Molecular detection of Histoplasma capsulatum in insectivorous and frugivorous bats in Southeastern Brazil

Molecular detection of Histoplasma capsulatum in insectivorous and frugivorous bats in... Abstract Bats are considered to play a significant role in the epidemiology of histoplasmosis, worldwide. We investigated the occurrence of H. capsulatum in lung samples from 89 bats, from urban areas in Southeastern Brazil, using nested PCR based on ribosomal DNA. Fungal DNA was detected in 31/89 samples (34.8%), of which 13/31 were Molossids (41.9%), 4/31 Eumops spp. (12.9%), 2/31 Artibeus lituratus (6.5%), and 12/31 others (38.7%). This is the first report of natural infection by H. capsulatum in A. lituratus in Southeastern Brazil, which reinforces the importance of these synanthropic animals in the epidemiology of histoplasmosis in urban areas. Histoplasma capsulatum, bats, molecular epidemiology, PCR, DNA Introduction Histoplasma capsulatum is a worldwide dimorphic fungus and the agent of histoplasmosis, an important zoonosis. The occurrence of the disease has been related to specific environmental conditions, that is, caves, abandoned or wrecked houses and buildings,1 as well as places where bird and bat feces are accumulated.2 It is usually asymptomatically in humans. However, the acute infections have been reported as outbreaks, characterized with intense clinical manifestations, that is, fever and respiratory problems. The disseminated progressive form of histoplasmosis affects, in particular, immunosuppressed individuals.3 In domestic animals, H. capsulatum infection has been described in dogs and cats associated with respiratory and/or gastrointestinal findings; however, it is usually asymptomatic as observed in humans.4 The enriched soil site offers an optimal condition for fungal growth and subsequent population exposure when the soil is disturbed, and the spores are aerosolized and inhaled,5 which highlight the importance of wild birds such as starlings, grackles, red-winged, blackbirds, and cowbirds and mammals such as raccoon, skunk, badger, rodents, armadillo, monkey, sloth, mara, paca, and bats due their feeding habits. H. capsulatum has been described in different wild animals.2,6–8 Bats become infected by inhalation, and the fungus may be disseminated systemically, including also to intestines. Once in feces, the fungus may be released to environment. Because the guano of bats are nitrogen-rich and act as a source of nutrients for the fungus in environment which allows the infection of new hosts.2,9 Given that bats play a significant role in the epidemiology of histoplasmosis, here we investigate the presence of H. capsulatum colonization in the lung of different bat species in the region of Botucatu, Southeastern Brazil. Methods In a cross-sectional study, we evaluated 89 bats from Southeastern Brazil, after tested for rabies on a routine epidemiological surveillance. All studied species and respective feeding habits are listed on Table 1. This study was approved by or local Ethics Committees, protocol number 170/2011. Table 1. The studied chiropterans according to their taxonomic classification, feeding habit and positive result in the polymerase chain reaction (PCR) for Histoplasma capsulatum. Family  Genus/Species  Feeding Habit  Number of Animals  Positivity to PCR  Molossidae  Molossidae  Insectivorous  6  2    Molossus spp.    5  3    Molossus molossus    21  8    Eumops spp.    4  1    Eumops glaucinus    3  1    Eumops perotis    6  2    Molossus rufus    2  0    Eumops auripendulus    1  0    Tadarida brasiliensis    1  0    Nyctinomops spp.    1  0  Phyllostomidae  Artibeus lituratus  Predominantly Frugivorous  6  2    Artibeus obscurus    1  0    Artibeus spp.    4  0    Platyrrhinus lineatus    1  1    Platyrrhinus spp.    1  0    Pygoderma bilabiatum    1  0    Carollia perspicillata    1  0    Desmodus rotundus  Hematophagous  1  0  Vespertilionidae  Myotis spp.  Insectivorous  5  0    Histiotus montanus    1  0  Other  Not identified    17  12  Total      89  31 (34.8%) (95%CI* 25.0 – 45.7)  Family  Genus/Species  Feeding Habit  Number of Animals  Positivity to PCR  Molossidae  Molossidae  Insectivorous  6  2    Molossus spp.    5  3    Molossus molossus    21  8    Eumops spp.    4  1    Eumops glaucinus    3  1    Eumops perotis    6  2    Molossus rufus    2  0    Eumops auripendulus    1  0    Tadarida brasiliensis    1  0    Nyctinomops spp.    1  0  Phyllostomidae  Artibeus lituratus  Predominantly Frugivorous  6  2    Artibeus obscurus    1  0    Artibeus spp.    4  0    Platyrrhinus lineatus    1  1    Platyrrhinus spp.    1  0    Pygoderma bilabiatum    1  0    Carollia perspicillata    1  0    Desmodus rotundus  Hematophagous  1  0  Vespertilionidae  Myotis spp.  