Using Digital PCR to Unravel the Occurrence of Piroplasmids, Bartonella spp., and Borrelia spp. in Wild Animals from Brazil
Abstract
:1. Introduction
2. Materials and Methods
2.1. Samples Analyzed
2.2. Molecular Assays
2.2.1. dPCR Assay (Multiplex for Piroplasmids, Bartonella spp., Borrelia spp., and Housekeeping Gene)
2.2.2. qPCR Assay (Multiplex for Piroplasmids, Bartonella spp., and Borrelia spp.)
2.2.3. Nested (n)PCR Assays for Piroplasmid Detection
2.3. Statistical Analysis
3. Results
3.1. Positivity for Each Technique
3.2. Statistical Analyses
3.2.1. Level of Agreement Between Tests
Piroplasmids
Bartonella spp. and Borrelia spp.
3.2.2. Analysis of Differences Between Proportions
Piroplasmids
Bartonella spp. and Borrelia spp.
3.2.3. Sensitivity and Specificity
Piroplasmids
Bartonella spp. and Borrelia spp.
3.2.4. Matthews Correlation Coefficient (MCC)
Piroplasmids
Bartonella spp. and Borrelia spp.
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
dPCR | digital PCR |
qPCR | quantitative PCR |
nPCR | nested PCR |
ddPCR | droplet digital PCR |
MCC | Matthews correlation coefficient |
References
- Zanella, J.R.C. Zoonoses emergentes e reemergentes e sua importância para saúde e produção animal. Pesqui. Agropecu. Bras. 2016, 51, 510–519. [Google Scholar] [CrossRef]
- Shaw, S.E.; Birtles, R.J.; Day, M.J. Review: Arthropod-transmitted infectious diseases of cats. J. Feline Med. Surg. 2001, 3, 193–209. [Google Scholar] [CrossRef] [PubMed]
- André, M.R. Diversity of Anaplasma and Ehrlichia/Neoehrlichia agents in terrestrial wild carnivores worldwide: Implications for human and domestic animal health and wildlife conservation. Front. Vet. Sci. 2018, 5, 293–297. [Google Scholar] [CrossRef] [PubMed]
- Birtles, R.J.; Raoult, D. Comparison of partial citrate synthase gene (gltA) sequences for phylogenetic analysis of Bartonella species. Int. J. Syst. Bacteriol. 1996, 46, 891–897. [Google Scholar] [CrossRef]
- Breitschwerdt, E.B. Bartonellosis: One health perspectives for an emerging infectious disease. ILAR J. 2014, 55, 46–58. [Google Scholar] [CrossRef]
- Kosoy, M.; McKee, C.; Albayrak, L.; Fofanov, Y. Genotyping of Bartonella bacteria and their animal hosts: Current status and perspectives. Parasitology 2018, 145, 543–562. [Google Scholar] [CrossRef]
- Margos, G.; Gofton, A.; Wibberg, D.; Dangel, A.; Marosevic, D.; Loh, S.M.; Oskam, C.; Fingerle, V. The genus Borrelia reloaded. PLoS ONE 2018, 13, e0208432. [Google Scholar] [CrossRef]
- Trevisan, G.; Cinco, M.; Trevisini, S.; di Meo, N.; Chersi, K.; Ruscio, M.; Forgione, P.; Bonin, S. Borreliae Part 1: Borrelia Lyme Group and Echidna-reptile Group. Biology 2021, 10, 1036. [Google Scholar] [CrossRef]
- Trevisan, G.; Cinco, M.; Trevisini, S.; di Meo, N.; Ruscio, M.; Forgione, P.; Bonin, S. Borreliae Part 2: Borrelia Relapsing Fever Group and unclassified Borrelia. Biology 2021, 10, 1117. [Google Scholar] [CrossRef]
- França, R.T.; Da Silva, A.S.; Loretti, A.P.; Mazzanti, C.M.; Lopes, S.T. Canine rangeliosis due to Rangelia vitalii: From first report in Brazil in 1910 to current day—A review. Ticks Tick-Borne Dis. 2014, 5, 466–474. [Google Scholar] [CrossRef]
- Alvarado-Rybak, M.; Solano-Gallego, L.; Millán, J. A review of piroplasmid infections in wild carnivores worldwide: Importance for domestic animal health and wildlife conservation. Parasites Vectors 2016, 9, 538. [Google Scholar] [CrossRef] [PubMed]
- Maggi, R.C.; Harms, C.A.; Breitschwerdt, E.B. Bartonelosis. An emerging disease of humans, domestic animals, and wildlife. In New Directions in Conservation Medicine: Applied Cases of Ecological Health, 1st ed.; Aguirre, A.A., Ostfeld, R.S., Daszak, P., Eds.; Oxford University Press: New York, NY, USA, 2012; p. 239. [Google Scholar]
- Kumar, A.; O’Bryan, J.; Krause, P.J. The global emergence of human babesiosis. Pathogens 2021, 10, 1447. [Google Scholar] [CrossRef] [PubMed]
- Dias, B.F.S. First National Report for the Convention on Biological Diversity, Brazil. 1999. Available online: https://antigo.mma.gov.br/estruturas/chm/_arquivos/chapter2a.pdf (accessed on 15 May 2025).
