (Venezuela) isolates (LIEM176 and MT1), the length was ~252 bp due to a large deletion encompassing both ND7 and COIII genes [37,38].

2.3. Sequencing of ND7 and COIII Mitochondrial Genes from T. vivax Isolates and Phylogenetic Analysis

The ND7/COIII PCR-amplified products (20 µl) were subjected to electrophoresis on 2% agarose gels, stained with GelRed® (Biotium, Fremont, CA, USA), visualized by UV trans-illumination, and amplicons from each T. vivax isolate purified, cloned, and sequenced in an ABI 3500 Genetic Analyzer (Applied Biosystems). Sequences of ND7/COIII genes were submitted to BLASTn and aligned with homologues retrieved from GenBank using the Clustal X program [49]. Unfortunately, mitochondrial sequences could not be determined for all isolates, particularly those derived from very low parasitemic and asymptomatic cattle or tiny amounts of tsetse samples.
An analysis of phylogenetic relationships among the isolates of T. vivax was inferred based on ND7 and COIII sequences (~1069 bp) using the Neighbor-Net method with Kimura 2 parameters implemented in Splits Tree4 V4.10 [50]. Internode support was estimated by 1000 bootstrap replicates using the same parameters optimized for network inferences. For comparative purposes, alongside sequences here determined for 36 isolates, we included all ND7 and COIII sequences from T. vivax available in GenBank from: two cattle isolates from Venezuela (MT1 and LIEM176), one cattle isolate from Argentina (IB), and the reference African T. vivax Y486 from cattle [37,38]. To assess erosion/deletions for ND7/COIII gene sequences, we based our comparisons on sequences from T. vivax Y486, the solely isolate with complete ND7/COIII sequences available in GenBank (Accession number MT090068). The isolates showing very large ND7 deletions were excluded from the phylogenetic analysis. DNA sequences determined in the present study were submitted to GenBank (PP842950–PP843011). All sequences used for phylogenetic inferences are shown in Supplementary Table S1.

2.4. Analyses of ND7/COIII Transcripts from African and South American T. vivax

Transcripts of ND7/COIII proteins were searched in the transcriptomes of T. vivax from Venezuela (LIEM176 and MT-1), Brazil (TviLinsSP), and Nigeria (Y486 and its clone IL1392) [30,37,38]. We assessed ND7 transcripts by tBLASTn search using as a query the entire ND7 protein (387 amino acids) sequence from T. vivax Y486 (GenBank AJO53306). However, because the entire COIII protein sequence is not available, we used a partial (103 aa) COIII sequence from T. vivax Y486 (AJO53307) as a query. Reads from transcriptomes of Venezuelan isolates were downloaded from the SRA database (GenBank accession numbers: SRR527235 from LIEM176, SRR527163 from MT-1, and ERR027070 from T. vivax Y486). In addition, we also downloaded reads from transcriptomes of different forms of T. vivax IL1392, a clone of T. vivax Y486, grown in vitro: epimastigotes (ERR236850–ERR236854), epimastigotes plus 20% of metacyclic-like trypomastigotes (ERR236855–ERR236858), and bloodstream forms from experimentally infected mice (ERR236859–ERR236862).

3. Results

3.1. Variability in Length of PCR-Amplified ND7/COIII Genes: From Absence to Highly Variable Deletions in South American T. vivax from Diverse Geographical Origin

Our preliminary investigation of mitochondrial erosion through analysis of the length variability of PCR-amplified contiguous ND7/COIII genes included 25 T. vivax isolates from South America. Preliminary analysis of the amplicons revealed three distinct lengths. Among the 13 Brazilian isolates, 10 exhibited an amplicon of ~1.1 kb, like the intact genes of African T. vivax, suggesting the absence of deletions. For other Brazilian isolates (3), and isolates from Colombia (1), Argentina (4), Ecuador (1), and Venezuela (2), amplicons of ~900 bp revealed two distinct mutation profiles (see below). Notably, four isolates from Venezuela displayed a large deletion, resulting in a smaller amplicon of ~300 bp length (Table 1, Figure 1).

