The aim of this study was to detect vector-borne pathogens (Anaplasmataceae family, Rickettsia genus, and Bartonella genus) in bats from Misiones (Argentina). Thirty-three specimens were captured over 8 days using mist nets. Twenty (60.6%) blood samples were positive (11/13 Artibeus lituratus, 4/10 Desmodus rotundus, 4/8 Carollia perspicillata, and 1/2 Myotis nigricans) by PCR for the gltA gene fragment of Bartonella. All samples were negative by PCR for the Anaplasmataceae family and Rickettsia genus.
The phylogenetic analysis showed seven Bartonella genotypes. The three genotypes obtained from A. lituratus, 2 from C. perspicillata, and 1 from D. rotundus were related to Bartonella spp. from New World bats, while the sequence obtained from M. nigricans was related to Old World bats.
We identified a considerable diversity of Bartonella genotypes in a small number of bats, thus further research is required to better understand the complex bat–pathogen interaction.
El objetivo de este estudio fue detectar patógenos transmitidos por vectores (familia Anaplasmataceae, género Rickettsia y género Bartonella) en murciélagos de Misiones (Argentina). Se capturaron 33 ejemplares con redes de niebla. Veinte (60,6%) muestras de sangre fueron positivas a una reacción en cadena de la polimerasa (PCR) para un fragmento del gen gltA de Bartonella (11/13 en muestras tomadas de ejemplares de la especie de murciélago Artibeus lituratus, 4/10 en Desmodus rotundus, 4/8 en Carollia perspicillata y 1/2 en Myotis nigricans). Todas las muestras resultaron negativas para la familia Anaplasmataceae y el género Rickettsia.
El análisis filogenético demostró siete genotipos de Bartonella. Los tres genotipos obtenidos de A. lituratus, 2 de C. perspicillata y 1 de D. rotundus estuvieron relacionados con Bartonella spp. de murciélagos del Nuevo Mundo, mientras que la secuencia obtenida de M. nigricans se relacionó con secuencias del Viejo Mundo. Es necesario estudiar un mayor número de ejemplares para comprender mejor la compleja interacción entre murciélagos, vectores y patógenos.
Bats are the second most diverse order of mammals globally. There are more than 1400 species distributed throughout the world except in the poles2. Bats are susceptible to different microorganisms, including viruses, bacteria, fungi and parasites, as well as to pathogens of importance in human and animal health15.
The province of Misiones (Argentina) is part of the Paranaense jungle and Campos and Malezales ecoregions due to the characteristics of its climate, soil and vegetation14. Misiones has the greatest diversity of bat species in the country, belonging to the families Noctilionidae, Phyllostomidae, Vespertilionidae and Molossidae2,3,13,17, with the presence of 42 species in the province out of a total of 70 species in Argentina. These families include carnivorous, piscivorous, frugivorous, insectivorous, nectarivorous and hematophagous bats2.
Bats have been implicated as reservoir hosts for diverse microorganisms, some of which are pathogenic to humans. Vector-borne bacteria (Bartonella, Rickettsia, Borrelia and Neorickettsia risticii) have been detected in blood and organ tissues of bats around the world15. Host-switching and variations in host-specificity of ectoparasites are important factors in these pathogens, with a potential risk of occurrence of epidemiological cycles of transmission between bats, humans and domestic animals15.
The only reports of vector-borne bacterium in bats from Argentina was in the specie Tadaridabrasiliensis (Molossidae family) from Buenos Aires City5; therefore, the aim of the present study was to detect vector-borne pathogens (Anaplasmataceae family, genus Rickettsia, and genus Bartonella) in different species of bats from Misiones (Argentina).
Sampling was performed during 8 days throughout the months of November 2015 and April 2016 in AICOM A-A003 (Spanish acronym for Area of Importance for Bat Conservation) known as Osununú/Teyú Cuaré, which involves the Teyú Cuaré Provincial Park and Osununú Private Reserve, which is located near San Ignacio village, Misiones Province, Argentina (27°17′04.53″S–55°35′19.44″). Bat capture was conducted using six mist nets with a length of 6–12m and a height of 4m, which remained open from 6 pm to 4 am on dark moon nights20. Thirty-three bats were captured and identified according to Barquez and Díaz, 20091: Artibeus lituratus (13), Desmodus rotundus (10), Carollia perspicillata (8) and Myotis nigricans (2). The four species of bats studied are widely distributed throughout South America; however, M. nigricans and A. lituratus have not been reported in Chile and Uruguay, respectively6. Artibeus lituratus and C. perspicillata are frugivorous, M. nigricans are insectivorous and D. rotundus is one of the three South American species that feeds exclusively on blood2.