Insectivorous  5  0    Histiotus montanus    1  0  Other  Not identified    17  12  Total      89  31 (34.8%) (95%CI* 25.0 – 45.7)  *95%CI = 95% confidence interval. View Large Table 1. The studied chiropterans according to their taxonomic classification, feeding habit and positive result in the polymerase chain reaction (PCR) for Histoplasma capsulatum. Family  Genus/Species  Feeding Habit  Number of Animals  Positivity to PCR  Molossidae  Molossidae  Insectivorous  6  2    Molossus spp.    5  3    Molossus molossus    21  8    Eumops spp.    4  1    Eumops glaucinus    3  1    Eumops perotis    6  2    Molossus rufus    2  0    Eumops auripendulus    1  0    Tadarida brasiliensis    1  0    Nyctinomops spp.    1  0  Phyllostomidae  Artibeus lituratus  Predominantly Frugivorous  6  2    Artibeus obscurus    1  0    Artibeus spp.    4  0    Platyrrhinus lineatus    1  1    Platyrrhinus spp.    1  0    Pygoderma bilabiatum    1  0    Carollia perspicillata    1  0    Desmodus rotundus  Hematophagous  1  0  Vespertilionidae  Myotis spp.  Insectivorous  5  0    Histiotus montanus    1  0  Other  Not identified    17  12  Total      89  31 (34.8%) (95%CI* 25.0 – 45.7)  Family  Genus/Species  Feeding Habit  Number of Animals  Positivity to PCR  Molossidae  Molossidae  Insectivorous  6  2    Molossus spp.    5  3    Molossus molossus    21  8    Eumops spp.    4  1    Eumops glaucinus    3  1    Eumops perotis    6  2    Molossus rufus    2  0    Eumops auripendulus    1  0    Tadarida brasiliensis    1  0    Nyctinomops spp.    1  0  Phyllostomidae  Artibeus lituratus  Predominantly Frugivorous  6  2    Artibeus obscurus    1  0    Artibeus spp.    4  0    Platyrrhinus lineatus    1  1    Platyrrhinus spp.    1  0    Pygoderma bilabiatum    1  0    Carollia perspicillata    1  0    Desmodus rotundus  Hematophagous  1  0  Vespertilionidae  Myotis spp.  Insectivorous  5  0    Histiotus montanus    1  0  Other  Not identified    17  12  Total      89  31 (34.8%) (95%CI* 25.0 – 45.7)  *95%CI = 95% confidence interval. View Large Lung samples (200 mg) were homogenized using an automated machine (Biospec Products). DNA extraction was processed using the Illustra Tissue and Cells genomic Prep Mini Spin Kit (GE Healthcare, Barrington, IL, USA), according to the manufacturer's instructions and for each 10 lungs evaluated, a positive (culture of H. capsulatum) and negative (ultra-pure water) controls were included. Polymerase chain reaction (PCR) and nested-PCR were carried out in 25 μl, containing the reaction buffer (10 mM Tris HCl pH 8.0, 50 mM KCl, 1.5 mM MgCl2), 0.2 mM dNTP, 20 μM each primer, 0.2 units of Taq Platinum (Invitrogen), and 10 ng genomic DNA. Molecular detection was carried out with universal primers ITS4 (5΄-TCCTCCGCTTATTGATATGC-3΄) and ITS5 (5΄-GGAAGTAAAAGTCGTAACAACG-3΄),10 and the inner primers were histoI (5΄- GCCGGACCT TTCCTCCTGGGGAGC-3΄) and histoII (5΄’- CAA GAATTTCACCTCTGACAGCCGA-3΄).11 Positive (DNA of culture of H. capsultatum) and negative (ultra-pure water and also DNA of Paracoccidioides brasiliensis) controls were employed in all PCR reactions. Amplicons were sequenced in a Genetic Analyzer 3500 automated sequencer (Applied Biosystems®, Foster City, CA, USA). The sequences were aligned using MEGA7 software12 and compared to other sequences deposited at GenBank (http://www.ncbi.nlm.nih.gov/BLAST). The frequency of infected animals was described based on percentages and their respective 95% confidence intervals (95% CI) were calculated using the software Stata v. 11.0 (StataCorp LP, College Station, TX, USA), considering a significance level (α) of 5%. Results Among 89 bat lung samples herein evaluated, we found 31 (34.8% 95% CI 25.7–45.2) positive for fungal detection. Table 1 describes the results according to the species and feeding habits. The five representative PCR products samples obtained for Molossus molossus, Molossus spp., Eumops spp., Artibeus lituratus, and Platyrrhinus lineatus were sequenced and compared, showing 99% similarity with Histoplasma capsulatum (accession number