Baptista Rosas Et Al FunEcol 2012

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Molecular detection of Coccidioides spp. from environmentalsamples in Baja California: linking Valley Fever to soil andclimate conditions

Ra�ul C. BAPTISTA-ROSASa,b, Jovani CATAL�AN-DIBENEb, Adriana L. ROMERO-OLIVARESb,Alejandro HINOJOSAc, Tereza CAVAZOSd, Meritxell RIQUELMEe,*aSchool of Health Sciences, Autonomous University of Baja California (UABC), Ensenada, 22890 Baja California, MexicobMolecular Ecology & Biotechnology Graduate Program, UABC, 22800 Baja California, MexicocGeographic Information Systems and Remote Sensing Laboratory, Department of Geology, Center for Scientific Research and Higher

Education of Ensenada (CICESE), Ensenada, 22860 Baja California, MexicodDepartment of Physical Oceanography, Center for Scientific Research and Higher Education of Ensenada (CICESE), Ensenada,

22860 Baja California, MexicoeDepartment of Microbiology, CICESE, 22860 Baja California, Mexico

a r t i c l e i n f o

Article history:

Received 26 April 2010

Revision received 5 July 2011

Accepted 17 July 2011

Available online -

Corresponding editor:

Mat Fisher

Keywords:

Coccidioidomycosis

Ecological niche

Molecular diagnosis

* Corresponding author. Tel.: þ52 646 175050E-mail address: [email protected] (M.

1754-5048/$ e see front matter ª 2011 Elsevdoi:10.1016/j.funeco.2011.08.004

Please cite this article in press as: Baptistain Baja California: linking Valley Fever to

a b s t r a c t

Coccidioidomycosis is an important human fungal infection of American deserts and nearby

semi-arid regions with highly endemic areas distributed along the United States-Mexico

border. Despite the increasing incidence in the last 20 yr, reports of positive isolations of

the causal agent, Coccidioides spp. from environmental samples have been scarce. To resolve

this paradox, it is extremely important to first identify the fundamental ecological niche of

this fungus. Soil samples (n¼ 90) including those from heteromyids’ active burrows, latrines

and other mammals’ dens were collected using an oriented sampling method from areas of

Baja California, Mexico previously predicted as putative endemic “hotspots”. The total

genomic DNA obtained from the collected samples was subjected to a nested PCR followed

by a diagnostic PCR designed to amplify the internal transcribed spacer (ITS) 2 region of

Coccidioides spp. From the 42 amplicons obtained and sequenced (37 from Valle de las Palmas

(VDP) and five from San Jose de la Zorra (SJZ)), 32 were confirmed to belong to Coccidioides spp.

No Coccidioides spp. were found in soils collected in Ensenada.

VDP and SJZ have different soil characteristics but share a Mediterranean climate having

less than 250 mm of precipitation per year, as well as a dry period of at least 6 months. The

development of Coccidioides spp. is probably related to the structure of the microbial pop-

ulation adapted to these conditions in the semi-arid-mediterranean ecotone.

ª 2011 Elsevier Ltd and The British Mycological Society. All rights reserved.

Introduction SouthernCalifornia inoneof themost important endemic areas

Coccidioidomycosis, also known as Valley Fever, is an endemic

fungal disease caused by the dimorphic Ascomycetes Cocci-

dioides spp. Coccidioides immitis is found in San Joaquin Valley in

0; fax: þ52 646 1750595Riquelme).ier Ltd and The British M

-Rosas RC, et al., Molecusoil and climate condit

in theUnited States,whereasCoccidioides posadasii is localized in

SouthernArizonaand inSouthAmerica,where caseshave been

found mostly in Argentina, Venezuela and Brazil. In Mexico,

most of the cases have been reported in the states of Sonora,

ycological Society. All rights reserved.

lar detection of Coccidioides spp. from environmental samplesions, Fungal Ecology (2011), doi:10.1016/j.funeco.2011.08.004

mailto:[email protected]

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http://www.elsevier.com/locate/funeco

http://dx.doi.org/10.1016/j.funeco.2011.08.004



2 R.C. Baptista-Rosas et al.

Nuevo Leon, Coahuila and Baja California. Specifically, in

northern Baja California, most reported cases are of C. posadasii

with only a few reported cases of C. immitis (Laniado-Labor�ın

et al. 1991; Fisher et al. 2001; Casta~n�on-Olivares et al. 2007).