Blood samples were taken from the brachial vein, not exceeding 1% of the total body weight, using non-heparinized 1ml tuberculin syringes with 27GX1/2″ needles7. DNA was extracted using the High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany), following the manufacturer's instructions. For each sample, three individual polymerase chain reactions (PCR) were performed for the screening of the Anaplasmataceae family, genus Rickettsia, and genus Bartonella8,9,18. Anaplasma centrale, Rickettsia conorii, and Bartonella clarridgeiae were used as positive control, respectively. Nuclease-free water was used as negative control.
Amplicons were randomly selected (representing a minimum of 50% of the findings per bat species) to be sequenced. Purification from agarose was performed using the Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Irvine, USA) and sequenced with a 3500 Genetic Analyzer sequencer (Applied Biosystems, Foster City, USA). The sequences were edited using BioEdit Sequence Alignment Editor and aligned with the Clustal W program, and compared with sequences deposited in GenBank. A phylogenetic analysis was performed using the maximum-likelihood (ML) method and the best-fitting substitution model was determined with the Bayesian Information Criterion using the ML model test implemented in MEGA 6.0. Support for the topologies was tested by bootstrapping over 1000 replications and gaps were excluded from the comparisons.
All samples tested negative by the PCRs for the 16S rRNA fragment of the Anaplasmataceae family and 23S-5S intergenic spacer fragment of the Rickettsia genus. Previous studies have detected N. risticii (Anaplasmataceae family) in bats of the Myotis genus from USA and T. brasiliensis in bats from Buenos Aires City (Argentina)5. Moreover, only two studies reported Rickettsia spp. in bats by PCR, including one in T. brasiliensis from Buenos Aires City (Argentina)5.
Twenty (60.6%) samples tested positive by PCR for the gltA gene fragment of Bartonella and 11 (55.0%) were sequenced (Table 1). The sequences obtained exhibited 81.5–100% identity among them (Table 1 and Fig. 1). The ML phylogenetic tree generated with gltA sequences evidenced seven different potential genotypes of Bartonella (3 from A. lituratus, 2 from C. perspicillata, 1 from D. rotundus and 1 from M. nigricans), which are associated with different groups related to previous findings in bats (Fig. 1).
Bats testing positive for the genus Bartonella.
| Bat species | Family | Number of specimens | Positives for Bartonella | Sequences obtained | Identity with each other | |
|---|---|---|---|---|---|---|
| n | % | % | ||||
| Artibeus lituratus | Phyllostomidae | 13 | 11 | 84.6 | 6 | 81.5–100 |
| Desmodus rotundus | Phyllostomidae | 10 | 4 | 40.0 | 2 | 100 |
| Carollia perspicillata | Phyllostomidae | 8 | 4 | 50.0 | 2 | 85.3 |
| Myotis nigricans | Vespertilionidae | 2 | 1 | 50.0 | 1 | – |
| Total | 33 | 20 | 60.6 | 11 | 81.5–100 | |
Maximum-likelihood tree constructed using the Tamura 3-parameter model (+G) from partial sequences of Bartonella gltA. Numbers represent bootstrap support generated from 1000 replications (only bootstrap support >70 is shown). GenBank accession numbers are in brackets. Additional information (host and country) is provided.
To our knowledge, this is the first finding of Bartonella spp. in the above species of bats from Argentina and the first detection in M. nigricans. Based on the gltA molecular marker, we found a high diversity of Bartonella in the bat species studied, which is consistent with previous reports. Bartonella spp. found in the Phyllostomidae family were related to previous findings from the New World family (South and Central America), while the Bartonella sp. from Vespertilionidae (M. nigricans) was grouped with sequences from that family from the Old World.
There are numerous reports of Bartonella spp. in different species of bats from America4,12,16,19. It should be noted that these studies showed different samples (blood, organs) and methodologies (PCR, culture, molecular characterization), as well as a variable number of samples studied4,12,16,19.