- Soares, J.F.; Dall’Agnol, B.; Costa, F.B.; Krawczak, F.S.; Comerlato, A.T.; Rossato, B.C.D.; Linck, C.M.; Sigahi, E.K.O.; Teixeira, R.H.F.; Sonne, L.; et al. Natural infection of the wild canid, Cerdocyon thous, with the piroplasmid Rangelia vitalii in Brazil. Vet. Parasitol. 2014, 202, 156–163. [Google Scholar] [CrossRef] [PubMed]
- de Quadros, R.M.; Soares, J.F.; Xavier, J.S.; Pilati, C.; da Costa, J.L.; Miotto, B.A.; Milettti, L.C.; Labruna, M.B. Natural infection of the wild canid Lycalopex gymnocercus by the protozoan Rangelia vitalii, the agent of canine rangeliosis. J. Wildl. Dis. 2015, 51, 787–789. [Google Scholar] [CrossRef] [PubMed]
- Silveira, J.A.G.; Rabelo, M.L.; Ribeiro, M.F.B. Detection of Theileria and Babesia in brown brocket deer (Mazama gouazoubira) and marsh deer (Blastocerus dichotomus) in the State of Minas Gerais, Brazil. Vet. Parasitol. 2011, 177, 61–66. [Google Scholar] [CrossRef]
- Soares, H.S.; Arlei, M.; Barbieri, R.M.; Minervino, A.H.H.; Moreira, T.R.; Gennari, S.M.; Labruna, M.B. Parasites and wildlife novel piroplasmid and Hepatozoon organisms infecting the wildlife of two regions of the Brazilian Amazon. Int. J. Parasitol. Parasites Wildl. 2017, 6, 115–121. [Google Scholar] [CrossRef]
- Calchi, A.C.; Vultão, J.G.; Alves, M.H.; Yogui, D.R.; Desbiez, A.L.J.; do Amaral, R.B.; Santi, M.; Teixeira, M.M.G.; Werther, K.; Machado, R.Z.; et al. Multi-locus sequencing reveals a novel Bartonella in mammals from the Superorder Xenarthra. Transbound. Emerg. Dis. 2020, 67, 2020–2033. [Google Scholar] [CrossRef]
- Muñoz-Leal, S.; Ramirez, D.G.; Luz, H.R.; Faccini, J.L.; Labruna, M.B. “Candidatus Borrelia ibitipoquensis,” a Borrelia valaisiana–related genospecies characterized from Ixodes paranaensis in Brazil. Microb. Ecol. 2020, 80, 682–689. [Google Scholar] [CrossRef]
- Gonçalves, L.R.; Paludo, G.; Bisol, T.B.; Perles, L.; de Oliveira, L.B.; de Oliveira, C.M.; da Silva, T.M.V.; Nantes, W.A.G.; Duarte, M.A.; Santos, F.M.; et al. Molecular detection of piroplasmids in synanthropic rodents, marsupials, and associated ticks from Brazil, with phylogenetic inference of a putative novel Babesia sp. from white-eared opossum (Didelphis albiventris). Parasitol. Res. 2021, 120, 3537–3546. [Google Scholar] [CrossRef]
- Ikeda, P.; Menezes, T.R.; Torres, J.M.; de Oliveira, C.E.; Lourenco, E.C.; Herrera, H.M.; Machado, R.Z.; André, M.R. First molecular detection of piroplasmids in non-hematophagous bats from Brazil, with evidence of putative novel species. Parasitol. Res. 2021, 120, 301–310. [Google Scholar] [CrossRef]
- Do Amaral, R.B.; Cardozo, M.V.; Varani, A.M.; Gonçalves, L.R.; Furquim, M.E.C.; Dias, C.M.; Santana, M.S.; de Assis, W.O.; da Silva, A.R.; Herrera, H.M.; et al. Bartonella machadoae sp. nov. isolated from wild rodents in the Pantanal wetland. Acta Trop. 2022, 229, 106368. [Google Scholar] [CrossRef] [PubMed]
- Do Amaral, R.B.; Cardozo, M.V.; Varani, A.M.; Furquim, M.E.C.; Dias, C.M.; Assis, W.O.; da Silva, A.R.; Herrera, H.M.; Machado, R.Z.; André, M.R. First report of Bartonella spp. in marsupials from Brazil, with a description of Bartonella harrusi sp. nov. and a new proposal for the taxonomic reclassification of species of the genus Bartonella. Microorganisms 2022, 10, 1609. [Google Scholar] [CrossRef] [PubMed]
- Mongruel, A.C.B.; Medici, E.P.; Canena, A.C.; Calchi, A.C.; Perles, L.; Rodrigues, B.C.B.; Soares, J.F.; Machado, R.Z.; André, M.R. Theileria terrestris nov. sp.: A novel Theileria in lowland tapirs (Tapirus terrestris) from two different biomes in Brazil. Microorganisms 2022, 10, 2319. [Google Scholar] [CrossRef] [PubMed]
- Weck, B.C.; Serpa, M.C.A.; Labruna, M.B.; Muñoz-Leal, S. A novel genospecies of Borrelia burgdorferi sensu lato associated with cricetid rodents in Brazil. Microorganisms 2022, 10, 204. [Google Scholar] [CrossRef]