3.2. The ND7 and COIII Genes Range from Intact to a Degree of Mutations in South American T. vivax

To accurately assess the extent of mutations in the ND7/COIII genes, we determined the DNA sequences from PCR amplicons and compared them with T. vivax Y486. The analysis uncovered four mitochondrial profiles: TviMit-0 to TviMit-3 (Table 1; Figure 2). Ten Brazilian isolates from Pará (Northern) and Paraiba (Northeaster) displayed intact DNA sequences of ND7/COIII genes. This is the first report of non-disrupted ND7/COIII sequences in South American T. vivax. The determined sequences had 1058 bp (~674 and ~312 nucleotides from ND7 and COIII, respectively, plus a ~72 bp intergenic region). No deletions were detected in both genes when compared to TviY486 (MT090068), and all isolates with this profile were referred to as TviMit-0 regardless of sequence divergences. While African isolates showed SNP divergence across both ND7 and COIII genes, Brazilian TviMit-0 isolates exhibited high similarity (~99.94%) (Figure 2).
The ND7/COIII sequences from 15 South American isolates of T. vivax differed in mutation profiles as indicated by amplicon lengths. Among Brazilian T. vivax, we identified TviMit-1, corresponding to PCR-amplicons of ~900 bp. Sequence analysis confirmed a length of ~896 bp, with a ~112 bp deletion beginning at position 597 of the 3′ end of the ND7 gene and finishing before the COIII gene, which remains intact. This deletion also involved the majority (51 out of 72 nucleotides) of the intergenic region (5′ end and center regions), resulting in ~163 bp deletion. TviMit-1 was found in Brazilian isolates from Paraiba, São Paulo, and Mato Grosso do Sul, with sequences identical to those reported from the isolate (IB) from Argentina [38]. Four new Argentinian isolates shared the TviMit-1 profile, so far, the single pattern detected in this country. A previously described synonymous substitution (A > G) at the position 595 of the ND7 gene in the isolate IB [38] was found in 7 new TviMit-1 isolates from Brazil and Argentina. All TviMit-1 isolates shared identical COIII sequences (Table 1; Figure 1 and Figure 2).
The mitochondrial profile TviMit-2 is reported here for the first time, identified through sequencing analysis revealing two different amplicons of ~900 bp. The TviMit-2 profile was characterized by ND7/COIII sequences slightly longer (918 bp) than TviMit-1 (~896 bp) and by a deletion of ~142 bp starting at position 501, affecting a region preceding the 3′-end of ND7 and finishing before the gene end. TviMit-2 was identified in two isolates from Venezuela [11] and one isolate from neighboring Colombia. Gene erosion in TviMit-2 did not involve COIII genes (Table 1; Figure 1 and Figure 2). We were unable to obtain sequences from the T. vivax isolate from Ecuador, generating ~900 bp amplicon; hence, this isolate remained designed as TviMit-1/Mit-2.
The TviMit-3 profile was characterized by amplicons of ~300 bp, with a major deletion of ~750 bp starting at position 267 of ND7 and spanning to the 3′-end of ND7 (deletion of ~440 bp length) plus 248 nucleotides of the 5′-end of COIII. The entire intergenic region (~72 bp length) was lost in TviMit-3. This was the only mutation profile involving COIII genes. This profile was previously reported from LIEM176 and MT1 isolates from Venezuela [37] and was here identified in four new isolates from Venezuelan Llanos. The frameshift insertion of “GA” at position 303–304 of the COIII gene reported for the isolate MT1 was found in three out of four Venezuelan TviMit-3 isolates. However, the missense substitution T > G at position 248 of the ND7 gene reported in the MT1 isolate [38] was not detected in any other South American T. vivax; all sequences showed a G in this position, likewise all African isolates (Table 1; Figure 1 and Figure 2).

3.3. Only Intact ND7/COIII Sequences Detected in West and East African Isolates of T. vivax

Consistent with data from T. vivax Y486 (Nigeria), all other African T. vivax isolates consistently exhibited a single PCR-amplicon of ~1.1 kb, indicating the preservation of ND7 and COIII genes. The African isolates originated from tsetse flies in Mozambique (3), Ethiopia (3), and Uganda (2); a Nyala antelope in Mozambique (1); and cattle in Burkina Faso (1). Consequently, all African isolates were of the TviMit-0 profile regardless of their geographical origin and phylogenetic lineage (Table 1; Figure 1 and Figure 2). Our sequencing analyses revealed abundant SNPs differing among ND7 and COIII genes from T. vivax Y486 and other African isolates, particularly those from Central and East Africa. However, detected polymorphisms are not expected to hinder the functionality of the proteins, as the isolates were obtained from tsetse flies (Figure 2).