This type of geographical distribution of the disease was

established over 40 yr ago according to the epidemiological data

at the time (Maddy&Coccozza1964; reviewedbyBaptista-Rosas

&Riquelme2007). Recent epidemiological researchhas reported

incidence rates of 150 cases/100 000 population in Kern county,

California (Vugia et al. 2009), with outbreaks of more than 500

cases/100 000 population (Zender & Talamantes 2006;

Flaherman et al. 2008). Similarly, there are reports of 159

cases/100 000 population in Maricopa County, Arizona

(Komatsu et al. 2003). Altogether there are estimates of more

than 200 000 cases annually in the United States alone (Buckley

2008), which represent about a six-fold increase since 1995. The

incidence of the disease inMexico is unknown because it is not

mandatory to officially report and register the cases. However,

ongoing studies have identified highly populated areas along

the United States-Mexico border endemic for coccidioidomy-

cosis (Ampel et al. 1998; Hector & Laniado-Laborin 2005; Fla-

herman et al. 2008). The highly endemic areas in southern

California and Arizona share biogeographical and bioclimatic

characteristics with some areas of Baja California previously

identified as potential hotspots for the presence of the fungus

(Baptista-Rosas et al. 2007). These similarities suggest that the

disease may have a comparable epidemiological distribution

across the border.

Coccidioidomycosis cannot be transmitted from person to

person, but acquired by inhalation of the arthrospores from the

environment where the fungus inhabits; therefore the fungus

should be isolated from endemic areas (Ajello et al. 1965; Ajello

1967; Lacy & Swatek 1974). However, the scarce environmental

evidence for Coccidioides spp. seems to be in disagreement with

the high incidence rates obtained for the disease. Only a few

positive isolations from environmental samplings have been

obtained in highly endemic areas in the United States (Stewart

&Meyer 1932; Emmons 1942;Maddy 1965). For instance, only 13

soil isolations were reported in California and one in Arizona

after extensive samplingusingmice intraperitoneal inoculation

and culture isolation (Swatek 1970). More recently, only four

positive isolations out of 720 soil samples were obtained in

California combining microbiological selective isolation tech-

niques and PCR diagnosis (Greene et al. 2000) and no positive

detections were obtained using a semi-selective method and

direct genomic DNA isolation followed by PCR from 150 soil

samples in Arizona (Tabor et al. 2002). In other studies, 62

environmental positive isolates from11 sites in theTucson area

were obtained by intraperitoneal inoculation of soil extracts

into female BALB/cmice (Mandel et al. 2007). InMexico, by using

this last methodology only two positive records have been re-

ported; one fromHermosillo, Sonora (Sotomayor et al. 1960), and

another one from Valle de las Palmas (VDP), 17miles south of

Tecate, Baja California (Cairns et al. 2000).

We suspect that the low number of positive isolations in

previous studies couldbedue inpart to anon-directed sampling

strategy given the poor characterization of Coccidioides ecolog-

ical niche and its role in the structural dynamics of the micro-

biological community of soil deserts, rather than the methods

used to process the collected samples. Even though the

Please cite this article in press as: Baptista-Rosas RC, et al., Molecuin Baja California: linking Valley Fever to soil and climate condit

requirements and conditions for Coccidioides growth in the

laboratory have been well characterized, the total range of

suitable environmental conditions for its development in the

natural habitat has not been well defined (Barker et al. 2007;

Fisher et al. 2007). Thus suggesting that efforts should be

invested towards the identification of Coccidioides spp. ecologi-

cal’s niche (Maddy 1957, 1958; Fisher et al. 2007). Furthermore,

the climatic characteristics of the region are also important

factors for Coccidioides spp. development (Maddy & Coccozza

1964; Comrie 2005). In the last two decades increasing rates of

prevalence and incidence of this disease have been directly

correlated with rainfall in usually dry regions in Arizona (Park

et al. 2005; Zender & Talamantes 2006). The available evidence

shows that outbreaks of the disease appear a year or two after

an abnormal weather-related rainfall increase, following

a period of prolonged drought (Comrie & Glueck 2007; Baptista-

Rosas et al. 2010).