- Calchi, A.C.; Yogui, D.R.; Alves, M.H.; Desbiez, A.L.J.; Kluyber, D.; Vultão, J.G.; Arantes, P.V.C.; de Santi, M.; Werther, K.; Teixeira, M.M.G.; et al. Molecular detection of piroplasmids in mammals from the Superorder Xenarthra in Brazil. Parasitol. Res. 2023, 122, 3169–3180. [Google Scholar] [CrossRef]
- De Mello, V.V.C.; Calchi, A.C.; de Oliveira, L.B.; Coelho, T.F.S.B.; Lee, D.A.B.; Franco, E.O.; Machado, R.Z.; André, M.R. Molecular survey of piroplasmids and hemosporidians in vampire bats, with evidence of distinct Piroplasmida lineages parasitizing Desmodus rotundus from the Brazilian Amazon. Parasitologia 2023, 3, 248–259. [Google Scholar] [CrossRef]
- De Oliveira, A.F.X.; Calchi, A.C.; Stocco, A.V.; Stocco, N.V.; Costa, A.C.; Mureb, E.N.; Pires, J.R.; Guimarães, A.; Raimundo, J.M.; Balthazar, D.A.; et al. Expanding the universe of Piroplasmids: Morphological detection and phylogenetic positioning of putative novel piroplasmids in black-eared opossums (Didelphis aurita) from southeastern Brazil, with description of “South American Marsupialia Group” of Piroplasmida. Parasitol. Res. 2023, 122, 1519–1530. [Google Scholar] [CrossRef]
- Krawczak, F.D.S.; Calchi, A.C.; Neves, L.C.; Dias, S.A.; da Silva, B.B.F.; Paula, W.V.F.; de Paula, L.G.F.; Tavares, M.A.; Pádua, G.T.; de Lima, N.J.; et al. Phylogenetic inferences based on distinct molecular markers confirm a novel Babesia species (Babesia goianiaensis nov. sp.) in capybaras (Hydrochoerus hydrochaeris) and associated ticks. Microorganisms 2023, 11, 2022. [Google Scholar] [CrossRef]
- Alabí Córdova, A.S.; Fecchio, A.; Calchi, A.C.; Dias, C.M.; Machado, R.Z.; André, M.R. Molecular evidence of Bartonella spp. in tropical wild birds from the Brazilian Pantanal, the largest wetland in South America. Vet. Res. Commun. 2024, 48, 1631–1640. [Google Scholar] [CrossRef]
- Calchi, A.C.; Braga, L.D.Q.V.; Bassini-Silva, R.; Castro-Santiago, A.C.; Herrera, H.M.; Soares, J.F.; Barros-Battesti, D.M.; Machado, R.Z.; Rocha, F.L.; André, M.R. Phylogenetic inferences based on distinct molecular markers reveals a novel Babesia (Babesia pantanalensis nov. sp.) and a Hepatozoon americanum-related genotype in crab-eating foxes (Cerdocyon thous). Exp. Parasitol. 2024, 262, 108786. [Google Scholar] [CrossRef]
- Calchi, A.C.; Duarte, J.M.B.; Castro-Santiago, A.C.; Bassini-Silva, R.; Barros-Battesti, D.M.; Machado, R.Z.; André, M.R. Genetic diversity of Theileria spp. in deer (Artiodactyla: Cervidae) from Brazil. Parasitol. Res. 2024, 123, 384. [Google Scholar] [CrossRef] [PubMed]
- Mongruel, A.C.B.; Medici, E.P.; Canena, A.C.; Machado, R.Z.; Clay, K.; Labruna, M.B.; André, M.R. First molecular detection of Borrelia sp. in tapirs (Tapirus terrestris). Vet. Res. Commun. 2024, 48, 2767–2774. [Google Scholar] [CrossRef] [PubMed]
- Calchi, A.C.; May-Júnior, J.A.; Baggio-Souza, V.; Berger, L.; Fagundes-Moreira, R.; Mallmann-Bohn, R.; Braga, L.Q.V.; Kirnew, M.D.; Silveria, M.F.; Ampuero, R.A.N.; et al. Diversity of Cytauxzoon spp. (Piroplasmida: Theileriidae) in Wild Felids from Brazil and Argentina. Pathogens 2025, 14, 148. [Google Scholar] [CrossRef] [PubMed]
- Pacheco, T.A.; Lee, D.A.B.; Maia, M.O.; Semedo, T.B.F.; de Mendonça, R.F.B.; Pedroni, F.; Dutra, V.; Nakazato, L.; Rossi, R.V.; André, M.R.; et al. Molecular survey of Hepatozoon spp. and piroplasmids in rodents and marsupials from midwestern Brazil, with evidence of a novel Piroplasmida clade (“South American Rodentia”) in the echimyid rodent Thrichomys pachyurus. Parasitol. Res. 2025, 124, 19. [Google Scholar] [CrossRef]
- Feng, Z.; Shu, Y. An Overview of Digital PCR. Bing Du Xue Bao 2017, 33, 103–107. [Google Scholar]
