Novel Tick-Borne Anaplasmataceae Genotypes in Tropical Birds from the Brazilian Pantanal Wetland

Despite numerous reports of Anaplasmataceae agents in mammals worldwide, few studies have investigated their occurrence in birds. The present study aimed to investigate the occurrence and molecular identity of Anaplasmataceae agents in birds from the Pantanal wetland, Brazil. Blood samples were collected from 93 different species. After DNA extraction, samples positive for the avian β-actin gene were subjected to both a multiplex quantitative real-time (q)PCR for Anaplasma and Ehrlichia targeting the groEL gene and to a conventional PCR for Anaplasmataceae agents targeting the 16S rRNA gene. As a result, 37 (7.4%) birds were positive for Anaplasma spp. and 4 (0.8%) for Ehrlichia spp. in the qPCR assay; additionally, 13 (2.6%) were positive for Anaplasmataceae agents in the PCR targeting the 16S rRNA gene. The Ehrlichia 16S rRNA sequences detected in Arundinicola leucocephala, Ramphocelus carbo, and Elaenia albiceps were positioned closely to Ehrlichia sp. Magellanica. Ehrlichia dsb sequences detected in Agelasticus cyanopus and Basileuterus flaveolus grouped with Ehrlichia minasensis. The 16S rRNA genotypes detected in Crax fasciolata, Pitangus sulphuratus and Furnarius leucopus grouped with Candidatus Allocryptoplasma. The 23S-5S genotypes detected in C. fasciolata, Basileuterus flaveolus, and Saltator coerulescens were related to Anaplasma phagocytophilum. In conclusion, novel genotypes of Anaplasma, Ehrlichia, and Candidatus Allocryptoplasma were detected in birds from the Pantanal wetland.

The identification of birds was performed by an skilled ornithologist using several field guides.Bird taxonomy follows the Birdtree project (see https://birdtree.org/ taxonomy) (accessed on 10 April 2024) [37].Representative bird specimens were deposited in the Bird Collection of UFMT, Cuiabá, Brazil.All birds and blood samples were collected under appropriate permits in Brazil.
The identification of birds was performed by an skilled ornithologist using several field guides.Bird taxonomy follows the Birdtree project (see https://birdtree.org/taxonomy) (accessed on 10 April 2024) [37].Representative bird specimens were deposited in the Bird Collection of UFMT, Cuiabá, Brazil.All birds and blood samples were collected under appropriate permits in Brazil.

Molecular
Screening for Anaplasma spp.and Ehrlichia spp.Targeting the groEL Gene by a Multiplex Quantitative Real-Time (q)PCR DNA avian blood samples that were positive for the endogenous avian β-actin gene were then screened for Anaplasma spp.and Ehrlichia spp.using a multiplex qPCR targeting the groEL gene, as previously described [39].The reactions consisted of 1 µL of sample DNA, 0.2 µM of each oligonucleotide primer and hydrolysis probe, Master Mix 2x (GoTaq ™ Probe qPCR Master Mix, Promega Corporation, Madison, WI, USA), and sterilized ultrapure water (Nuclease-Free Water, Promega ® , Madison, WI, USA) to complete a final volume of 10 µL.Amplification reactions were carried out in a CFX96 Thermal Cycler (BioRad ® , Hercules, CA, USA) (Supplementary Table S2).Samples were tested in duplicates.
Quantification of the number of target DNA copies/µL was performed using G-Blocks (G-Blocks, Integrated DNA Technologies ® , Coralville, IA, USA) containing the target sequences.Serial dilutions were made to construct standards with different concentrations of G-Blocks DNA containing the target sequence (2.0 × 10 7 copies/µL to 2.0 × 10 0 copies/µL) in order to obtain the efficiency and correlation coefficient of reactions.The number of target gene copies was determined according to the formula (Xg/µL DNA/(plasmid size (bp) × 660)) × 6.022 ×1023 × plasmid copies/µL.All analyses were carried out in accordance with standards established by MIQE ("Minimum Information for Publication of Quantitative real-time PCR Experiments") [40].Quantification cycles (Cq) with a difference of no more 0.5 were considered positive; otherwise, they were repeated in triplicates.Cq is considered as the fraction of a PCR cycle where the target in the samples is quantified [41].
2.2.3.Molecular Screening for Anaplasmataceae Agents Targeting the 16S rRNA Gene by Conventional PCR Avian DNA samples that were positive for the endogenous avian β-actin gene were also screened for Anaplasmataceae agents using the previously described PCR targeting the 16S rRNA gene [42].Oligonucleotides sequences and thermal conditions are shown in Supplementary Table S2.
For Anaplasma molecular characterization, positive samples in the screening PCR assays were subjected to additional previously described conventional PCR assays based on the 16S rRNA gene [20,42,[49][50][51][52][53], and the intergenic fragment 23S-5S (ITS) [54], as well as to a nested PCR based on the groEL gene [55].Samples in which sequences related to Candidatus Allocryptoplasma spp.were detected were subjected to additional previously described conventional PCR assays targeting the groEL and sucA genes [4].The primer oligonucleotides and thermal sequences used in PCR assays are presented in Supplementary Table S2.
Ehrlichia canis (Jaboticabal strain) DNA (obtained from DH82-infected cells [56]) and Anaplasma sp.DNA (obtained from cattle from Mozambique [57]) were used as positive controls in all conventional and nested PCR assays for Ehrlichia spp.and Anaplasma spp., respectively.Sterilized ultra-pure water (Invitrogen ® , Carlsbad, CA, USA) was used as a negative control in all PCR assays.

