High frequency of Paracoccidioides brasiliensis infection in armadillos ...

Medical Mycology 2003, 41, 217 /223

High frequency of Paracoccidioides brasiliensis infection in armadillos (Dasypus novemcinctus): an ecological study E. BAGAGLI*, M. FRANCO$, S. DE M. G. BOSCO*, F. HEBELER-BARBOSA*, L. A. TRINCA% & M. R. MONTENEGRO§ *Departamento de Microbiologia e Imunologia, Instituto de Biocieˆncias, UNESP, Botucatu, Brasil; $Departamento de Patologia, Escola Paulista de Medicina, UNIFESP, Saõ Paulo, Brasil; %Departamento de Bioestatı´stica, Instituto de Biocieˆncias, UNESP, Botucatu, Brasil; §Departamento de Patologia, Faculdade de Medicina, UNESP, Botucatu, Saõ Paulo, Brasil

The fungus Paracoccidioides brasiliensis has been isolated from nine-banded armadillos (Dasypus novemcinctus) in different regions where paracoccidiodomycosis (PCM) is endemic. The link between PCM and these animals has provided the first valuable clue in the effort to elucidate the ecological niche of P. brasiliensis. The present study was aimed at correlating P. brasiliensis infection in armadillos with local ecological features and, if possible, the presence of the fungus in the soil in the Botucatu hyperendemic area of PCM. In this region the mean temperature ranges from 14.8 to 25.88C and the annual average precipitation is 1520 mm. The sites where 10 infected animals (positive group) were collected were studied and compared with the sites where five uninfected animals were found. The occurrence of the fungus in soil samples collected from the positive armadillos’ burrows and foraging sites was investigated by the indirect method of animal inoculation. Environmental data from the sites of animal capture, such as temperature, rainfall, altitude, vegetation, soil composition, presence of water and proximity of urban areas, were recorded. All 37 soil samples collected from the sites had negative fungal cultures. Positive animals were found much more frequently in sites with disturbed vegetation, such as riparian forests and artificial Eucalyptus or Pinus forests, in altitudes below 800 m, near water sources. The soil type of the sites of positive animals was mainly sandy, with medium to low concentrations of organic matter. The pH was mainly acidic at all the sites, although the concentrations of aluminum cations (HAl) were lower at the sites where positive animals were found. Positive armadillos were also captured in sites very close to urban areas. Our data and previous studies indicate that P. brasiliensis occurs preferentially in humid and shady disturbed forests in a strong association with armadillos. /

Keywords armadillos, Dasypus brasiliensis, paracoccidioidomycosis

novemcinctus,

ecology,

Paracoccidioides

Introduction Received December 2001; Accepted 7 May 2002; Final revision received 16 December 2002 Correspondence: Eduardo Bagagli, Departamento de Microbiologia e Imunologia, Instituto de Biocieˆncias, UNESP/Botucatu, Distrito de Rubiaõ Jr, Botucatu, Saõ Paulo, Brasil (CEP) 18.618-000. E-mail: [email protected]

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The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidiodomycosis (PCM), the most important systemic mycosis in Latin America [1]. The disease is characterized by the development of granulomatous lesions in the lungs, skin, mucous DOI: 10.1080/13693780310001597368

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membranes and other organs. It may be progressive and lethal if not treated, or may cause important sequelae such as permanent pulmonary fibrosis [2]. Although PCM was described in the early 20th century and many of its pathological and clinical aspects are well known, other important features of the agent are poorly understood. The ecological niche of P. brasiliensis has not been determined, although it is assumed that the fungus exists as a saprobe in the environment, where the infective propagula are produced [3]. The fungus has been rarely isolated from soil and related materials [4 /9], and from the feces of fruit bats (Artibeus lituratus ) and penguins (Pygoscelis adeliae ) [10,11]. Many other attempts have yielded negative results [12,13]. The prolonged latency of the disease, along with the frequent migration of the inhabitants of the endemic area and the lack of reported outbreaks, has also contributed to the difficulty in locating sites where the fungus occurs in nature [9,14/16]. The confirmation that the nine-banded armadillo (Dasypus novemcinctus ) is a natural reservoir of P. brasiliensis has opened new avenues for studying its ecological niche [17 /21]. Several features of the ninebanded armadillos suggest that these animals represent good sentinels for locating the whereabouts of the fungus in nature. In having a low body temperature and a weak immunological system, they are particularly susceptible to infectious diseases [22,23], including PCM. Armadillo PCM is common and is widely distributed in different endemic areas [15,16]. Also, the armadillos remain constantly within restricted localities for several consecutive years [24,25]. The aims of the present paper were to investigate the main ecological features associated with P. brasiliensis infection in armadillos, with emphasis on soil composition

and vegetation, and to attempt to isolate the fungus from soil and debris collected from the habitats of positive animals. Our results and those of previous studies [26 /28] indicate that P. brasiliensis occurs preferentially in a limited range of habitats, typified by the presence of disturbed and humid vegetation, and situated near water sources.