3.4. Phylogenetic Inferences Based on Mitochondrial Sequences Corroborated the Lineages TVL (East and Central Africa) and TVV (All Isolates from South America and West Africa Plus One Isolate from East Africa)

This study reports the first phylogenetic analysis of T. vivax from both South America and Africa using mitochondrial genes. Congruent with phylogenetic inferences based on nuclear genes, mitochondrial analysis showed a cohesive cluster corresponding to the TVV lineage, formed by slightly different sequences of isolates from West Africa and South America and a more divergent genotype from Ethiopia. Although all South American isolates formed a tight assemblage, the distinct mitochondrial profiles evidenced four clusters: (1) Brazilian isolates characterized by intact genes (TviMit-0); (2) Brazilian and Argentinean isolates of TviMit-1 profile; (3) Venezuelan and Colombian isolates of TviMit-2 (Table 1, Figure 3). The Venezuelan isolates of TviMit-3, exhibiting very large deletions in ND7/COIII genes, were excluded from the analysis. As expected, phylogenetic analysis including their small sequences generated a very long branch exclusive of TviMit-3. Also consistent with phylogenetic inferences using nuclear genes, relatively homogeneous TVV was placed distant from the more heterogeneous TVL lineage, which exhibited two main branches clearly separating sequences from Uganda and Mozambique (Table 1; Figure 2 and Figure 3).

3.5. Differences Between ND7 and COIII Transcripts of African and South American Isolates of T. vivax

Because of extensive RNA editing, mitochondrial DNA sequences of trypanosomatids may correspond to transcripts encoding proteins with different sequences [34]. Therefore, we assessed ND7 and COIII transcripts in transcriptomes from African and South American T. vivax. Intact transcripts were identified in transcriptomes from all life stages of T. vivax Y486, showing high e-values (cutoff 1 × 10−6) in hits with 100% similarity and coverage of 82% and 95% for ND7 and COIII, respectively. Interestingly, transcripts from T. vivax clone IL1392 exhibited two regions that did not match the ND7 (positions 163–181 and 230–232) and COIII (positions 1–9 and 42–53) queries of T. vivax Y486, from which this clone derived. Despite these mutations, both proteins remain functional in both isolates, as evidenced by their known ability to develop in tsetse flies and laboratory animals [32,33].
In contrast to African T. vivax, transcriptomes of blood forms from South American isolates showed highly truncated transcripts with substantial deletions, which hinder the transcription of functional proteins. Consistent with extensive deletion, the Venezuelan isolate LIEM176 (TviMit-3) exhibited small transcripts, with hits limited to ~250 bp at the 5′ end of ND7, displaying 100% similarity compared with T. vivax Y486 only at positions 49–66, and to ~50 bp at the 3′ end of COIII transcripts. In contrast, the transcriptome of blood forms of T. vivax TviLinsSP from Brazil, assigned to TviMit-1, showed transcripts of ND7 and COIII proteins with substantial coverage and sequence identity compared to T. vivax Y486. However, no hits were found for ~100 bp at the 3′ end of ND7, consistent with a deletion and with a non-functional ND7 protein in this isolate (Table 1, Figure 2).