- Mirabile, A.; Sangiorgio, G.; Bonacci, P.G.; Bivona, D.; Nicitra, E.; Bonomo, C.; Musso, N. Advancing pathogen identification: The role of digital PCR in enhancing diagnostic power in different settings. Diagnostics 2024, 14, 1598. [Google Scholar] [CrossRef]
- Chen, B.; Jiang, Y.; Cao, X.; Liu, C.; Zhang, N.; Shi, D. Droplet digital PCR as an emerging tool in detecting pathogens nucleic acids in infectious diseases. Clin. Chim. Acta 2021, 517, 156–161. [Google Scholar] [CrossRef]
- Wichianchot, S.; Hongsrichan, N.; Maneeruttanarungroj, C.; Pinlaor, S.; Iamrod, K.; Purisarn, A.; Donthaisong, P.; Karanis, P.; Nimsuphan, B.; Rucksaken, R. A newly developed droplet digital PCR for Ehrlichia canis detection: Comparisons to conventional PCR and blood smear techniques. J. Vet. Med. Sci. 2022, 84, 831–840. [Google Scholar] [CrossRef]
- Tsokana, C.N.; Symeonidou, I.; Sioutas, G.; Gelasakis, A.I.; Papadopoulos, E. Current applications of digital PCR in veterinary parasitology: An overview. Parasitologia 2023, 3, 269–283. [Google Scholar] [CrossRef]
- Hoshino, T.; Inagaki, F. Molecular quantification of environmental DNA using microfluidics and digital PCR. Syst. Appl. Microbiol. 2012, 35, 390–395. [Google Scholar] [CrossRef]
- Gutiérrez-Aguirre, I.; Rački, N.; Dreo, T.; Ravnikar, M. Droplet digital PCR for absolute quantification of pathogens. Methods Mol. Biol. 2015, 1302, 331–347. [Google Scholar] [CrossRef] [PubMed]
- Li, H.; Bai, R.; Zhao, Z.; Tao, L.; Ma, M.; Ji, Z.; Jian, M.; Ding, Z.; Dai, X.; Bao, F.; et al. Application of droplet digital PCR to detect the pathogens of infectious diseases. Biosci. Rep. 2018, 38, BSR20181170. [Google Scholar] [CrossRef] [PubMed]
- Capobianco, J.A.; Clark, M.; Cariou, A.; Leveau, A.; Pierre, S.; Fratamico, P.; Strobaugh, T.P., Jr.; Armstrong, C.M. Detection of Shiga toxin-producing Escherichia coli (STEC) in beef products using droplet digital PCR. Int. J. Food Microbiol. 2020, 319, 108499. [Google Scholar] [CrossRef] [PubMed]
- Yang, R.; Paparini, A.; Monis, P.; Ryan, U. Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples. Int. J. Parasitol. 2014, 44, 1105–1113. [Google Scholar] [CrossRef]
- Dewantoro, A.; Anggundari, W.C.; Prasetya, B. Review of digital PCR potential for surveillance of emerging disease from wastewater. IOP Conf. Ser. 2021, 926, 012065. [Google Scholar] [CrossRef]
- Wu, J.; Tang, B.; Qiu, Y.; Tan, R.; Liu, J.; Xia, J.; Zhang, J.; Huang, J.; Qu, J.; Sun, J.; et al. Clinical validation of a multiplex droplet digital PCR for diagnosing suspected bloodstream infections in ICU practice: A promising diagnostic tool. Crit. Care 2022, 26, 243. [Google Scholar] [CrossRef]
- King, J.L.; Smith, A.D.; Mitchelli, E.A.; Allen, M.S. Validation of droplet digital PCR for the detection and absolute quantification of Borrelia DNA in Ixodes scapularis ticks. Parasitology 2017, 144, 359–367. [Google Scholar] [CrossRef]
- Das, S.; Hammond-Mckibben, D.; Guralski, D.; Lob, S.; Fiedler, R.P.N. Development of a sensitive molecular diagnostic assay for detecting Borrelia burgdorferi DNA from the blood of Lyme disease patients by digital PCR. PLoS ONE 2020, 15, e0235372. [Google Scholar] [CrossRef]
- Maggi, R.G.; Breitschwerdt, E.B.; Quorollo, B.; Miller, J.C. Development of a multiplex droplet digital PCR assay for the detection of Babesia, Bartonella, and Borrelia species. Pathogens 2021, 10, 1462. [Google Scholar] [CrossRef]
- Maggi, R.G.; Richardson, T.; Breitschwerdt, E.B.; Miller, J.C. Development and validation of a droplet digital PCR assay for the detection and quantification of Bartonella species within human clinical samples. J. Microbiol. Methods 2020, 176, 106022. [Google Scholar] [CrossRef]
- Lesiczka, P.M.; Modry, D.; Sprong, H.; Fonville, M.; Pikula, J.; Piacek, V.; Hrazdilova, K. Detection of Anaplasma phagocytophilum in European brown hares (Lepus europaeus) using three different methods. Zoonoses Public Health 2021, 68, 917–925. [Google Scholar] [CrossRef] [PubMed]