Material and methods Armadillos Fifteen adult nine-banded armadillos (eight male and seven female) and their habitats were studied. Two animals were 1/2 years old, whereas the remainder were older than 3 years, according to estimations based on the body size and weight [29]. The study was carried out in the Botucatu hyperendemic region of PCM [30], Saõ Paulo State, Brazil, within an area of approximately 5000 km2 (Fig. 1). The animals were captured under a license from the Brazilian Federal Environmental Protection Agency (IBAMA), which regulates and protects wildlife in the country. The isolation of the fungus from the animals and histopathological examination were carried out as previously described [15,18]. P. brasiliensis was isolated from 10 animals (positive group).

Attempts to isolate P. brasiliensis from soil samples Thirty-seven samples of soil and debris from the positive armadillo sites (interior of animal burrows, at depths of 0.5, 1.0 and 1.5 m) and foraging places (at surface and 10 /20 cm deep) were collected in 80-ml sterile plastic containers, maintained at 20/258C and processed the same day or 1 day after collection. Two grams of the samples were weighed out and diluted in

Fig. 1 Geographic location of P. brasiliensis -positive and -negative armadillos (D. novemcinctus ), and the urban area most proximal to each finding.

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Paracoccidioides brasiliensis infection in armadillos

50 ml of physiological saline containing 1000 U of penicillin G and 1 mg ml1 of streptomycin in Erlenmeyer flasks. After vigorous manual stirring for 5-min, the soil was allowed to settle for 1 h. The interface between the supernatant and the sediment was removed with a sterile pipette and used for animal inoculation. From each sample, 0.2 ml were inoculated intratesticularly in each of five adult golden hamsters and 1.0 ml intraperitoneally in each of five adult Swiss mice. Four and eight weeks after inoculation, the animals were anesthetized and killed by exsanguination, and their organs (lungs, spleen, liver and mesenteric lymph nodes) were removed and cultured in Sabouraud glucose agar tubes containing chloramphenicol (50 mg ml1) and in Mycosel agar plates (MycoselTM Agar, Becton Dickinson and Company, Cockeysville, MD, USA) supplemented with gentamicin (40 mg ml1). The Sabouraud tubes were incubated at 258C and the Mycosel plates at 32 /358C, with weekly observations for up to 10 weeks.

Ecological studies For each of the sites where an animal was captured, ecological and climatological features were obtained, such as (a) the medium temperature and pluvial precipitation, obtained at the Seçaõ de Climatologia of the Instituto de Agronomia de Campinas (IAC), Saõ Paulo; (b) latitude and longitude, obtained using the GPS (Global Position System, GPS 45 Personal NavigatorTM, Garmin Corporation, Olathe, KS, USA); (c) altitude, obtained from geomorphologic maps of Instituto Brasileiro de Geografia e Estatı´stica (IBGE), scale 1:50,000; (d) type of vegetation, classified according to the vegetation maps of IBGE as either riparian forest, semideciduous forest or artificial Pinus or Eucalyptus forest mixed with natural herbs and scrubs; (e) vegetation density, classified as low, medium or high; (f) height of leaf litter layer, classified as low (0 /20 mm), medium (21 /40 mm) and high (41 /60 mm); (g) anthropogenic disturbance of the sites, classified as low, medium or high; (h) microclimatological humidity, classified as the distance between the burrow and the nearest water source, namely as high (up to 15 m), medium (15 /70 m) and low (higher than 70 m); (i) distances from the nearest urban area, obtained using the GPS (Garmin GPS 45 Personal NavigatorTM) device’s Distance and Sums function, and classified as very near (up to 3 km), near (3 /6 km) and distant (higher than 6 km); (j) chemical and physical soil analyses, at the depths of 0/5, 0/20 and 20 /40 cm, including pH determined in 0.01 M CaCl2 (1:1), amount of organic matter, macroelements (P, K, Ca – 2003 ISHAM, Medical Mycology, 41, 217 /223

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and Mg), hydrogen-plus aluminum cations (H/Al, a parameter that indicates the potential acidity of the soil), saturated base, the total of cation exchange capacity and the percentage of base saturation, microelements (B, Cu, Fe, Mn and Zn), percentages of clay, sand and silt, according to standard international protocols (IAC System).