Bartonella spp. were previously reported in A. lituratus from Costa Rica and Brazil4,12,19. Two sequences obtained in our study from A. lituratus (M122 and M129, GenBank accession numbers MZ005985 and MZ019482, respectively) showed 100% identity among them and with Bartonella spp. associated to A. lituratus from Costa Rica (MH234317, MH234324 and MH234326). Two other sequences from the same bat species (M117 and M118, GenBank accession numbers MZ005987 and MZ005986, respectively) exhibited 100% identity among them and 99.6% with Bartonella spp. of Artibeus jamaicensis from Guatemala (MN529479) and the bat fly from Mexico (MF988085). The remaining two sequences obtained from A. lituratus (M124 and M126, GenBank accession numbers MZ005984 and MZ005983, respectively) showed 95.4% identity with Bartonella sp. from Sturnira lilium (Mexico) (KY629850) and 100% identity with Bartonella sp. from A. lituratus (Costa Rica) (MH234318), respectively.
In C. perspicillata, Bartonella spp. were detected in Guatemala, Costa Rica, Peru and Brazil4,12,19. In our analysis, one sequence obtained (M139, GenBank accession number MZ019484) is closely related to a group of sequences from Carollia spp. and Artibeus spp. from Peru, Brazil, Costa Rica and Guatemala, and the other (M116, GenBank accession number MZ005990) is related to a group of sequences from different bat species (D. rotundus and A. lituratus) and bat flies from Peru, Brazil, Costa Rica and Belize (Fig. 1).
Bartonella spp. associated to D. rotundus were found in Brazil, Peru, Belize, Mexico, Costa Rica and Guatemala4,12,19. The sequences found in D. rotundus (M110 and M112, GenBank accession numbers MZ005988 and MZ005989, respectively) showed 100% identity among them, and with Bartonella sp. of D. rotundus of Peru (MG799415).
The only sequence obtained from M. nigricans (M134, GenBank accession number MZ019483) showed 96.5% identity with the Bartonella spp. found in Nycteribia kolenatii bat fly from Romania (MK140280) and Belgium (MK140265), and Myotis blythii from Georgia (MK140355), among others. In America, Bartonella spp. were detected in Myotis chiloensis from Chile16, Myotis sp. from Peru19, Myotis keaysi from Costa Rica19 and Myotis lucifugus and Myotis grisescens from USA10. All these findings are not phylogenetically related to the Bartonella sp. found in M. nigricans in our study. Finally, the previous report of Bartonella sp. in bats (species T. brasiliensis, family Molossidae) from Argentina5 is not phylogenetically related to the findings of the present study.
With regard to their significance in human and animal health, the species Bartonella found in our study are of unknown pathogenicity. However, other authors have detected a Bartonella sp. in bats that has been associated with endocarditis in humans10.
Different clades of Bartonella found in bats are related to families, superfamilies and suborders of bats, suggesting co-divergence between bats and Bartonella11,15. In our study, a significant diversity of Bartonella species was identified in a small number of bats sampled, revealing a complex host-pathogen interaction. Thus, further research is required to better understand the circulation of vector-borne pathogens in bats and their ectoparasites. Sampling at different times of the year should be conducted to gain deeper insight into the host-pathogen relationship, as well as into the occurrence of spillover of Bartonella species into other wild and domestic animals, and the potential risk for human populations.
FundingThis work was partially supported by The Rufford Foundation (Small Grant).
Authors’ contributionsA.P. and A.D.R. fieldwork; M.N.D.S. and G.L.C. lab work and phylogenetic analysis; A.P., M.N.D.S. and G.L.C. writing – original draft preparation; I.L.R, M.N.D.S. and G.L.C. writing – review and editing; F.J.B. and F.E.G.D. resources administration and supervision. All authors reviewed the manuscript.
Conflicts of interestThe authors have no relevant financial or non-financial interests to disclose.
Data availabilityThe DNA sequences generated and analyzed during the current study are available in the GenBank repository [https://www.ncbi.nlm.nih.gov/nucleotide/].
A.P. and A.R. would like to thank Temaiken Foundation and Raúl Flores for his collaboration in the fieldwork.