4. Discussion

The introduction of T. vivax from Africa to the New World appears to have resulted in a genetic bottleneck, resulting in high genetic homogeneity in the nuclear gene sequences of South American populations, in contrast to the relevant genetic diversity observed in African T. vivax [24,25,45,48]. However, unlike highly conserved nuclear genomes, mitochondrial genomes exhibited mutations and gene erosion, suggesting that the integrity of kDNA relies on cyclical development in tsetse flies [37,38].
In order to offer a more comprehensive understanding of mitochondrial gene mutations and, consequently, on the evolution of the T. vivax mitochondrial genome, we characterized contiguous ND7/COIII genes from 25 South American and 11 African isolates from lineages TVV and TVL. The ND7 subunit of respiratory Complex I and COIII proteins play a crucial role in mitochondrial respiratory chain complexes, being pivotal for the survival of trypanosomes in insect hosts [42,51,52]. Both ND7 and COIII transcripts undergo extensive editing in trypanosomes, including African T. vivax [37,38]. To assess mutations in ND7/COIII genes, we initially evaluated the length of DNA amplicons generated by PCR amplification of these contiguous genes. Subsequent sequencing analyses suggested that isolates from West (Nigeria and Burkina Faso), Central (Uganda), and East (Mozambique and Ethiopia) Africa possess intact genes like T. vivax Y484, the only African T. vivax isolate characterized prior to our current study [37,38].
Compared to African T. vivax, South American isolates exhibited substantial divergence in PCR-amplicon lengths of ND7/COIII genes. Ten Brazilian isolates, among the 25 South American isolates studied, produced amplicons compatible with intact genes referred to as TviMit-0. Our sequence analysis confirmed the integrity of ND7/COIII genes in 10 TviMit-0 isolates from Brazil, six from the Paraiba state, four of them from severely sick sheep during an outbreak, and two from asymptomatic sheep sampled one year later in the same farm where the outbreak occurred [5]. Four BrazilianTviMit-0 isolates were from asymptomatic water buffaloes in the endemic Amazon region [44]. TviMit-1 profile was identified in three Brazilian isolates: two from fatal infections in outbreaks in naïve herds (northeastern and southeastern regions) and one from asymptomatic cattle in the endemic The Pantanal in Central Brazil. Additionally, four new isolates from fatal acute infections in cattle from Argentina [7] were assigned to TviMit-1, and their sequences were identical to that of T. vivax IB from this country [38].
Despite sharing ND7/COIII PCR-amplicons of ~900 bp, some isolates differed in sequences from those of TviMit-1 and were referred to as TviMit-2. This profile was identified for the first time in two isolates from buffaloes with fatal infections in Venezuela [11] and one isolate from asymptomatic cattle in Colombia. TviMit-3 profile (~300 bp) was identified in four new isolates from Venezuela. This extensive mutation is unique in involving deletions in both ND7 and COIII, as formerly reported for the isolates LIEM176 and MT1 from cattle in the Venezuelan states of Trujillo and Monagas, respectively [37,38]. TviMit-3 was detected across the Venezuelan Llanos (Anzoátegui, Apure, and Cojedes) in cattle, buffalo, and sheep, in asymptomatic and severely sick animals [11,45]. To date, TviMit-3 is exclusive and widespread across Venezuela, where TviMit-2 was also detected.
Reflecting potential association between mutation profiles and geography, TviMit-2 and TviMit-3 were exclusive to the western side of the Andes, in Venezuela and Colombia, while TviMit-0 and TviMit-1 have been reported on the eastern side of the Andean Mountains. In Brazil, TviMit-0 was found in Pará, Northern region, and adjacent Northeastern regions. Interestingly, movement of livestock from Pará to the nearby state of Maranhão, and subsequently through the northeastern region, was suggested as the source of T. vivax causing the first outbreak of acute disease in naïve cattle in northeastern Brazil [4]. TviMit-1 was found in the Southeastern and Southern regions, the latter bordering Argentina, where only TviMit-1 was identified in the northern endemic Chaco region, known for the enzootic stability of T. vivax, to the central regions, where outbreaks of fatal disease occur in highly susceptible dairy cattle [7]. Similarly, the movement of horses and cattle from the Pantanal region has been implicated as a source of T. vivax to outbreaks in dairy cattle in São Paulo (southeastern) and Rio Grande do Sul, southern Brazil [53].
Therefore, three distinct deletion patterns (TviMit-1 to TviMit-3), alongside the absence of deletions in ND7/COIII genes (TviMit-0), were identified in South American T. vivax, regardless of whether the isolates originated from endemic or outbreak areas, from sick (including fatal infections) or asymptomatic livestock, and from animals varying from low to high parasitemia. The substantial mutations particularly affected genes nonessential for the life of T. vivax confined to blood forms in American landscapes devoid of tsetse flies. Drastic erosions of ND7/COIII genes and transcripts, particularly ND7, limit T. vivax to blood forms since both genes are essential for trypanosome development within insects. Consistent with this constraint, attempts to infect tsetse flies with three isolates from Colombia were unsuccessful, whereas T. vivax from Nigeria and Kenya cyclically developed in tsetse flies [54,55]. The absence of selective pressures to maintain minicircle diversity and the full array of gRNA genes in South American T. vivax may have led to gradual minicircle homogenization, impairing mitochondrial gene editing and, consequently, functional proteins. A similar process was proposed for T. evansi. However, all T. evansi isolates have completely lost their maxicircles, consequently lacking all mitochondrial genes, and are exclusively mechanically transmitted in Africa, the Americas, and Asia [42,43].
Our phylogenetic analysis based on mitochondrial genes supported TVV and TVL main lineages of T. vivax and corroborated closer relationships between South American and West African isolates as previously demonstrated using nuclear genes [24,25,26,30,48]. Our analysis segregated TVV isolates into four clusters, all distant from the heterogenous TVL isolates from Uganda, Ethiopia, and Mozambique. In these countries, as well as in Kenya, Tanzania, and Zambia, TVL and TVV genotypes share both ruminant hosts and tsetse flies [25,27,28,29]. A recent phylogenomic study placed an isolate from Uganda closer to Brazilian isolates of TVV [30]. Uganda and Ethiopia are historically major centers for cattle production in Africa and served as a source for livestock movement through the historical Sahelian corridor toward West Africa as well as toward southeastern regions [56].
Although 15 of the 25 South American isolates of T. vivax examined in the present study showed erosion of ND7/COIII mitochondrial genes, 10 Brazilian isolates displayed intact ND7/COIII genes, suggesting some pressure for mitochondrial preservation. Nevertheless, although mechanically transmitted by flies other than tsetse flies, both outside the tsetse belt and outside Africa [19,20,21], cyclical development of T. vivax is restricted to tsetse flies [54,57].
It was previously hypothesized that T. vivax mitochondrial genes may have diverged soon after the introduction to the American continent, adapting to exclusive mechanical transmission and accumulating mutations at an amazingly rapid rate. Alternatively, it was suggested that the drastic mitochondrial mutations observed in South American isolates likely began in Africa [37,38]. The identification of non-mutated, or at least less mutated, isolates taken into account that our analyses were restricted to ND7/COIII genes suggests the introduction of African T. vivax with whole mitochondrial genomes into South America. Regarding intact ND7/COIII genes detected in Brazilian isolates, it is essential to note that the importation of live animals from Africa to Brazil began about five centuries ago and has been prohibited for over ~70 years, highlighting uncertainties about the time required for the parasite to accumulate mutations leading to mitochondrial erosion.
While our study marks the first documentation of T. vivax isolates with intact ND7 and COIII genes in South American isolates, future research focused on whole mitochondrial genomes, including isolates without deletions, along with African isolates from regions infested with and free of tsetse flies, is essential for a better understanding of mitochondrial genome evolution in T. vivax.