Statistical analysis The Pearson correlation coefficient was used to study the correlation between the various variables measured at different soil depths. As high coefficients were observed, the mean was used for all variables. Each sample point was then classified as having a high or a low level for each soil variable when the median value for the variable was used as a cut-off point. The association between animal infection and each ecological variable was studied with the two-tailed Fisher’s exact test [31]. We considered strong evidence of association a p -value smaller than 0.05, whereas some indications of possible association were inferred at values between 0.05 and 0.10. The Statistical Analysis System software (SAS Inc, Release 6.12, 1996) [32] was used to perform the analysis.

Results Attempts to isolate P. brasiliensis from soil All 37 soil samples around the living habitat of the 10 positive armadillos gave negative cultures.

Environmental features of the study sites The mean temperature fluctuated between 14.8 and 25.88C, and the average annual rainfall was 1520 mm for the whole area. Table 1 shows values for the association analyses between animal infection and the various ecological variables. Strong evidence of associations was observed between animal infection and altitude (P /0.03), perturbation of the vegetation (P /0.03) and microclimatological humidity (P /0.02). Infected armadillos were significantly associated with sites below 800 m, in which the vegetation was moderately to highly disturbed, and close to sources of water (70 m or closer). Some evidence of associations was also observed between infected animals and types of vegetation (P / 0.06) and vegetation debris (P /0.08). Higher percentages of infected animals were found in sites from riparian forests and artificial Pinus and Eucalyptus forests with low amounts of debris.

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Table 1

Association between armadillo infection by P. brasiliensis and ecological features

Variable

Level

Low ( 5/710) Medium (710 /800) High (/800) Vegetation type Riparian Semideciduous Artificial (Eucalyptus , Pinus ) Vegetation density Low Medium High Vegetation debris (mm) (0 /20] (20 /40] /40 Vegetation perturbation Low Medium High Humidity: Distance from nearest water source (m) (0 /15] (15 /70] /70 Distance from nearest urban area (km) (0 /3] (3 /6] /6 Low ( 5/4.43) pH (1 /14 scale) High (/4.43) Organic matter (g dm 3) Low ( 5/34.67) High (/34.67) 3 P (mg dm ) Low ( 5/9.33) High (/9.33) Low ( 5/42.33) H/Al (mmolc dm 3) High (/42.33) 3 K (mmolc dm ) Low ( 5/1.33) High (/1.33) Ca (mmolc dm 3) Low ( 5/12.67) High (/12.67) 3 Mg (mmolc dm ) Low ( 5/6.00) High (/6.00) Low ( 5/24.33) Saturated Base (mmolc dm 3) High (/24.33) 3 Low ( 5/83.67) Cation Exchange Capacity (mmolc dm ) High (/83.67) Base Saturation (%) Low ( 5/36.67) High (/36.67) 3 B (mg dm ) Low ( 5/0.13) High (/0.13) Low ( 5/3.25) Cu (mg dm 3) High (/3.25) Fe (mg dm 3) Low ( 5/100.00) High (/100.00) Low ( 5/17.75) Mn (mg dm 3) High (/17.75) Zn (mg dm 3) Low ( 5/2.3) High (/2.3) Sand (%) Low ( 5/62.5) High (/62.5) Clay (%) Low ( 5/34.00) High (/34.00) Silt (%) Low ( 5/3.00) High (/3.00) Altitude (m)

Number of Total p -value infected number animals of animals 7 3 0 6 0 4 2 8 0 2 7 1 0 7 3 8 2 0 5 3 2 5 5 8 2 5 5 8 2 5 5 5 5 5 5 5 5 7 3 7 3 5 5 5 5 6 4 6 4 5 5 3 7 8 2 7 3

8 4 3 7 3 5 2 11 2 3 8 4 3 8 4 9 3 3 6 3 6 6 9 6 9 8 7 9 6 8 7 9 6 8 7 8 7 8 7 8 7 9 6 8 7 8 7 9 6 8 7 8 7 9 6 9 6

0.0310

0.056

0.187

0.080

0.030

0.020

0.156

0.580 0.089 1.000 0.089 1.000 0.580 1.000 1.000 0.119 0.580 0.580 1.000 0.608 1.000 1.000 0.026 0.089 0.329

p -values derive from the Fisher’s exact test.



Concerning various soil features, few of them showed association with positive or negative animals. The most important association was related to the physical aspect of the soil, in terms of percentage of sand and clay. Positive animals were significantly associated with sandy soil (P /0.02), whereas some evidence of possible association was observed with soil containing low concentrations of organic matter (P /0.08), of clay (P /0.09) and of H/Al (P /0.09). The pH was acid in sites where both positive and negative animals were captured. The nearest distance to urban areas from the capture sites varied from 0.5 to 7.6 km (mean of 3.4 km) for the group of positive animals, and from 2.0 to 7.9 km (mean of 6.58 km) for the negative group. However, evidence of an association between distance to urban area and infection was not obtained.