KP132276.1). Discussion The reduction, fragmentation, and transformation of natural habitats represent a threat to the global biodiversity, and bats are one of the most affected species.13 The population of insectivorous and frugivorous bats has significantly grown in urban areas due to the lack of planning for urbanization, especially concerning the development of architectonic and landscape projects.14 The reduction of the natural habitats for bats associated to the presence of shelters and absence of predators contributes to this process. Consequently, the close contact among bats, men and domestic animals has increased.3,15 The detection of chiropterans infected with H. capsulatum is important when considering that these animals were originally captured from urban areas, when a bat displays diurnal behavior or entered a house. Among the bats herein evaluated, we found 18 (20.2%) positive for rabies, but no coinfection with H. capsulatum was observed (data not shown). By our results we observed that fungal infection was greater than viral one (34.8% vs.20.2%, respectively), which highlights the importance of monitoring H. capsulatum in bats. The fact that infected bats come from urban areas increases the importance of histoplasmosis surveillance, beside literature reports noting that the majority of human histoplasmosis is associated with harborages, such as caves, where the exposure to the fungus present in soil with guano can be more intense than in residences roofs, for example.9 Every year, many people become infected with H. capsulatum, especially in the Americas. The human infection usually occurs in endemic areas during daily or labor-related activities, especially due to inhalation of fungal spores present in caves and old buildings and houses.16 In an investigation of 2,850 cases of histoplasmosis resulted from 105 reported outbreaks in United States and Puerto Rico during 1938 to 2013, 23% of these outbreaks were related to bats or bat droppings, and birds or bird droppings was related to 56%. In this way, excavation or clearing foliage in a bird-roosting site may contribute to occurrence of the histoplasmosis, associated to a contaminated environment by animal excrements.17 In Brazil, 28 outbreaks were reported since 1958 in nine states with 255 patients.18,19 For São Paulo State, two outbreaks were reported in Ubatuba, and one in Arapeí, Paraíba Valley, in 2007, involving 35 individuals who visited a cave populated by bats. The access to the caves was interdicted after the H. capsulatum isolation.20 Dias et al.2 isolated H. capsulatum from 87/2,427 (3.6%) insectivorous bats of different molossid species in São Paulo, Brazil. In our study, high positivity was observed for insectivorous bats from Molossidae (17/31; 95% CI 36.0–72.7), due to the abundance of this animals in the urban habitat, commonly inhabiting ceilings, basements and attics, and though tending to be closer to the human population. Frugivorous bats are also found in urban areas which may explain the first report of the infection in an Artibeus lituratus in São Paulo State. A. lituratus is widely distributed throughout the Americas and can be observed in urban centers.2 Many studies in literature among zoonotic and potentially zoonotic pathogens in bats are related to viral infections, such as rabies (and other lyssaviruses), severe acute respiratory syndrome (SARS-CoV), Nipah, Hendra, Ebola, and Marburg viruses.21 More studies involving molecular epidemiology of H. capsulatum in urban areas are required, especially to understand the range of host species and the places of the occurrence of the fungus. These studies may allow establishing surveillance plans aiming to prophylactic measures to control histoplasmosis dissemination. Among other actions, sanitary measures aiming to raise the public awareness about the role of bats in the transmission of histoplasmosis are necessary, as well as surveillance inquiries for H. capsulatum infection in bats linked to rabies control activities. Bats are, undoubtedly, important animals in nature by predating insects and pollinizing vegetation; however, they are also considered synanthropic animals living close to man and harboring several pathogens. Detecting H. capsulatum infection in bats from urban areas is an important finding from a public health prospective, considering the proximity of these animals with human habitations and their role in the maintenance and dissemination of the pathogenic fungus in the environment. Acknowledgements This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP n. 2011/10978-2). Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Huhn GD, Austin C, Carr M et al.   Two outbreaks of occupationally acquired histoplasmosis: More than workers at risk. Environ Health Perspect . 2005; 113: 585– 589. Google Scholar CrossRef Search ADS PubMed  2. Dias MAG, Oliveira RMZ, Giudice MC et al.   Isolation of Histoplasma capsulatum from bats in the urban area of São Paulo State, Brazil. Epidemiol Infect . 2011; 139: 1642– 1644. Google Scholar CrossRef Search ADS PubMed  3. Wheat LJ, Azar MM, Bahr NC, Spec A, Relich RF, Hage C. Histoplasmosis. Infect Dis Clin North Am . 2016; 30: 207– 227. Google Scholar CrossRef Search ADS PubMed  4. Brömel C, Sykes JE, Wheat LJ et al.   Histoplasmosis in dogs and cats. Clin Tech Small Anim Pract . 2005; 20: 227– 232. Google Scholar CrossRef Search ADS PubMed  5. Hock RHUC. The Malaysian Medical Association's role in public health control for reduction of bird dropping hazards in Sarawak. Aust N Z J Public Health . 2009; 33: 194– 195. Google Scholar CrossRef Search ADS PubMed  6. Naiff RD, Barret TV, Naiff MF, Ferreira LCL, Arias JR. New records of Histoplasma capsulatum from wild animals in the Brazilian Amazon. Rev Inst Med Trop São Paulo . 1996; 38: 273– 277. Google Scholar CrossRef Search ADS PubMed  7. Taylor ML, Chávez-Tapia CB, Reyes-Montes MR. Molecular typing of Histoplasma capsulatum isolated from infected bats, captured in Mexico. Fungal Genet Biol . 2000; 30: 207– 212. Google Scholar CrossRef Search ADS PubMed  8. González-González AE, Aliouat-Denis CM, Ramírez-Bárcenas JA et al.   Histoplasma capsulatum and Pneumocystis spp. co-infection in wild bats from Argentina, French Guyana, and Mexico. BMC Microbiol . 2014; 14: 1– 8. Google Scholar CrossRef Search ADS   9. Hoff GL, Bigler WJ. The role of bats in the propagation and spread of histoplasmosis: a review. J Wildl Dis . 1981; 17: 191– 196. Google Scholar CrossRef Search ADS PubMed  10. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR Protocols: A Guide to Methods and Applications . San Diego, CA: Academic Press, 1990: 315– 322. Google Scholar CrossRef Search ADS   11. Bialek R, Feucht A, Aepinus C, Robertson VJ, Hohle R. Evaluation of Two Nested PCR Assays for detection of Histoplasma capsulatum DNA in human tissue. J Clin Microbiol . 2002; 40: 1644– 1647. Google Scholar CrossRef Search ADS PubMed  12. Kumar S, Stecher G, Tamura K. “ MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets.” Mol Biol Evol . 2016; 33: 1870– 1874. Google Scholar CrossRef Search ADS PubMed  13. Avila-Flores R, Fenton MB. Use of spatial features by foraging insectivorous bats in a large urban landscape. J Mammal . 2005; 86: 1193– 1204. Google Scholar CrossRef Search ADS   14. Savard JPL, Clergeau P, Mennechez G. Biodiversity concepts and urban ecosystems. Landsc Urban Plan . 2000; 48: 131– 142. Google Scholar CrossRef Search ADS   15. García-Morales R, Moreno CR, Badano EI et al.   A deforestation impacts on bat functional diversity in tropical landscapes. PloS One . 2016; 11: e0166765. Google Scholar CrossRef Search ADS PubMed  16. Kauffman CA. Histoplasmosis: a clinical and laboratory update. Clin Microbiol Rev . 2007; 20: 115– 132. Google Scholar CrossRef Search ADS PubMed  17. Benedict K, Mody RK. Epidemiology of histoplasmosis outbreaks, United States, 1938–2013. Emerg Infect Dis . 2016; 22: 370– 378. Google Scholar CrossRef Search ADS PubMed  18. Oliveira FM, Unis G, Severo LC. Microepidemia de histoplasmose em Blumenau, Santa Catarina. J Bras Pneumol . 2006; 32: 375– 378. Google Scholar CrossRef Search ADS PubMed  19. Passos AN, Kohara VS, Freitas RS, Vicentini AP. Immunological assays employed for the elucidation of a histoplasmosis outbreak in São Paulo, SP. Braz J Microbiol . 2014; 45: 1357– 1361. Google Scholar CrossRef Search ADS PubMed  20. Vicentini-Moreira AP, Kohara VS, Passos NA et al.   Histoplasmosis microepidemics in the city of Arapeí, São Paulo. Bepa . 2008; 5: 8– 11. 21. Hayman DTS, Bowen RA, Cryan PM et al.   Ecology of zoonotic infectious diseases in bats: current knowledge and future directions. Zoonoses Public Health . 2013; 60: 2– 21. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

Molecular detection of Histoplasma capsulatum in insectivorous and frugivorous bats in Southeastern Brazil