5. Conclusions

This study represents the most comprehensive investigation to date on mitochondrial genes in T. vivax from both South America and Africa. All African isolates exhibited intact ND7 and COIII genes despite showing relevant polymorphisms. However, in South America, for the first time, some Brazilian isolates showed intact genes, whereas other isolates exhibited eroded genes. A degree of mutation profiles was observed in all isolates from Venezuela, Argentina, Ecuador, and Colombia. Our findings highlighted the complex epidemiology and evolutionary dynamics of mitochondrial genomes in T. vivax populations introduced for centuries from Africa into American landscapes devoid of tsetse flies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/parasitologia4040035/s1, Table S1: Isolates of Trypanosoma vivax employed for phylogenetic relationships using ND7/COIII sequences.

Author Contributions

Conceptualization, H.A.G., E.F.P.C. and M.M.G.T.; Data curation, C.M.F.R.; Formal analysis, C.M.F.R.; Funding acquisition, E.F.P.C. and M.M.G.T.; Investigation, H.A.G., C.M.F.R., C.L.P., D.S. and Z.B.; Methodology, A.R.-B. and M.A.C.-L.; Project administration, H.A.G. and M.M.G.T.; Resources, A.S.F., A.M.B., A.R.-B., M.A.C.-L., C.L.P., Z.B., D.S., A.B.K., F.P. and M.M.G.T.; Supervision, F.P., E.F.P.C. and M.M.G.T.; Validation, A.R.-B.; Visualization, A.C.R. and F.P.; Writing—original draft, H.A.G. and C.M.F.R.; Writing—review and editing, H.A.G., A.C.R., A.B.K. and M.M.G.T. All authors have read and agreed to the published version of the manuscript.