The current state of thedisease and its correlation to thearid

and semi-arid climate conditions of northern Mexico have not

been studiedwith the attention they deserve. Bearing all this in

mind, we have conducted an oriented environmental sampling

strategy in the most likely fundamental ecological niche for

Coccidioides in two semi-arid areas of Baja California previously

predicted as endemic hotspots (Baptista-Rosas et al. 2007) and

used nested PCR followed by diagnostic PCR to explore the

presence of Coccidioides spp. These studies are part of a long-

term multidisciplinary project aimed to study the impact of

global climate changes in the distribution of the Valley Fever

fungus in Baja California.

Methods

Soil sampling strategies

Using available coccidioidomycosis epidemiological informa-

tion and data on ecological niche modelling obtained by

combining theGeneticAlgorithmforRuleSetProduction (GARP)

and Geographical Information Systems (Baptista-Rosas et al.

2007), we designed two sampling polygons in predicted

endemic areas of Baja California, Mexico: San Jos�e de la Zorra

(SJZ), 38 km north of the Ensenada; and Valle de las Palmas

(VDP), 17 km south of Tecate (Fig 1). As reference, we collected

soil samples from Ensenada (Fig 1). We conducted an oriented

soil sampling strategy (at least 10 samples per polygon) within

16 km2 polygons set around areas with evidence of small

mammals’ activity (Fig 6). For VDP and SJZ, soil samples from

heteromyids’ active burrows, latrines and other mammals’

dens were included. Soil samples from about 10 cm under the

surface were collected also for all locations. For each sampling

site, 20 g of soil were collected. The shovel was cleaned with

hydroxide chloride in between samples. A clean plastic

disposable spoon was used for each sample, which was

collected into a sterile 90ml specimen collection cup.

Sampling dates were selected according to a bioclimatic

analysis defined by the relationship between the number of

reported cases of the disease and the monthly mean precipi-

tation from 1971 to 1995, where most of the reported cases of

the disease were registeredmainly in the driest months of the

year after seasonal rains (Baptista-Rosas et al. 2010). Therefore


Fig 1 eMap of Mexico showing the location of the three sampling sites, Valle de las Palmas, San Jos�e de la Zorra, and

Ensenada, in the state of Baja California. Potential spatial distribution of Coccidioides spp. in North America

by GARP modelling (Baptista-Rosas et al. 2007), using climate and topography data as input variables, is shown in

grey.

Molecular detection of Coccidioides spp. from environmental samples 3

samplingwas conducted about 2e3 months after the seasonal

rains. Soil samples (n¼ 100) were collected between 2006 and

2010. Seventy samples were collected for VDP (18 in May 2007,

10 in Jun. 2008, 23 in Jan. and May 2009, and 19 in Jun. 2010), 20

for SJZ (10 in May 2006, and 10 in Jun. 2010) and 10 for Ense-

nada (Jun. 2010) as control.

Sampling points were registered with a Global Positioning

System (GPS) in geographical coordinates. Samples were kept

and transported at environmental temperature in darkness

until processed in the laboratory within a week.

Genomic DNA extraction from environmental samples

Total genomicDNAwasobtaineddirectly fromthesoil samples.

Extraction and purification of total DNA were comparatively

carried out with a Power soil DNA isolation kit (Mobio Labora-

tories Inc. Carlsbad, CA, USA), a ZR Soil Microbe DNA MiniPrep

kit (Zymo Research) and an UltraClean soil DNA isolation kit

(Mobio Laboratories Inc. Carlsbad, CA, USA) according to the

supplier instructions. DNA extraction was quantified by spec-

trophotometry at 260 nm and verified by 1 % agarose gel

electrophoresis.