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Abstract

Abstract Bats are considered to play a significant role in the epidemiology of histoplasmosis, worldwide. We investigated the occurrence of H. capsulatum in lung samples from 89 bats, from urban areas in Southeastern Brazil, using nested PCR based on ribosomal DNA. Fungal DNA was detected in 31/89 samples (34.8%), of which 13/31 were Molossids (41.9%), 4/31 Eumops spp. (12.9%), 2/31 Artibeus lituratus (6.5%), and 12/31 others (38.7%). This is the first report of natural infection by H. capsulatum in A. lituratus in Southeastern Brazil, which reinforces the importance of these synanthropic animals in the epidemiology of histoplasmosis in urban areas. Histoplasma capsulatum, bats, molecular epidemiology, PCR, DNA Introduction Histoplasma capsulatum is a worldwide dimorphic fungus and the agent of histoplasmosis, an important zoonosis. The occurrence of the disease has been related to specific environmental conditions, that is, caves, abandoned or wrecked houses and buildings,1 as well as places where bird and bat feces are accumulated.2 It is usually asymptomatically in humans. However, the acute infections have been reported as outbreaks, characterized with intense clinical manifestations, that is, fever and respiratory problems. The disseminated progressive form of histoplasmosis affects, in particular, immunosuppressed individuals.3 In domestic animals, H. capsulatum infection has been described in dogs and cats associated with respiratory and/or gastrointestinal findings; however, it is usually asymptomatic as observed in humans.4 The enriched soil site offers an optimal condition for fungal growth and subsequent population exposure when the soil is disturbed, and the spores are aerosolized and inhaled,5 which highlight the importance of wild birds such as starlings, grackles, red-winged, blackbirds, and cowbirds and mammals such as raccoon, skunk, badger, rodents, armadillo, monkey, sloth, mara, paca, and bats due their feeding habits. H. capsulatum has been described in different wild animals.2,6–8 Bats become infected by inhalation, and the fungus may be disseminated systemically, including also to intestines. Once in feces, the fungus may be released to environment. Because the guano of bats are nitrogen-rich and act as a source of nutrients for the fungus in environment which allows the infection of new hosts.2,9 Given that bats play a significant role in the epidemiology of histoplasmosis, here we investigate the presence of H. capsulatum colonization in the lung of different bat species in the region of Botucatu, Southeastern Brazil. Methods In a cross-sectional study, we evaluated 89 bats from Southeastern Brazil, after tested for rabies on a routine epidemiological surveillance. All studied species and respective feeding habits are listed on Table 1. This study was approved by or local Ethics Committees, protocol number 170/2011. Table 1. The studied chiropterans according to their taxonomic classification, feeding habit and positive result in the polymerase chain reaction (PCR) for Histoplasma capsulatum. Family  Genus/Species  Feeding Habit  Number of Animals  Positivity to PCR  Molossidae  Molossidae  Insectivorous  6  2    Molossus spp.    5  3    Molossus molossus    21  8    Eumops spp.    4  1    Eumops glaucinus    3  1    Eumops perotis    6  2    Molossus rufus    2  0    Eumops auripendulus    1  0    Tadarida brasiliensis    1  0    Nyctinomops spp.    1  0  Phyllostomidae  Artibeus lituratus  Predominantly Frugivorous  6  2    Artibeus obscurus    1  0    Artibeus spp.    4  0    Platyrrhinus lineatus    1  1    Platyrrhinus spp.    1  0    Pygoderma bilabiatum    1  0    Carollia perspicillata    1  0    Desmodus rotundus  Hematophagous  1  0  Vespertilionidae  Myotis spp.  Insectivorous  5  0    Histiotus montanus    1  0  Other  Not identified    17  12  Total      89  31 (34.8%) (95%CI* 25.0 – 45.7)  Family  Genus/Species  Feeding Habit  Number of Animals  Positivity to PCR  Molossidae  Molossidae  Insectivorous  6  2    Molossus spp.    5  3    Molossus molossus    21  8    Eumops spp.    4  1    Eumops glaucinus    3  1    Eumops perotis    6  2    Molossus rufus    2  0    Eumops auripendulus    1  0    Tadarida brasiliensis    1  0    Nyctinomops spp.    1  0  Phyllostomidae  Artibeus lituratus  Predominantly Frugivorous  6  2    Artibeus obscurus    1  0    Artibeus spp.    4  0    Platyrrhinus lineatus    1  1    Platyrrhinus spp.    1  0    Pygoderma bilabiatum    1  0    Carollia perspicillata    1  0    Desmodus rotundus  Hematophagous  1  0  Vespertilionidae  Myotis spp.  