Funding

Grants to MMGT and EPC from CNPq (PROAFRICA and PROSUL programs) Brazilian agency supported this research. CMFR and ACR were post-doctoral fellowships from CAPES and CNPq, respectively. HAG has been supported by fellowships from CNPq (Process: 166104/2015-9), FAPESP (Process: 2016/03028-1) and by the Collaborating Researcher Program from the Institute of Biomedical Sciences, University of São Paulo (Process: 2022-10093).

Institutional Review Board Statement

Animals were handled in accordance with international animal care standards. All protocols were approved by the respective institutions involved in the study, including the following Protocols: UFERSA-CEUA (Protocol No. 23091.1901/10-98), and Protocol from the the University of São Paulo, Brazil (CEPICB Protocol No. 317/09). Laboratory work was conducted in Brazil in compliance with the recommendations of the Ethical Committee of the Biomedical Institute at the University of São Paulo (Approved Protocol No. 009, page 3 of Book 3).

Informed Consent Statement

Trypanosoma vivax isolates characterized in this survey were from our previous studies. Samples from tsetse flies, as well as blood samples from livestock, were collected in strict adherence to established ethical standards and regulatory guidelines, ensuring the welfare and humane treatment of animals.

Data Availability Statement

DNA sequences determined in the present study for ND7 and COIII gene were submitted to GenBank (Accession numbers PP842950–PP843011).

Acknowledgments

We would like to thank many people who provided invaluable assistance during the field studies conducted in various countries, where T. vivax isolates from livestock and tsetse flies analyzed in the present study were obtained.

Conflicts of Interest

The authors declare no conflicts of interest with respect to the conduct, authorship, and/or publication of this study.