Coccidioides molecular diagnosis

To reduce possibilities of non-specific amplification, a nested

PCR method, designed by Martin & Rygiewicz (2005) to amplify

fungal ITS1-5.8s-ITS2 900 bp DNA sequences belonging to the


subkingdom Dikarya, was conducted (Fig 2). Initially different

conditions were assessed: Genomic DNA used as template was

used at different concentrations, and DMSO or BSAwere added

to the reactions. Successful amplifications were attained when

using 1 ml of the total genomic DNA obtained from the soil

extractionas template, andnoDMSOorBSAwereadded.For the

first amplification step we used forward primer NSA3

(50-AAACTCTGTCGTGCTGGGGATA-30) and reverse primerNLC2

(50-GAGCTGCATTCCCAAACAACTC-30). For the second step we

used forward primer NSI1 (50-GATTGAATGGCTTAGTGAGG-30)and reverse primer NLB4 (50-GGATTCTCACCCTCTATGAC-30)using as template 1 ml of the previous step product. For each

reaction (20 ml final volume)we used 2.5 mMMgCl2, 0.6 mMeach

primer, 0.2 mM each dNTP, 1� GoTaq Flexi polymerase 5�buffer (Promega) and 1 U GoTaq Flexi polymerase (Promega) on

aMultigene II thermal cycler (Labnet International Inc.) or anMJ

Mini� Gradient Thermal Cycler (Bio Rad), both equipped with

a heated lid. An initial denaturation and enzyme activation step

of 2 min at 95 �C was followed by 35 cycles: 45 and 30 s at 95 �Cfor thefirst andsecondnestedPCRreactions respectively, 40 s at

55 or 60 �C for the first and second nested PCR reactions

respectively, and 60 s at 72 �C, and a final 5 min extension at

72 �C. As controls for the first and second nested PCRs, we used

C. posadasiiDNA kindly provided by G. Gonzalez andM. Orbach,

and Neurospora crassa strain N1 (FGSC #988) DNA. Nested water

controls were also included for each reaction set. For amplifi-

cation of a Coccidioides spp. specific ITS2 region (171 bp for

C. immitis and 179 bp forC. posadasii), we used as template 1 ml of


Fig 2 eMolecular detection of Coccidioides spp. (A) Illustration of a region of the Coccidioides genome containing the internal

transcribed spacer 1 (ITS1), the 5.8S ribosomal DNA and the internal transcribed spacer 2 (ITS2). Primers NSA3-NLC2 and

NSI1-NLB4 for nested PCR allow the amplification of a 900e1000 bp ITS1-5.8s-ITS2 region of Ascomycetes and

Basidiomycetes. Primers ITS2F and ITS2R are designed to amplify a 170 bp ITS2 region. 18s (Ribosomal Small Subunit

DNA), 28s (Ribosomal Large Subunit DNA). Image not to scale. (B) Gel electrophoresis of the obtained amplicons of the

second nested PCR for the samples collected in Jun. 2010. (C) Gel electrophoresis of the obtained amplicons of the

diagnostic PCR for the samples collected in Jun. 2010. For both B and C, lanes 52e70 correspond to samples from VDP,

lanes 71e80 to samples from Ensenada, and lanes 91e100 to samples from SJZ. Lane A contains the molecular marker

(GeneRuler 1 kb DNA Ladder in B, and O’GeneRuler 100 bp DNA Ladder Plus in C, both from Fermentas), lane B is a water

control, in lane C genomic DNA of N. crassa was used as template, and in lane D genomic DNA of C. immitis was used as

template.


a 1/100 diluted second nested PCR product, and primers ITS2F

(50-CGA GGT CAA ACC GGA TA-30) and ITS2R (50-CCT TCA AGC

ACGGCTT-30) (Binnicker et al. 2007). An initial denaturation and

enzyme activation step of 10min at 95 �C was followed by

45 cycles: 10 s at 95 �C, 15 s at 59 �C, and 20 s at 72 �C, and a final

10 min extension at 72 �C. As controls for the diagnostic PCRs,

the control reactionsof thenestedPCRswereusedas templates.