Gel Electrophoresis and Amplicon Purification
PCR products were separated in a 1% agarose gel stained with ethidium bromide (0.5 µL/mL) in TBE buffer (pH 8).A molecular-weight size marker of 100 pb was employed to verify the size of the PCR products (Kasvi, Campina, São José dos Pinhais, PR, Brazil).Agarose gels were visualized under an ultra-violet transilluminator Chemidoc (BioRad ® , Hercules, CA, USA) and image analyzer software, Image Lab (BioRad ®, Hercules, CA, USA).Amplicons were purified with the Wizard SV Gel and PCR cleanup system kit (Promega, Madison, WI, USA), following manufacturer's recommendations.
While the Cq values of Anaplasma-positive samples ranged from 28.94 to 39.24, Ehrlichiapositive samples showed Cq values ranging from 33.24 to 37.89.Even after re-testing the positive samples in triplicates, achievement of the estimated quantification was possible in only one positive sample for Anaplasma spp.probably due to Monte Carlo effect [40]: BAP87 (232) from a specimen of Legatus leucophaius from Poconé, with an average Cq of 37.96 and an average quantification of 5.845 × 10 −1 copies/ µL.All samples positive in the qPCR were negative in additional conventional PCR assays based on the 16S rRNA, dsb, gltA, sodB, omp-1, rpoB, groEL, and ftsZ genes and 23S-5S (ITS) intergenic region, mostly likely due the low bacteremia at the time of blood sampling.S4.