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Figure 1. Geographical origin of Trypanosoma vivax isolates from tsetse cyclical transmission in African countries and mechanical transmission in South American endemic and outbreak areas. These isolates were obtained from wild animals and livestock with infections ranging from severely sick to asymptomatic and tsetse flies. Sampling areas in South America include Venezuela (states of Tru, Trujillo; Apu, Apure; Anz, Anzoátegui; Mon, Monagas), Brazil (states of PA, Pará; PB, Paraíba; MS, Mato Grosso do Sul; SP, São Paulo), and other countries (see Table 1 for sampling details).
Figure 1. Geographical origin of Trypanosoma vivax isolates from tsetse cyclical transmission in African countries and mechanical transmission in South American endemic and outbreak areas. These isolates were obtained from wild animals and livestock with infections ranging from severely sick to asymptomatic and tsetse flies. Sampling areas in South America include Venezuela (states of Tru, Trujillo; Apu, Apure; Anz, Anzoátegui; Mon, Monagas), Brazil (states of PA, Pará; PB, Paraíba; MS, Mato Grosso do Sul; SP, São Paulo), and other countries (see Table 1 for sampling details).
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Figure 2. Schematic representation of polymorphisms, including mutations and deletions, on the contiguous NADH dehydrogenase subunit 7 (ND7) and cytochrome oxidase subunit III (COIII) genes from Trypanosoma vivax isolates from South America and Africa. TviMit-0 to TviMit-3 correspond to the four mitochondrial profiles determined for the isolates characterized in this and in a previous study [37,38]. The vertical bars represent single nucleotide polymorphisms. Symbols indicating the origin of the isolates are shown in Figure 1.
Figure 2. Schematic representation of polymorphisms, including mutations and deletions, on the contiguous NADH dehydrogenase subunit 7 (ND7) and cytochrome oxidase subunit III (COIII) genes from Trypanosoma vivax isolates from South America and Africa. TviMit-0 to TviMit-3 correspond to the four mitochondrial profiles determined for the isolates characterized in this and in a previous study [37,38]. The vertical bars represent single nucleotide polymorphisms. Symbols indicating the origin of the isolates are shown in Figure 1.
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Figure 3. Phylogenetic relationships of Trypanosoma vivax isolates from Africa and South America based on NADH dehydrogenase subunit 7 (ND7) and cytochrome oxidase subunit III (COIII) sequences. The branching pattern supports distant lineages TVV and TVL, predominantly comprising isolates from South America/West Africa and East Africa, respectively. Within TVV, the isolates cluster according to TviMit-0, TviMit-1, and TviMit-2 mitochondrial profiles. The analysis was performed using the Neighbour-Net method with the K2 parameter, and nodal support was estimated with 500 bootstrap replicates. SA, South America; WA, West Africa (Nigeria); EA, East Africa (Eth, Ethiopia, Ug, Uganda, Mz, Mozambique). Brasilian States: PB, Paraíba; PA, Pará; SP, São Paulo; MS, Mato Grosso do Sul.
Figure 3. Phylogenetic relationships of Trypanosoma vivax isolates from Africa and South America based on NADH dehydrogenase subunit 7 (ND7) and cytochrome oxidase subunit III (COIII) sequences. The branching pattern supports distant lineages TVV and TVL, predominantly comprising isolates from South America/West Africa and East Africa, respectively. Within TVV, the isolates cluster according to TviMit-0, TviMit-1, and TviMit-2 mitochondrial profiles. The analysis was performed using the Neighbour-Net method with the K2 parameter, and nodal support was estimated with 500 bootstrap replicates. SA, South America; WA, West Africa (Nigeria); EA, East Africa (Eth, Ethiopia, Ug, Uganda, Mz, Mozambique). Brasilian States: PB, Paraíba; PA, Pará; SP, São Paulo; MS, Mato Grosso do Sul.
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Table 1. Trypanosoma vivax isolates from South America used in this study, livestock species, geographic origin, and clinical conditions of the infected animals, and ND7/COIII mitochondrial profiles.
Table 1. Trypanosoma vivax isolates from South America used in this study, livestock species, geographic origin, and clinical conditions of the infected animals, and ND7/COIII mitochondrial profiles.
T. vivax
Isolates		Host
Species		OriginDate of
Isolation		Clinical SignsRef.ND7/COIII
Genes
(kb) #		Mitochon Profile $
Brazil
TviBrPA20buffaloPA2009asymptomatic[45]∼1.1TviMit-0
TviBrPA24buffaloPA2009asymptomatic[45]∼1.1TviMit-0
TviBrPA28buffaloPA2009asymptomatic[45]∼1.1TviMit-0
TviBrPA48buffaloPA2009asymptomatic[45]∼1.1TviMit-0
TviBrPB27sheepPB2008high parasitemia low PCV/NS-Fatal[5,45]∼1.1TviMit-0
TviBrPB28sheepPB2008high parasitemia low PCV/NS-Fatal[5,45]∼1.1TviMit-0
TviBrPB30sheepPB2008high parasitemia low PCV/NS-Fatal[5,45]∼1.1TviMit-0
TviBrPB47sheepPB2008high parasitemia low PCV/NS-Fatal[5,45]∼1.1TviMit-0
TviBrPB50sheepPB2009asymptomatic[5,45]∼1.1TviMit-0
TviBrPB52sheepPB2009asymptomatic[5,45]∼1.1TviMit-0
TviLinsSPcattleSP2008high parasitemia low PCV/NS-Fatal[10,24,45]∼0.9TviMit-1
TviBrCacattlePB2007high parasitemia low PCV/NS-Fatal[10,24,45]∼0.9TviMit-1
TviBrMicattleMS2000asymptomatic[10,24,45]∼0.9TviMit-1
Argentina
TviArSF119cattleSF2017high parasitemia low PCV/Fatal[7]∼0.9TviMit-1
TviArSF126cattleSF2017high parasitemia low PCV/Fatal[7]∼0.9TviMit-1
TviArSF135cattleSF2017high parasitemia low PCV/Fatal[7]∼0.9TviMit-1
TvArFs08cattleFs2017high parasitemia low PCV/Fatal[7]∼0.9TviMit-1
IBcattle--2020-[38]∼0.9TviMit-1
Colombia
TviCoBoy01cattleBoy2016asymptomatic--∼0.9TviMit-2
Venezuela
TviVzAp10buffaloApu2015high parasitemia low PCV/Fatal[11]∼0.9TviMit-2
TviVzAp11buffaloApu2015high parasitemia low PCV/Fatal[11]∼0.9TviMit-2
TviVzAp5sheepApu2006moderate parasitemia low PCV[11,45]∼0.30TviMit-3
TviVzAnz1cattleAnz2006asymptomatic[11,45]∼0.30TviMit-3
TviVzAnz3cattleAnz2006asymptomatic[11,45]∼0.30TviMit-3
TviVzCojEX buffaloCoj2006asymptomatic[11,45]∼0.30TviMit-3
MT1cattleMon2007severe anemia, fever, lymphadenopathy[37]∼0.30TviMit-3
LIEM176cattleTru2002unknown (parasitemic)[37]∼0.30TviMit-3
Ecuador
TviEc01cattleMan2018pale mucosa, fever, weakness[12]∼0.9TviMit-1/Mit-2
Africa
TviEthG65tsetseEth2016-[47]∼1.1TviMit-0
TviEthG106tsetseEth2016-[47]∼1.1TviMit-0
TviEthG79tsetseEth2016-[47]∼1.1TviMit-0
TviMzNyNyalaMz2008asymptomatic[24]∼1.1TviMit-0
TviUgG34tsetseUg2016-[47]∼1.1TviMit-0
TviUgG36tsetseUg2016-[47]∼1.1TviMit-0
TviMzG13tsetseMz2014-[25]∼1.1TviMit-0
TviMzG52tsetseMz2014-[25]∼1.1TviMit-0
TviMzG70tsetseMz2014-[25]∼1.1TviMit-0
Y486cattle Nig1976-[46]∼1.1TviMit-0
TviBfL445cattle Bf2008-[45,48]∼1.1-
States: PA, Pará; PB, Paraiba; MS, Mato Grosso do Sul; Boy, Boyacá; Apu, Apure; Anz, Anzoategui; Coj, Cojedes; Mon, Monagas; Tru, Trujillo; Man, Manabí; Sf, Santa Fe; Fs, Formosa. African Countries: Eth, Ethiopia; Mz, Mozambique; Ug, Uganda; Nig, Nigeria; Bf, Burkina Faso; PVC, packed cell volume; NS, nervous system. #, length of ND7/COIII PCR-amplicon; $, mitochondrial profiles as in Figure 2.
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Garcia, H.A.; Rodrigues, A.C.; Rodrigues, C.M.F.; Florentin, A.S.; Bethencourt, A.M.; Reyna-Bello, A.; Chávez-Larrea, M.A.; Pereira, C.L.; Bengaly, Z.; Sheferaw, D.; et al. From Intact to Highly Degraded Mitochondrial Genes in Trypanosoma vivax: New Insights into Introduction from Africa and Adaptation to Exclusive Mechanical Transmission in South America. Parasitologia 2024, 4, 390-404. https://doi.org/10.3390/parasitologia4040035