- Basanisi, M.G.; La Bella, G.; Nobili, G.; Raele, D.A.; Cafiero, M.A.; Coppola, R.; Damato, A.M.; Fraccalvieri, R.; Sottili, R.; La Salandra, G. Detection of Coxiella burnetii DNA in sheep and goat milk and dairy products by droplet digital PCR in south Italy. Int. J. Food Microbiol. 2022, 366, 109583. [Google Scholar] [CrossRef] [PubMed]
- Ramírez, J.D.; Herrera, G.; Hernández, C.; Cruz-Saavedra, L.; Muñoz, M.; Flórez, C.; Butcher, R. Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples. PLoS Negl. Trop. Dis. 2018, 12, e0007063. [Google Scholar] [CrossRef] [PubMed]
- Ramírez, J.D.; Herrera, G.; Muskus, C.; Mendez, C.; Duque, M.C.; Butcher, R. Development of a digital droplet Polymerase Chain Reaction (ddPCR) assay to detect Leishmania DNA in samples from Cutaneous Leishmaniasis patients. Int. J. Infect. Dis. 2019, 79, 1–3. [Google Scholar] [CrossRef]
- Pereira, D.C.A.; Teixeira-Neto, R.G.; Lopes, V.V.; Pena, H.P.; Paz, G.F.; Custodio, C.H.X.; Belo, V.S.; Fonseca-Júnior, A.A.; da Silva, E.S. Development of quantitative PCR and digital PCR for the quantification of Leishmania infantum in dogs. Mol. Cell. Biochem. 2023, 478, 2445–2450. [Google Scholar] [CrossRef]
- Koepfli, C.; Nguitragool, W.; Hofmann, N.E.; Robinson, L.J.; Ome-Kaius, M.; Sattabongkot, J.; Felger, I.; Mueller, I. Sensitive and accurate quantification of human malaria parasites using droplet digital PCR (ddPCR). Sci. Rep. 2016, 6, 39183. [Google Scholar] [CrossRef]
- Srisutham, S.; Saralamba, N.; Malleret, B.; Rénia, L.; Dondorp, A.M.; Imwong, M. Four human Plasmodium species quantification using droplet digital PCR. PLoS ONE 2017, 12, e0175771. [Google Scholar] [CrossRef]
- Mahendran, P.; Liew, J.W.K.; Amir, A.; Ching, X.T.; Lau, Y.L. Droplet digital polymerase chain reaction (ddPCR) for the detection of Plasmodium knowlesi and Plasmodium vivax. Malar. J. 2023, 19, 241. [Google Scholar] [CrossRef]
- Wilson, M.; Glaser, K.C.; Adams-Fish, D.; Boley, M.; Mayda, M.; Molestina, R.E. Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani. Exp. Parasitol. 2015, 149, 24–31. [Google Scholar] [CrossRef]
- Kao, Y.F.; Peake, B.; Madden, R.; Cowan, S.R.; Scimeca, R.C.; Thomas, J.E.; Miller, C.A. A probe-based droplet digital polymerase chain reaction assay for early detection of feline acute cytauxzoonosis. Vet. Parasitol. 2021, 29, 109413. [Google Scholar] [CrossRef]
- Murthy, S.; Suresh, A.; Dandasena, D.; Singh, S.; Subudhi, M.; Bhandari, V.; Bhanot, V.; Arora, J.S.; Sharma, P. Multiplex ddPCR: A promising diagnostic assay for early detection and drug monitoring in bovine theileriosis. Pathogens 2023, 12, 296. [Google Scholar] [CrossRef] [PubMed]
- Jefferies, R.; Ryan, U.M.; Jardine, J.; Broughton, D.K.; Robertson, I.D.; Irwin, P.J. Blood, bull terriers and babesiosis: Further evidence for direct transmission of Babesia gibsoni in dogs. Aust. Vet. J. 2007, 85, 459–463. [Google Scholar] [CrossRef] [PubMed]
- Fleiss, J.L. Measuring nominal scale agreement among many raters. Psychol. Bull. 1971, 76, 378–382. [Google Scholar] [CrossRef]
- Cohen, J. A coefficient of agreement for nominal scales. Educ. Psychol. Meas. 1960, 20, 37–46. [Google Scholar] [CrossRef]
- McHugh, M.L. Interrater reliability: The kappa statistic. Biochem. Med. 2012, 22, 276–282. [Google Scholar] [CrossRef]
- McNemar, Q. Note on the sampling error of the difference between correlated proportions or percentages. Psychometrika 1947, 12, 153–157. [Google Scholar] [CrossRef]
- Revelle, W. psych: Procedures for Psychological, Psychometric, and Personality Research, R Package Version 2.3.12; Northwestern University: Evanston, IL, USA, 2024. Available online: https://CRAN.R-project.org/package=psych (accessed on 28 February 2025).