Molecular
When combining the results obtained by the multiplex qPCR (groEL) and cPCR (16S rRNA gene) assays and comparing the positivity for Anaplasma spp./CandidatusAllocryptoplasma spp.and Ehrlichia spp., a higher positivity for Anaplasma spp./CandidatusAllocryptoplasma spp.(9.7%; 37/382) and Ehrlichia spp.(2.3%; 9/382) was found among birds sampled in the state of Mato Grosso.On the other hand, a positivity of 2.5% (3/118) for Anaplasma spp. was found among birds sampled in the state of Mato Grosso do Sul.
When performing conventional PCR assays to characterize Anaplasmataceae agents with the 16S rRNA [52], dsb gene [43], and 23S-5S rRNA intergenic region (ITS) [54], three sequences (ranging from 832 to 884 bp) were obtained in the semi-nested PCR protocol targeting a fragment of 800 bp of the 16S rRNA gene [52].The positive samples were obtained from one C. fasciolata, one F. leucopus, and one A. leucocephala from Nossa Senhora do Livramento.
Two sequences (ranging from 401 to 402 bp) were obtained in the PCR protocol targeting the dsb gene.The two positive samples were obtained from one A. cyanopus from Nossa Senhora do Livramento (MT) and one B. flaveolus from Santo Antonio de Leverger (MT).
Three sequences (ranging from 832 to 884 bp) were obtained in the PCR protocol targeting the 23S-5S rRNA intergenic region (ITS).The positive samples were obtained from one C. fasciolata from Nossa Senhora do Livramento (MT) and one S. coerulescens and one B. flaveolus from Santo Antonio de Leverger (MT).The BLAST results for the obtained 16S rRNA, dsb, and ITS sequences are shown in Supplementary Table S4.
Sequences showing high identity to Candidatus Allocryptoplasma sp. and obtained in the PCR protocols described by Parola [42] or Eberhardt [52] targeting the 16S rRNA gene were submitted to additional PCR assays based on the sucA and groEL genes [4].As a result, one C. fasciolata from Nossa Senhora do Livramento (MT) showed positivity in the PCR targeting the groEL gene, but due to low intensity of the obtained band, it was not possible to retrieve a readable sequence.
the PCR targeting the groEL gene, but due to low intensity of the obtained band, it was not possible to retrieve a readable sequence.
evolutionary model based on an alignment of 1439 bp of 16S rRNA gene sequences from Anaplasmataceae agents, containing 57 homologous sequences for the 16S rRNA gene from the genera Anaplasma, Candidatus Allocryptoplasma sp., and Aegyptianella sp.Ehrlichia minasensis and Ehrlichia ewingii sequences were used as outgroups (NR148800 and NR_044747) and are indicated with (***).The sequences obtained in this project are highlighted in red.Bootstraps lower than 50 are not shown.
In the present study, the molecular occurrence of Ehrlichia/Anaplasma in birds inferred by both conventional (16S rRNA) and real-time (groEL) PCR assays used as screening tests was 9.2% (46/500), falling within previously reported prevalence in birds, which ranges from 0.73% [25] up to 43.5% [28].Such difference in molecular prevalence for Ehrlichia/Anaplasma in birds among different studies might be due to the variety of avian species tested, the sensitivity/specificity of PCR protocols used, geographic region, level of infestation by tick vectors, and susceptibility of the birds sampled with regard to the development of detectable bacteremia, among others.
Herein, 3.7% of sampled birds were positive in the qPCR for Anaplasma spp.based on the groEL gene.Unfortunately, all samples positive in the qPCR were negative in conventional PCR assays, which is similar to the results reported by [29] among carnivorous birds from Brazil.
In the present study, the molecular occurrence of Ehrlichia/Anaplasma in birds inferred by both conventional (16S rRNA) and real-time (groEL) PCR assays used as screening tests was 9.2% (46/500), falling within previously reported prevalence in birds, which ranges from 0.73% [25] up to 43.5% [28].Such difference in molecular prevalence for Ehrlichia/Anaplasma in birds among different studies might be due to the variety of avian species tested, the sensitivity/specificity of PCR protocols used, geographic region, level of infestation by tick vectors, and susceptibility of the birds sampled with regard to the development of detectable bacteremia, among others.
Herein, 3.7% of sampled birds were positive in the qPCR for Anaplasma spp.based on the groEL gene.Unfortunately, all samples positive in the qPCR were negative in conventional PCR assays, which is similar to the results reported by [29] among carnivorous birds from Brazil.
In the phylogenetic analysis based on the 16S rRNA gene, the genotypes detected in specimens of C. fasciolata (#330 BAP146 (PP326053)) and F. leucopus (#341 BAP428 (PP373692)) were positioned within the clade of Candidatus Allocryptoplasma sp.Previous studies have shown that Candidatus Cryptoplasma sp., recently named as Candidatus Allocryptoplasma sp.[4], presents itself as a monophyletic clade [4,72].When comparing the phylogenetic tree of the present study with that one described by Ouass [4], it is possible to observe the proximity of Candidatus Allocryptoplasma and Anaplasma spp.While in the phylogeny of the present study, it was possible to observe a polyphyletic clade of Candidatus Allocryptoplasma, a monophyletic profile was observed for the available sequences of Candidatus Allocryptoplasma sp. in the studies developed by Ouass [4] and Mendoza-Roldan [72].This discrepancy can be explained by the inclusion of distinct and novel genotypes detected in the present study, which indicates that the phylogenetic positioning of such agents is far from being resolved.
Candidatus Allocryptoplasma has already been reported in Ixodes ricinus from Morocco and Tunisia [73], Ixodes pacificus from the United States of America [3], Ixodes ricinus from Serbia [74], Ixodes ricinus from France [75], Amblyomma tholloni and Haemaphysalis parmata from Uganda and Amblyomma coelebs from French Guiana [4], Lacerta viridis lizards and Apodemus agrarius rodents from Slovakia [76], Podarcis lizards from Italy [71], as well as in an Amblyomma dissimile tick from Brazil [77].Candidatus Allocryptoplasma spp.present apparent worldwide distribution, whose implications for animal and human health are still unknown.Since the majority of Candidatus Allocryptoplasma sequences were obtained from ticks (Ixodes ricinus, Ixodes pacificus, Amblyoma tholloni, Amblyomma dissimile, and Haemaphysalis longicornis) so far, it has been suggested that ticks might play an important role in the transmission of such agents to hosts.When it comes to vertebrate hosts, Candidatus Allocryptoplasma spp. or closely related agents have only been detected in reptiles [72,78] and rodents [76].The present work showed, for the first time, the occurrence of Candidatus Allocryptoplasma spp. or a closely related agent in birds.Altogether, these findings emphasize the need to unravel the diversity of Anaplasmataceae agents in non-classical vertebrate hosts such as birds.
Taking into account that the birds analyzed in the present work originated from a previous study that investigated tick infestation [34], only 2/37(5.55%)birds positive for Anaplasma spp.were parasitized by ticks at the sampling time: R. carbo #417 (F073) was infested by an Amblyomma longirostris nymph, and Eucometis penicillata #51 (SL025) was found parasitized by an Amblyomma nudosum nymph.Future studies should be performed in order to unravel the tick species involved in the transmission of these novel Anaplasmataceae genotypes among birds.