AMA Style

Garcia HA, Rodrigues AC, Rodrigues CMF, Florentin AS, Bethencourt AM, Reyna-Bello A, Chávez-Larrea MA, Pereira CL, Bengaly Z, Sheferaw D, et al. From Intact to Highly Degraded Mitochondrial Genes in Trypanosoma vivax: New Insights into Introduction from Africa and Adaptation to Exclusive Mechanical Transmission in South America. Parasitologia. 2024; 4(4):390-404. https://doi.org/10.3390/parasitologia4040035

Chicago/Turabian Style

Garcia, Herakles A., Adriana C. Rodrigues, Carla M. F. Rodrigues, Andrea S. Florentin, Angélica M. Bethencourt, Armando Reyna-Bello, María Augusta Chávez-Larrea, Carlos L. Pereira, Zacharia Bengaly, Desie Sheferaw, and et al. 2024. "From Intact to Highly Degraded Mitochondrial Genes in Trypanosoma vivax: New Insights into Introduction from Africa and Adaptation to Exclusive Mechanical Transmission in South America" Parasitologia 4, no. 4: 390-404. https://doi.org/10.3390/parasitologia4040035

APA Style

Garcia, H. A., Rodrigues, A. C., Rodrigues, C. M. F., Florentin, A. S., Bethencourt, A. M., Reyna-Bello, A., Chávez-Larrea, M. A., Pereira, C. L., Bengaly, Z., Sheferaw, D., Kato, A. B., Paiva, F., Camargo, E. F. P., & Teixeira, M. M. G. (2024). From Intact to Highly Degraded Mitochondrial Genes in Trypanosoma vivax: New Insights into Introduction from Africa and Adaptation to Exclusive Mechanical Transmission in South America. Parasitologia, 4(4), 390-404. https://doi.org/10.3390/parasitologia4040035

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