Insectivorous  5  0    Histiotus montanus    1  0  Other  Not identified    17  12  Total      89  31 (34.8%) (95%CI* 25.0 – 45.7)  *95%CI = 95% confidence interval. View Large Table 1. The studied chiropterans according to their taxonomic classification, feeding habit and positive result in the polymerase chain reaction (PCR) for Histoplasma capsulatum. Family  Genus/Species  Feeding Habit  Number of Animals  Positivity to PCR  Molossidae  Molossidae  Insectivorous  6  2    Molossus spp.    5  3    Molossus molossus    21  8    Eumops spp.    4  1    Eumops glaucinus    3  1    Eumops perotis    6  2    Molossus rufus    2  0    Eumops auripendulus    1  0    Tadarida brasiliensis    1  0    Nyctinomops spp.    1  0  Phyllostomidae  Artibeus lituratus  Predominantly Frugivorous  6  2    Artibeus obscurus    1  0    Artibeus spp.    4  0    Platyrrhinus lineatus    1  1    Platyrrhinus spp.    1  0    Pygoderma bilabiatum    1  0    Carollia perspicillata    1  0    Desmodus rotundus  Hematophagous  1  0  Vespertilionidae  Myotis spp.  Insectivorous  5  0    Histiotus montanus    1  0  Other  Not identified    17  12  Total      89  31 (34.8%) (95%CI* 25.0 – 45.7)  Family  Genus/Species  Feeding Habit  Number of Animals  Positivity to PCR  Molossidae  Molossidae  Insectivorous  6  2    Molossus spp.    5  3    Molossus molossus    21  8    Eumops spp.    4  1    Eumops glaucinus    3  1    Eumops perotis    6  2    Molossus rufus    2  0    Eumops auripendulus    1  0    Tadarida brasiliensis    1  0    Nyctinomops spp.    1  0  Phyllostomidae  Artibeus lituratus  Predominantly Frugivorous  6  2    Artibeus obscurus    1  0    Artibeus spp.    4  0    Platyrrhinus lineatus    1  1    Platyrrhinus spp.    1  0    Pygoderma bilabiatum    1  0    Carollia perspicillata    1  0    Desmodus rotundus  Hematophagous  1  0  Vespertilionidae  Myotis spp.  Insectivorous  5  0    Histiotus montanus    1  0  Other  Not identified    17  12  Total      89  31 (34.8%) (95%CI* 25.0 – 45.7)  *95%CI = 95% confidence interval. View Large Lung samples (200 mg) were homogenized using an automated machine (Biospec Products). DNA extraction was processed using the Illustra Tissue and Cells genomic Prep Mini Spin Kit (GE Healthcare, Barrington, IL, USA), according to the manufacturer's instructions and for each 10 lungs evaluated, a positive (culture of H. capsulatum) and negative (ultra-pure water) controls were included. Polymerase chain reaction (PCR) and nested-PCR were carried out in 25 μl, containing the reaction buffer (10 mM Tris HCl pH 8.0, 50 mM KCl, 1.5 mM MgCl2), 0.2 mM dNTP, 20 μM each primer, 0.2 units of Taq Platinum (Invitrogen), and 10 ng genomic DNA. Molecular detection was carried out with universal primers ITS4 (5΄-TCCTCCGCTTATTGATATGC-3΄) and ITS5 (5΄-GGAAGTAAAAGTCGTAACAACG-3΄),10 and the inner primers were histoI (5΄- GCCGGACCT TTCCTCCTGGGGAGC-3΄) and histoII (5΄’- CAA GAATTTCACCTCTGACAGCCGA-3΄).11 Positive (DNA of culture of H. capsultatum) and negative (ultra-pure water and also DNA of Paracoccidioides brasiliensis) controls were employed in all PCR reactions. Amplicons were sequenced in a Genetic Analyzer 3500 automated sequencer (Applied Biosystems®, Foster City, CA, USA). The sequences were aligned using MEGA7 software12 and compared to other sequences deposited at GenBank (http://www.ncbi.nlm.nih.gov/BLAST). The frequency of infected animals was described based on percentages and their respective 95% confidence intervals (95% CI) were calculated using the software Stata v. 11.0 (StataCorp LP, College Station, TX, USA), considering a significance level (α) of 5%. Results Among 89 bat lung samples herein evaluated, we found 31 (34.8% 95% CI 25.7–45.2) positive for fungal detection. Table 1 describes the results according to the species and feeding habits. The five representative PCR products samples obtained for Molossus molossus, Molossus spp., Eumops spp., Artibeus lituratus, and Platyrrhinus lineatus were sequenced and compared, showing 99% similarity with Histoplasma capsulatum (accession number KP132276.1). Discussion The reduction, fragmentation, and transformation of natural habitats represent