Conclusions
Novel genotypes of Ehrlichia spp.(closely related to Ehrlichia Magellanica and E. minasensis), Anaplasma spp.(closely related to A. phagocytophilum), and Candidatus Allocryptoplasma spp.were molecularly detected in blood samples from tropical bird species sampled in the Pantanal wetland.More studies are needed to molecularly characterize and unravel the transmission dynamics of these Anaplasmataceae agents in tropical birds from Pantanal wetland in order to investigate the role of wild birds in the dispersion of such agents.Whole-genome sequencing approaches should be performed with the aim of shedding light on the real identity of these novel Anaplasmataceae agents infecting neotropical wild birds.This is the first molecular evidence of Candidatus Allocryptoplasma spp. in birds in the world.

Microorganisms 2024 , 16 Figure 1 .
Figure 1.Brazilian biomes and sampling sites in the Pantanal wetland in the states of Mato Grosso and Mato Grosso do Sul.

Figure 1 .
Figure 1.Brazilian biomes and sampling sites in the Pantanal wetland in the states of Mato Grosso and Mato Grosso do Sul.

Figure 2 .
Figure 2. Phylogenetic analysis generated by the maximum likelihood method and TIM2+F+I+G4 evolutionary model based on an alignment of 1439 bp of 16S rRNA gene sequences from Anaplasmataceae agents, containing 57 homologous sequences for the 16S rRNA gene from the genera Anaplasma, Candidatus Allocryptoplasma sp., and Aegyptianella sp.Ehrlichia minasensis and Ehrlichia ewingii sequences were used as outgroups (NR148800 and NR_044747) and are indicated with (***).The sequences obtained in this project are highlighted in red.Bootstraps lower than 50 are not shown.

Figure 3 .Figure 3 .
Figure 3. Phylogenetic analysis generated by the maximum likelihood method and GTR+I+G4+F evolutionary model based on an alignment of 1339 bp of 16S rRNA gene sequences from Ehrlichia spp.and Neoehrlichia spp., containing 33 homologous sequences.Anaplasma centrale (AF309869) and Anaplasma marginale (AF309867) sequences were used as outgroups and are indicated with (***).The sequences obtained in this project are highlighted in red.Bootstraps lower than 50 are not shown.The phylogenetic analysis based on an alignment of 409 bp of the dsb gene positioned the two sequences obtained in the present study into the same clade: The sequences obtained from A. cyanopus (#389 (BAP289)) (PP417932) from Nossa Senhora do Livramento and from B. flaveolus (#418 (F54)) (PP417933) from Poconé were closely related to Ehrlichia minasensis obtained from a common sloth (Bradypus variegatus) (MH212419) Figure 3. Phylogenetic analysis generated by the maximum likelihood method and GTR+I+G4+F evolutionary model based on an alignment of 1339 bp of 16S rRNA gene sequences from Ehrlichia spp.and Neoehrlichia spp., containing 33 homologous sequences.Anaplasma centrale (AF309869) and Anaplasma marginale (AF309867) sequences were used as outgroups and are indicated with (***).The sequences obtained in this project are highlighted in red.Bootstraps lower than 50 are not shown.The phylogenetic analysis based on an alignment of 409 bp of the dsb gene positioned the two sequences obtained in the present study into the same clade: The sequences obtained from A. cyanopus (#389 (BAP289)) (PP417932) from Nossa Senhora do Livramento and from B. flaveolus (#418 (F54)) (PP417933) from Poconé were closely related to Ehrlichia minasensis obtained from a common sloth (Bradypus variegatus) (MH212419) from Brazil, from cattle from the Philippines (LC641910) and Colombia (ON209405), from Rhipicephalus microplus ticks from Brazil (JX629808) and Colombia (KM015219), and from a Rhipicephalus sanguineus tick from Brazil (MT135769) (Figure 4).