PCR products were confirmed by 2 % agarose gel electro-

phoresis. Bands were excised from the gels and DNA was

purified with a QIAquick Gel Extraction Kit (Qiagen, German-

town, MD, USA) according to the supplier instructions.

Sequencing was carried out by Eton Bioscience Inc. and Retro-

gen Inc., San Diego, CA, USA. Resulting sequences were

manually edited using software CodonCode Aligner

(CodonCode Corporation Dedham, MA) and Mega 5.05 (Tamura

et al. in press), and analyzed in the Genebank (National Center

for Biotechnology Information, Rockville Pike, Bethesda MD,

USA) database.


Ecological landscape characterization of the sampling sites

Vegetation coverage in the sampled polygons was described

using Baja California Plant guides and the Autonomous

University of Baja California herbarium. Heteromyids were

captured at night with Sherman traps. Soil characteristics

were described according to the edaphology charts of the

FAOeUNESCO classification from 1989 (INIFAP-CONABIO

1995).

Bioclimatic and ombrothermic analysis

Values for maximum temperature (Tmax), minimum

temperature (Tmin) and accumulated precipitation (pp)

from 1971 to 2010 of historic and active meteorological

stations in Baja California were obtained from the data-

base of the National Weather Service of Mexico. For VDP

available data covered only from Apr. 1971 to Dec. 1995.



To characterize VDP at the bioclimatic level, monthly

mean temperatures (Tm) and pp were calculated using

those available data, which were plotted against the inci-

dence rates reported for coccidioidomycosis in Baja

California (Baptista-Rosas et al. 2007). A Gaussen ombro-

thermic chart was generated to estimate the soil evapo-

transpiration of the sampled sites and to explore a graphic

representation of regional climate showing evidence of

differences and similarities with respect to other areas.

The bioclimatic analysis by Gaussen ombrothermic charts

are a useful tool to evaluate the dry period in which pp is

less than twice the Tm: the rainfall curve will be below

the temperature curve and the area between the two

curves will indicate the duration and intensity of drought

(Peinado et al. 1994).

Results

Low levels of genomic DNA were obtained formost sampling sites

The best yield of total genomic DNAwas obtained by using the

UltraClean soil DNA isolation commercial kit (MoBio Labora-

tories Inc.). No additional cleaning of the extracted DNA was

needed prior to PCR.

Different amounts of total microbial genomic DNA were

obtained from the soil samples collected in the three different

locations chosen for this study: 6 ng ml�1 from VDP, 2 ng ml�1

from SJZ and 4 ng ml�1 from Ensenada. No genomic DNA could

be obtained from some of the samples, which were not further

processed.

Coccidioides spp. were identified in VDP and SJZ

After the first round of PCR no amplicon was seen for most of

the samples (data not shown). Nevertheless, 1 ml of the

product was used directly as template for the second round of

nested PCR. After this step, an amplicon of about 900 bp was

obtained in 71 of the 100 soil samples collected: 53 from VDP

(15 from May 2007, nine from Jun. 2008, eight from Jan. 2009,

eight from May 2009 and 14 from Jun. 2010), 12 from SJZ

(10 from May 2006 and two from Jun. 2010) and five from

Ensenada (Table 1; Fig 2B).

Regardless of the second nested PCR outcome, all samples

were further submitted to the diagnostic PCR. Amplicons of

about 170 bp were obtained in 43 samples from VDP, in five

samples from SJZ and two samples from Ensenada (Table 1;

Fig 2C).