- Gamer, M.; Lemon, J.; Fellows, I.; Singh, P. irr: Various Coefficients of Interrater Reliability and Agreement, R Package Version 0.84. Available online: https://cran.r-project.org/package=irr (accessed on 28 February 2025).
- Chicco, D.; Jurman, G. The advantages of the Matthews correlation coefficient (MCC) over F1 score and accuracy in binary classification evaluation. BMC Genom. 2020, 21, 6. [Google Scholar] [CrossRef]
- Lashnits, E.; Neupane, P.; Bradley, J.M.; Richardson, T.; Maggi, R.G.; Breitschwerdt, E.B. Comparison of serological and molecular assays for Bartonella species in dogs with hemangiosarcoma. Pathogens 2021, 10, 794. [Google Scholar] [CrossRef]
- Belmonte, F.R.; Martin, J.L.; Frescura, K.; Damas, J.; Pereira, F.; Tarnopolsky, M.A.; Kaufman, B.A. Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions. Sci. Rep. 2016, 6, 25186. [Google Scholar] [CrossRef]
- Massolo, A.; Gerber, A.; Umhang, G.; Nicholas, C.; Liccioli, S.; Mori, K.; Klein, C. Droplet digital PCR as a sensitive tool to assess exposure pressure from Echinococcus multilocularis in intermediate hosts. Acta Trop. 2021, 223, 106078. [Google Scholar] [CrossRef]
- Yu, Z.; Zhao, Z.; Chen, L.; Li, J.; Ju, X. Development of a droplet digital PCR for detection of Trichuriasis in sheep. J. Parasitol. 2020, 106, 603–610. [Google Scholar] [CrossRef] [PubMed]
- Calchi, A.C.; Moore, C.O.; Bartone, L.; Kingston, E.; André, M.R.; Breitschwerdt, E.B.; Maggi, R.G. Development of multiplex assays for the identification of zoonotic Babesia species. Pathogens 2024, 13, 1094. [Google Scholar] [CrossRef] [PubMed]
- Maggi, R.G.; Calchi, A.C.; Moore, C.O.; Kingston, E.; Breitschwerdt, E.B. Human Babesia odocoilei and Bartonella spp. co-infections in the Americas. Parasites Vectors 2024, 17, 302. [Google Scholar] [CrossRef] [PubMed]
- Souza, U.; Dall’Agnol, B.; Michel, T.; Webster, A.; Klafke, G.; Martins, J.R.; Reck, J. Detection of Bartonella sp. in deer louse flies (Lipoptena mazamae) on gray brocket deer (Mazama gouazoubira) in the neotropics. J. Zoo Wildl. Med. 2017, 48, 532–535. [Google Scholar] [CrossRef]
- Farias, I.F.; Moura, L.M.; Sá, J.C.B.; Souza, D.S.; Torres-Santos, P.T.; Oliveira, J.B.; Muñoz-Leal, S.; Horta, M.C. Evaluation of infection by Borrelia sp. in domestic and wild mammals and ticks from the Catimbau National Park, Pernambuco. Pesq. Vet. Bras. 2023, 43, e07203. [Google Scholar] [CrossRef]
- Jorge, F.R.; Muñoz-Leal, S.; De Oliveira, G.M.; Serpa, M.C.A.; Magalhães, M.M.; De Oliveira, L.M.; Moura, F.R.P.; Teixeira, B.M.; Labruna, M.H. Novel Borrelia genotypes from Brazil indicate a new group of Borrelia spp. associated with South American bats. J. Med. Entomol. 2023, 60, 213–217. [Google Scholar] [CrossRef]
- Mongruel, A.C.B.; Medici, E.P.; Canena, A.C.; Dias, C.M.; Machado, R.Z.; André, M.R. Molecular evidence of Bartonella spp. in wild lowland tapirs (Tapirus terrestris), the largest land mammals in Brazil. Comp. Immunol. Microbiol. Infect. Dis. 2023, 101, 102042. [Google Scholar] [CrossRef]