Figure 4 .
Figure 4. Phylogenetic analysis generated by the maximum likelihood method and TIM3+I+G4+F evolutionary model based on an alignment of 409 bp of dsb gene sequences, containing 53 homologous sequences.Ehrlichia muris and Ehrlichia sp. were used as outgroups (KU672594, KF523726, and AY236484) and are indicated with (***).The sequences obtained in this project are highlighted in red.Bootstraps lower than 50 are not shown.The phylogenetic analysis based on an alignment of 430 bp of the 23S-5S intergenic region (ITS) positioned the sequences obtained herein (C.fasciolata (#330 BAP146) (PP417934) and B. flaveolus (#507 F12) (PP417936) from Nossa Senhora do Livramento and S. coerulescens (#497 F58) (PP417935) from Santo Antonio de Leverger) in the same clade, grouping with sequences of A. phagocytophilum (KU588997; KU588996) from Ixodes pacificus ticks from the USA (Figure 5).

Figure 4 .
Figure 4. Phylogenetic analysis generated by the maximum likelihood method and TIM3+I+G4+F evolutionary model based on an alignment of 409 bp of dsb gene sequences, containing 53 homologous sequences.Ehrlichia muris and Ehrlichia sp. were used as outgroups (KU672594, KF523726, and AY236484) and are indicated with (***).The sequences obtained in this project are highlighted in red.Bootstraps lower than 50 are not shown.The phylogenetic analysis based on an alignment of 430 bp of the 23S-5S intergenic region (ITS) positioned the sequences obtained herein (C.fasciolata (#330 BAP146) (PP417934) and B. flaveolus (#507 F12) (PP417936) from Nossa Senhora do Livramento and S. coerulescens (#497 F58) (PP417935) from Santo Antonio de Leverger) in the same clade, grouping with sequences of A. phagocytophilum (KU588997; KU588996) from Ixodes pacificus ticks from the USA (Figure 5).

Figure 5 .
Figure 5. Phylogenetic analysis generated by the maximum likelihood method and evolutionary model TVM+G4+I+F based on an alignment of 430 bp of sequences from the 23S-5S intergenic region of Anaplasma sp., containing 26 homologous sequences from the 23S-5S intergenic region from Anaplasma sp.Two sequences from Ehrlichia spp.used as outgroups (OM863955 and OM863956) are indicated with (***).The sequences obtained in this project are highlighted in red.Bootstraps lower than 50 are not shown.

Figure 5 .
Figure 5. Phylogenetic analysis generated by the maximum likelihood method and evolutionary model TVM+G4+I+F based on an alignment of 430 bp of sequences from the 23S-5S intergenic region of Anaplasma sp., containing 26 homologous sequences from the 23S-5S intergenic region from Anaplasma sp.Two sequences from Ehrlichia spp.used as outgroups (OM863955 and OM863956) are indicated with (***).The sequences obtained in this project are highlighted in red.Bootstraps lower than 50 are not shown.
Screening for Anaplasmataceae Agents by a cPCR Based on the 16S rRNA Gene and Molecular Characterization Thirteen avian DNA samples were positive in the screening for Anaplasmataceae agents targeting the gene 16S rRNA (345 bp) [42]: Ehrlichia sp. was detected in one R. carbo from Poconé (MT), Ehrlichia sp. was detected in one E. albiceps from Poconé (MT), and Ehrlichia sp. was detected in one R. carbo from Santo Antonio de Leverger (MT).Anaplasma sp. was detected in one P. sulphuratus from Santo Antonio de Leverger (MT).Candidatus Allocryptoplasma spp. was detected in one C. fasciolata from Nossa Senhora do Livramento (MT).The BLAST results regarding the five 16S rRNA sequences (ranging from 179-281 bp) obtained in the Parola et al. (2000)'s protocol [42] are shown in Supplementary Table