Discussion In the present study, we extended the investigation of a previous report in which we showed that the ninebanded armadillo can frequently harbor P. brasiliensis and develop PCM [18]. All armadillos were captured within the same PCM hyperendemic area, and the fungus was isolated in 10/15 animals studied [15]. Comparatively, P. brasiliensis infection appears to be 103 /104 times more frequent in armadillos than in humans, as the Brazilian incidence of PCM is reported to be 10 cases per one million inhabitants [33]. Moreover, primary PCM exposure among the inhabitants of the Botucatu hyperendemic area, as estimated by the paracoccidioidin skin test, is as low as 13 /24% [34,35]. Furthermore, armadillo infection is broadly distributed, as it has been observed in different regions of Brazil (north, central and south) and Colombia [17 / 21]. The data from the published literature, with the inclusion of the present study and two additional reports from meetings [36,37], indicate that a total of 93 animals have been studied, and of these 36 (38.7%) harbored P. brasiliensis in their organs. The present evaluation of ecological associations revealed that P. brasiliensis- positive armadillos are found in places near water sources, with altitudes below 800 m, with different types of sandy soil, and disturbed vegetation types, including artificial Pinus and Eucalyptus forests, as well as tropical riparian forests. In one restricted site, where all three armadillos studied were P. brasiliensis negative, the altitude was higher (940 m), the semideciduous tropical forest was well preserved, and water sources were scarce. These data agree with several other observations that indicate that increased incidence rates of PCM and higher paracoccidioidin – 2003 ISHAM, Medical Mycology, 41, 217 /223

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sensitivity are associated with environmental changes [26,27] and the proximity of humid forests [28]. The association with human PCM and riparian sites is also reflected in the absence of P. brasiliensis in arid regions, such as northeasten Brazil [33]. Blastomyces dermatitidis is closely related to P. brasiliensis [38] and its solitary infections and outbreaks, in human and dogs in North America, have also been associated with sites located near waterways in riparian and humid habitats [15]. The fact that positive animals were found in sites close to urban areas may help to explain why the mycosis occurs in patients with no record of contact with rural areas. Furthermore, nine-banded armadillos are much consumed as a food supplement in several Latin American countries. It has been demonstrated that contact with armadillos is a risk factor for PCM [14]. Therefore, it is important to conduct educational campaigns to promote avoidance of direct contact with armadillos and their habitats, except by persons adequately protected against exposure to P. brasiliensis. The term ‘reservarea’ was coined by Borelli [39] to refer to areas that should correspond to the microniche of the fungus. The armadillo’s burrows and foraging places encompass several favorable ecological conditions for P. brasiliensis, and probably harbor its saprobic phase, as has already been suggested by Borelli [39,40]. Although our attempts to isolate the fungus from the soil of armadillo burrows and foraging places yielded negative results, several additional soil samples, collected from the same sites and processed elsewhere, have proven to be positive for fungal DNA amplification, by the polymerase chain reaction (PCR) with P. brasiliensis -specific pairs of primers (J. McEwen et al ., unpublished data). Currently, we are using these molecular PCR approaches as well as using the Remote Sensing and the Geographic Information System to assist our study of the ecological conditions prevalent in sites positive and negative for P. brasiliensis [41,42]. The integration of these studies is expected to help to define the niche of this pathogen. We hope to facilitate the detection of high-risk sites for the disease, and then carry out measures to prevent human infection. Armadillos are an old group of mammals (Xenarthra), established in South America since the Paleocene age, 65 million years ago. At that time South America was detached from North America [43]. P. brasiliensis, by contrast, is closely related to members of the family Onygenaceae [38], a subgroup of the order Onygenales that is believed to have appeared 150 million years ago (JW Taylor et al ., University of Berkeley, unpublished data). The observation that armadillos from different regions of Brazil as well as from Colombia can harbor

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P. brasiliensis in high frequency suggests that the fungal association with the animal is relatively ancient, certainly dating to a time considerably earlier than the advent of humans on the earth. Only recently, around 1880, did the nine-banded armadillo extend its range into Texas, and since then it has gradually moved to northward and eastward in the USA [44,45]. Because P. brasiliensis can occur in a wide range of ecological conditions and is strongly associated with armadillos, it is very important to monitor this recent northward extension, mainly in regions sharing environmental features similar to those of South American PCMendemic areas. Furthermore, the transport of these animals from South to North America should be strongly restricted.

Acknowledgements We thank He´lio Grassi Filho for soil analysis, the rural workers and technicians involved in the field and laboratory procedures and Angela Restrepo and He´lio J. Pereira Jr for helpful discussions. We also thank Juan McEwen (Corporacioń para Investigaciones Biolo´gicas, Medellıń, Colombia) and John W. Taylor (University of California, Berkeley, CA, USA) for sharing unpublished results. This work was supported by Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo (FAPESP).

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