a threat to the global biodiversity, and bats are one of the most affected species.13 The population of insectivorous and frugivorous bats has significantly grown in urban areas due to the lack of planning for urbanization, especially concerning the development of architectonic and landscape projects.14 The reduction of the natural habitats for bats associated to the presence of shelters and absence of predators contributes to this process. Consequently, the close contact among bats, men and domestic animals has increased.3,15 The detection of chiropterans infected with H. capsulatum is important when considering that these animals were originally captured from urban areas, when a bat displays diurnal behavior or entered a house. Among the bats herein evaluated, we found 18 (20.2%) positive for rabies, but no coinfection with H. capsulatum was observed (data not shown). By our results we observed that fungal infection was greater than viral one (34.8% vs.20.2%, respectively), which highlights the importance of monitoring H. capsulatum in bats. The fact that infected bats come from urban areas increases the importance of histoplasmosis surveillance, beside literature reports noting that the majority of human histoplasmosis is associated with harborages, such as caves, where the exposure to the fungus present in soil with guano can be more intense than in residences roofs, for example.9 Every year, many people become infected with H. capsulatum, especially in the Americas. The human infection usually occurs in endemic areas during daily or labor-related activities, especially due to inhalation of fungal spores present in caves and old buildings and houses.16 In an investigation of 2,850 cases of histoplasmosis resulted from 105 reported outbreaks in United States and Puerto Rico during 1938 to 2013, 23% of these outbreaks were related to bats or bat droppings, and birds or bird droppings was related to 56%. In this way, excavation or clearing foliage in a bird-roosting site may contribute to occurrence of the histoplasmosis, associated to a contaminated environment by animal excrements.17 In Brazil, 28 outbreaks were reported since 1958 in nine states with 255 patients.18,19 For São Paulo State, two outbreaks were reported in Ubatuba, and one in Arapeí, Paraíba Valley, in 2007, involving 35 individuals who visited a cave populated by bats. The access to the caves was interdicted after the H. capsulatum isolation.20 Dias et al.2 isolated H. capsulatum from 87/2,427 (3.6%) insectivorous bats of different molossid species in São Paulo, Brazil. In our study, high positivity was observed for insectivorous bats from Molossidae (17/31; 95% CI 36.0–72.7), due to the abundance of this animals in the urban habitat, commonly inhabiting ceilings, basements and attics, and though tending to be closer to the human population. Frugivorous bats are also found in urban areas which may explain the first report of the infection in an Artibeus lituratus in São Paulo State. A. lituratus is widely distributed throughout the Americas and can be observed in urban centers.2 Many studies in literature among zoonotic and potentially zoonotic pathogens in bats are related to viral infections, such as rabies (and other lyssaviruses), severe acute respiratory syndrome (SARS-CoV), Nipah, Hendra, Ebola, and Marburg viruses.21 More studies involving molecular epidemiology of H. capsulatum in urban areas are required, especially to understand the range of host species and the places of the occurrence of the fungus. These studies may allow establishing surveillance plans aiming to prophylactic measures to control histoplasmosis dissemination. Among other actions, sanitary measures aiming to raise the public awareness about the role of bats in the transmission of histoplasmosis are necessary, as well as surveillance inquiries for H. capsulatum infection in bats linked to rabies control activities. Bats are, undoubtedly, important animals in nature by predating insects and pollinizing vegetation; however, they are also considered synanthropic animals living close to man and harboring several pathogens. Detecting H. capsulatum infection in bats from urban areas is an important finding from a public health prospective, considering the proximity of these animals with human habitations and their role in the maintenance and dissemination of the pathogenic fungus in the environment. Acknowledgements This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP n. 2011/10978-2). Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Huhn GD, Austin C, Carr M et al.   Two outbreaks of occupationally acquired histoplasmosis: More than workers at risk. Environ Health Perspect . 2005; 113: 585– 589. Google Scholar CrossRef Search ADS PubMed  2. Dias MAG, Oliveira RMZ, Giudice MC et al.   Isolation of Histoplasma capsulatum from bats in the urban area of São Paulo State, Brazil. Epidemiol Infect . 2011; 139: 1642– 1644. Google Scholar CrossRef Search ADS PubMed  3. Wheat LJ, Azar MM, Bahr NC, Spec A, Relich RF, Hage C. Histoplasmosis. Infect Dis Clin North Am . 2016; 30: 207– 227. Google Scholar CrossRef Search ADS PubMed  4. Brömel C, Sykes JE, Wheat LJ et al.   Histoplasmosis in dogs and cats. Clin Tech Small Anim Pract . 2005; 20: 227– 232. Google Scholar CrossRef Search ADS PubMed  5. Hock RHUC. The Malaysian Medical Association's role in public health control for reduction of bird dropping hazards in Sarawak. Aust N Z J Public Health . 2009; 33: 194– 195. Google Scholar CrossRef Search ADS PubMed  6. Naiff RD, Barret TV, Naiff MF, Ferreira LCL, Arias JR. New records of Histoplasma capsulatum from wild animals in the Brazilian Amazon. Rev Inst Med Trop São Paulo . 1996; 38: 273– 277. Google Scholar CrossRef Search ADS PubMed  7. Taylor ML, Chávez-Tapia CB, Reyes-Montes MR. Molecular typing of Histoplasma capsulatum isolated from infected bats, captured in Mexico. Fungal Genet Biol . 2000; 30: 207– 212. Google Scholar CrossRef Search ADS PubMed  8. González-González AE, Aliouat-Denis CM, Ramírez-Bárcenas JA et al.   Histoplasma capsulatum and Pneumocystis spp. co-infection in wild bats from Argentina, French Guyana, and Mexico. BMC Microbiol . 2014; 14: 1– 8. Google Scholar CrossRef Search ADS   9. Hoff GL, Bigler WJ. The role of bats in the propagation and spread of histoplasmosis: a review. J Wildl Dis . 1981; 17: 191– 196. Google Scholar CrossRef Search ADS PubMed  10. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR Protocols: A Guide to Methods and Applications . San Diego, CA: Academic Press, 1990: 315– 322. Google Scholar CrossRef Search ADS   11. Bialek R, Feucht A, Aepinus C, Robertson VJ, Hohle R. Evaluation of Two Nested PCR Assays for detection of Histoplasma capsulatum DNA in human tissue. J Clin Microbiol . 2002; 40: 1644– 1647. Google Scholar CrossRef Search ADS PubMed  12. Kumar S, Stecher G, Tamura K. “ MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets.” Mol Biol Evol . 2016; 33: 1870– 1874. Google Scholar CrossRef Search ADS PubMed  13. Avila-Flores R, Fenton MB. Use of spatial features by foraging insectivorous bats in a large urban landscape. J Mammal . 2005; 86: 1193– 1204. Google Scholar CrossRef Search ADS   14. Savard JPL, Clergeau P, Mennechez G. Biodiversity concepts and urban ecosystems. Landsc Urban Plan . 2000; 48: 131– 142. Google Scholar CrossRef Search ADS   15. García-Morales R, Moreno CR, Badano EI et al.   A deforestation impacts on bat functional diversity in tropical landscapes. PloS One . 2016; 11: e0166765. Google Scholar CrossRef Search ADS PubMed  16. Kauffman CA. Histoplasmosis: a clinical and laboratory update. Clin Microbiol Rev . 2007; 20: 115– 132. Google Scholar CrossRef Search ADS PubMed  17. Benedict K, Mody RK. Epidemiology of histoplasmosis outbreaks, United States, 1938–2013. Emerg Infect Dis . 2016; 22: 370– 378. Google Scholar CrossRef Search ADS PubMed  18. Oliveira FM, Unis G, Severo LC. Microepidemia de histoplasmose em Blumenau, Santa Catarina. J Bras Pneumol . 2006; 32: 375– 378. Google Scholar CrossRef Search ADS PubMed  19. Passos AN, Kohara VS, Freitas RS, Vicentini AP. Immunological assays employed for the elucidation of a histoplasmosis outbreak in São Paulo, SP. Braz J Microbiol . 2014; 45: 1357– 1361. Google Scholar CrossRef Search ADS PubMed  20. Vicentini-Moreira AP, Kohara VS, Passos NA et al.   Histoplasmosis microepidemics in the city of Arapeí, São Paulo. Bepa . 2008; 5: 8– 11. 21. Hayman DTS, Bowen RA, Cryan PM et al.   Ecology of zoonotic infectious diseases in bats: current knowledge and future directions. Zoonoses Public Health . 2013; 60: 2– 21. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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Medical MycologyOxford University Press

Published: Dec 27, 2017

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