Agent | Gene | Size | Primer Sequences | Probe Sequences |
---|---|---|---|---|
Bartonella spp. | 16S-23S rRNA (ITS) | 90–120 bp | BsppITS325s 5′-CCTCAGATGATGATCCCAAGCCTTCTGGCG-3′ BsppITS543as 5′-AATTGGTGGGCCTGGGAGGACTTG-3′ | BsppITS500probe 5′-FAM-GTTAGAGCGCGCGCTTGATTAAG-BHQ1-3′ |
Borrelia spp. | 16S-23S rRNA (ITS) | 104 bp | BobulITS120s 5′-AGGTCATTTTGGGGGTTTAGCTCAGTTGGCT-3′ BobulITS200as 5′-AATGGAGGTTAA GGGACTCGAACCCT-3′ | BobulITS160probe 5′-HEX-CGGCTTTGCAAGCCGAGGGTCAAG-BHQ2-3′ |
Piroplasmids | 18S rRNA | 125–138 bp | Piro18S-238s 5′-TCGGTGATTCATAATAAACTRGCGAATCGC-3′ Piro18S-380as 5′-AATCGAACCCCAATTCCCCGTTACCCG-3′ | Piro18S-340probe 5′ CY5-GACGGTAGGGTATTGGCCTACCG-BHQ2-3′ |
Housekeeping gene | B-Raf Proto-Oncogene (BRAF) gene | 120 bp | CaFeBRAF-15s 5′-TCAYGA AGACCTCACAGTAAAAATAGG T-3′ CaFeBRAF-110as 5′-GATCCAGACAAC TGTTCA AACTGATG-3′ | CaFeBRAF-50 5′-TAMRA-GTCTAGCCACAGTGAAATCTCGATG-BHQ2-3′ |
Number of Positive Samples | |||||||
---|---|---|---|---|---|---|---|
Bartonella spp. | Borrelia spp. | Piroplasmids | |||||
Wild Animal Group | dPCR | qPCR | dPCR | qPCR | nPCR | dPCR | qPCR |
Deer (Total) | 56 (32.18%) | 12 (6.9%) | 25 (14.37%) | 0 | 137 (78.73%) | 154 (88.51%) | 51 (29.31%) |
Subulo gouazoubira | 6 | 2 | 6 | 0 | 22 | 19 | 2 |
Mazama rufa | 0 | 0 | 1 | 0 | 3 | 2 | 0 |
Mazama jucunda | 2 | 0 | 3 | 0 | 4 | 4 | 0 |
Blastocerus dichotomus | 45 | 10 | 15 | 0 | 102 | 124 | 47 |
Ozotocerus bezoarticus | 3 | 0 | 0 | 0 | 6 | 5 | 2 |
Desmodus rotundus (Total) | 46 (90.2%) | 17 (33.33%) | 14 (27.45%) | 2 (3.92%) | 42 (82.35%) | 47 (92.15%) | 16 (31.37%) |
Cerdocyon thous (Total) | 3 (27.27%) | 1 (9.09%) | 1 (9.09%) | 0 | 3 (27.27%) | 11 (100%) | 9 (81.81%) |
Felids (Total) | 6 (17.14%) | 1 (2.86%) | 7 (20%) | 0 | 29 (82.86%) | 34 (97.14%) | 19 (54.29%) |
Leopardus pardalis | 1 | 0 | 1 | 0 | 9 | 12 | 10 |
Panthera onca | 4 | 1 | 4 | 0 | 18 | 21 | 9 |
Puma concolor | 1 | 0 | 2 | 0 | 2 | 2 | 0 |
Xenarthra (Total) | 6 (13.64%) | 6 (13.64%) | 6 (13.67%) | 0 | 22 (50%) | 21 (47.73%) | 14 (31.82%) |
Myrmecophaga tridactyla | 0 | 2 | 1 | 0 | 12 | 10 | 6 |
Tamandua tetradactyla | 1 | 0 | 1 | 0 | 4 | 3 | 1 |
Euphractus sexcinctus | 3 | 1 | 1 | 0 | 1 | 3 | 2 |
Dasypus novemcinctus | 2 | 2 | 2 | 0 | 4 | 2 | 3 |
Priodontes maximus | 0 | 1 | 1 | 0 | 1 | 3 | 2 |
Birds (Total) | 4 (22.22%) | 1 (5.56%) | 2 (11.11%) | 0 | 8 (44.44%) | 13 (72.22%) | 6 (33.33%) |
Pseudoseisura unirufa | 0 | 0 | 0 | 0 | 1 | 1 | 1 |
Saltator coerulescens | 2 | 0 | 1 | 0 | 2 | 2 | 2 |
Turdus leucomelas | 1 | 0 | 0 | 0 | 1 | 1 | 0 |
Cercomacra melanaria | 0 | 0 | 0 | 0 | 1 | 1 | 0 |
Ramphoceluls carbo | 0 | 0 | 0 | 0 | 1 | 0 | 0 |
Basileuterus flaveolus | 0 | 0 | 0 | 0 | 1 | 1 | 0 |
Leptotila verreauxi | 0 | 1 | 0 | 0 | 1 | 2 | 1 |