All amplicons obtained in the diagnostic PCR (n¼ 50) were

gel purified and sequenced (Suppl. Fig 1). Six sequencing

reactions either failed or produced non-satisfactory data

(data not shown). Twelve were identified by BLAST as Apha-

noascus spp. Of these, seven corresponded to Aphanoascus

canadensis, all in VDP (four burrows, one soil, one large

mammal den and one latrine), three to Aphanoascus kerati-

nophilus (from VDP burrows) and two to Aphanoascus verru-

cosus (Ensenada soils). The remaining 32 were identified as

Coccidioides spp. (five from SJZ and 27 from VDP). All the five

samples identified as Coccidioides spp. in SJZ were from


burrows. Whereas in VDP, from the 27 samples identified as

Coccidioides spp, three were from soils, 17 from burrows, five

from large mammal dens, and two from heteromyid latrines

(Table 1).

Bioclimatic analysis

Our results indicate that the phenologic patterns that occur in

Baja California are similar to those described previously in the

main endemic areas for coccidioidomycosis in North America

(Kolivras & Comrie 2003; Park et al. 2005; Zender & Talamantes

2006; Baptista-Rosas et al. 2010).

Baja California’s climate is very diverse, ranging from

Mediterranean to arid, but generally, characterized by a dry

season between Apr. and Oct. with accumulated rainfall during

winter (Fig 3) (Garc�ıa 2003). Usually the western coast and the

mountains are moister and colder, while the Gulf of Baja Cal-

ifornia is extremely arid with pp below 80mmyr�1. The

northwestern corner of the Baja California Peninsula lies at the

transition zone between Mediterranean and desert climates.

Along the Pacific coast, the Mediterranean climate dominates

with a rainy season during winter and a long dry season from

May to Oct. (Fig 3). Tm ranges between 15.7 �C and 18.1 �C. Thedesert climate is found to the east of the Mediterranean region,

extending all the way to the coast of the Gulf of California and

to the south of the state; it is characterized by having a Tm

between 22.6 �C and 23 �C (Reyes Coca et al. 1990; Miranda

Reyes et al. 1991). The Mediterranean corner is rarely exposed

to tropical cyclones; summer precipitation is scarce and

consists of small showers associated with the North American

monsoon and convective systems; these are not hydrologically

significant as the showers are seasonal and the evapotranspi-

ration rates are high (Minnich et al. 2000). These variations in

the precipitation, as well as the microclimatic subtle differ-

ences in this ecosystem, change the soil chemistry and the

biota composition (Amundson et al. 1994).

The three sites analyzed are characterized by a Mediterra-

nean climate. SJZ, located at 320 m above sea level, has a Tm

of 17.3 �C (Tmax 39 �C and Tmin 9 �C) and about 230 mmyr�1

of rain. VDP is part of the Tijuana river watershed at 280 m

above sea level, with a Tm of 17.5 �C (Tmax 34.9 �C and Tmin

4.1 �C) and 212 mmyr�1 of rain, with most of the scarce rains

occurring in winter (Fig 4). Ensenada, located at sea level, has

a Tm of 18.1 �C (Tmax 21 �C and Tmin 12 �C) and 285 mmyr�1

of rainfall (Reyes Coca et al. 1990; Miranda Reyes et al. 1991;

Reyes Coca & Troncoso Gayt�an 2004).

Most cases of coccidioidomycosis reported in the state of

Baja California occur during the dry season after seasonal rains

(Fig 4). Analyses of the weather conditions for VDP and the

epidemiology of coccidioidomycosis in Baja California indi-

cated that most of the clinical cases in endemic areas occur

8e12 weeks after the last winter rains, during the dry season

between May and Jul., and a secondary peak occurs in Nov.

after the scarce summer rains (Fig 4) (Baptista-Rosas et al. 2010).

The bioclimatic characteristics of VDP correspond to a tran-

sition between infra-thermomediterranean belts with a dry

ombrothype (Fig 5). VDP has the seasonal climatic characteris-

tics (low seasonal rainfall, dry summers and high soil evapo-

transpiration rate) that favour the development and spread of

the fungus.


Table 1 e Molecular identification of Coccidioides spp. by PCR from environmental samples of Baja California, Mexico

Collectiondate

Samplea Characteristics PCR amplicons Sequencingresult

900 bp 170 bp

May 2007 1 VDP Burrow þ þþ Coccidioides spp.