Icterus cayanensis | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
Lepidocolaptes angustirostris | 0 | 0 | 1 | 0 | 0 | 1 | 0 |
Fumarius rufus | 0 | 0 | 0 | 0 | 0 | 1 | 0 |
Fumarius leucopus | 0 | 0 | 0 | 0 | 0 | 1 | 0 |
Cyamocorax chrysops | 1 | 0 | 0 | 0 | 0 | 1 | 1 |
Tapirus terrestris (Total) | 4 (16.67%) | 0 | 4 (16.67%) | 0 | 16 (66.67%) | 24 (100%) | 4 (16.67%) |
Rodents (Total) | 4 (44.44%) | 1 (11.11%) | 2 (22.22%) | 0 | 0 | 9 (100%) | 4 (44.44%) |
Oecomys mamorae | 3 | 0 | 0 | 0 | 0 | 4 | 3 |
Thrichomys fosteri | 1 | 1 | 1 | 0 | 0 | 4 | 1 |
Clyomis laticeps | 0 | 0 | 1 | 0 | 0 | 1 | 0 |
Total | 129 (35.26%) | 39 (10.66%) | 61 (16.67%) | 2 (0.55%) | 257 (70.22%) | 313 (85.52%) | 123 (33.61%) |
Test | Comparison | Kappa (k) | p-Value |
---|---|---|---|
Fleiss’ Kappa | dPCR vs. qPCR vs. nPCR | −0.0517 | 0.0865 |
Cohen’s Kappa | qPCR vs. nPCR | −0.0935 | 0.0185 |
nPCR vs. dPCR | 0.2010 | <0.0001 | |
dPCR vs. qPCR | 0.0896 | 0.0012 |
Comparation | χ2 | p-Value |
---|---|---|
qPCR vs. nPCR | 79.101 | <0.0001 |
nPCR vs. qPCR | 30.447 | <0.0001 |
dPCR vs. qPCR | 177.09 | <0.0001 |
Agent | Comparation | χ2 | p-Value |
---|---|---|---|
Bartonella spp. | qPCR vs. dPCR | 69.482 | <0.0001 |
Borrelia spp. | qPCR vs. dPCR | 55.148 | <0.0001 |
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Calchi, A.C.; Mongruel, A.C.B.; Cavalcanti, F.B.P.; Bartone, L.; Duarte, J.M.B.; Medici, E.P.; Kluyber, D.; Caiaffa, M.G.; Alves, M.H.; Desbiez, A.L.J.; et al. Using Digital PCR to Unravel the Occurrence of Piroplasmids, Bartonella spp., and Borrelia spp. in Wild Animals from Brazil. Pathogens 2025, 14, 567. https://doi.org/10.3390/pathogens14060567
Calchi AC, Mongruel ACB, Cavalcanti FBP, Bartone L, Duarte JMB, Medici EP, Kluyber D, Caiaffa MG, Alves MH, Desbiez ALJ, et al. Using Digital PCR to Unravel the Occurrence of Piroplasmids, Bartonella spp., and Borrelia spp. in Wild Animals from Brazil. Pathogens. 2025; 14(6):567. https://doi.org/10.3390/pathogens14060567
Chicago/Turabian StyleCalchi, Ana Cláudia, Anna Claudia Baumel Mongruel, Fernanda Beatriz Pereira Cavalcanti, Lilliane Bartone, José Maurício Barbanti Duarte, Emília Patrícia Medici, Danilo Kluyber, Mayara G. Caiaffa, Mario Henrique Alves, Arnaud Leonard Jean Desbiez, and et al. 2025. "Using Digital PCR to Unravel the Occurrence of Piroplasmids, Bartonella spp., and Borrelia spp. in Wild Animals from Brazil" Pathogens 14, no. 6: 567. https://doi.org/10.3390/pathogens14060567
APA StyleCalchi, A. C., Mongruel, A. C. B., Cavalcanti, F. B. P., Bartone, L., Duarte, J. M. B., Medici, E. P., Kluyber, D., Caiaffa, M. G., Alves, M. H., Desbiez, A. L. J., Coelho, T. F. S. B., Machado, R. Z., Breitschwerdt, E. B., Maggi, R. G., & André, M. R. (2025). Using Digital PCR to Unravel the Occurrence of Piroplasmids, Bartonella spp., and Borrelia spp. in Wild Animals from Brazil. Pathogens, 14(6), 567. https://doi.org/10.3390/pathogens14060567