2 VDP Burrow þ þþ Aphanoascus

keratinophilus

3 VDP Burrow þ �4 VDP Large mammal den � �5 VDP Large mammal den þ þþ Coccidioides spp.

6 VDP Large mammal den þ þþ Coccidioides spp.

7 VDP Burrow þþ �8 VDP Burrow þ �9 VDP Burrow þþ þþ Coccidioides spp.

10 VDP Large mammal den þ �11 VDP Large mammal den þþ þþ Coccidioides spp.

12 VDP Heteromyid latrine þþ þþ Coccidioides spp.

13 VDP Large mammal den þ þþ Coccidioides spp.

14 VDP Large mammal den þþ þþ Coccidioides spp.

15 VDP Burrow � þþ Coccidioides spp.

16 VDP Heteromyid latrine � þþ Coccidioides spp.

17 VDP Burrow þþ þþ Coccidioides spp.

18 VDP Dried grass þþ þþ Coccidioides spp.

Jun. 2008 19 VDP Burrow þ �20 VDP Burrow þþ þþ Coccidioides spp.

21 VDP Burrow þþ �22 VDP Burrow þ �23 VDP Burrow � þþ Coccidioides spp.

24 VDP Burrow þ þþ Failed

25 VDP Burrow þ þþ Coccidioides spp.



28 VDP Burrow þþ �Jan. 2009 29 VDP Burrow þ þþ Coccidioides spp.

30 VDP Burrow þþ �31 VDP Burrow þ þþ Failed

32 VDP Burrow þþ þþ Aphanoascus

canadensis




keratinophilus


canadensis


canadensis


keratinophilus

May 2009 39 VDP Burrow þþ �40 VDP Burrow � þþ Coccidioides spp.

41 VDP Burrow þ þþ Failed

42 VDP Burrow � þþ Failed

43 VDP Burrow þþ þþ Failed

44 VDP Burrow þþ þþ Failed


46 VDP Burrow þþ �47 VDP Burrow � �48 VDP Burrow þþ þþ Coccidioides spp.



51 VDP Burrow þ �Jun. 2010 52 VDP Burrow þþ �

53 VDP Soil � �54 VDP Burrow � �55 VDP Soil þþ þþ Coccidioides spp.

56 VDP Soil þþ �


Please cite this article in press as: Baptista-Rosas RC, et al., Molecular detection of Coccidioides spp. from environmental samplesin Baja California: linking Valley Fever to soil and climate conditions, Fungal Ecology (2011), doi:10.1016/j.funeco.2011.08.004

Table 1 e (continued)

Collectiondate

Samplea Characteristics PCR amplicons Sequencingresult

900 bp 170 bp


canadensis

58 VDP Heteromyid

latrine

þþ þþ Aphanoascus

canadensis

59 VDP Burrow þþ �60 VDP Soil þþ þþ Coccidioides spp.

61 VDP Burrow � �62 VDP Burrow � �63 VDP Soil þþ �64 VDP Burrow þþ �65 VDP Soil þþ �66 VDP Soil þþ þþ Aphanoascus

canadensis

67 VDP Burrow � �68 VDP Burrow þþ �69 VDP Large

mammal

den

þþ þþ Aphanoascus

canadensis

70 VDP Soil þþ �Jun. 2010 71 E Soil � þþ Aphanoascus

verrucosus

72 E Soil þþ þþ Aphanoascus

verrucosus

73 E Soil þþ �74 E Soil � �75 E Soil � �76 E Soil � �77 E Soil þþ �78 E Soil þþ �79 E Soil þþ �80 E Soil � �

May 2006 81 SJZ Burrow þ þþ Coccidioides spp.

82 SJZ Burrow þ þþ Coccidioides spp.

83 SJZ Burrow þ þþ Coccidioides spp.

84 SJZ Burrow þ �85 SJZ Burrow þ �86 SJZ Burrow þ �87 SJZ Burrow þ �88 SJZ Burrow þ �89 SJZ Burrow þ �90 SJZ Burrow þ �

Jun. 2010 91 SJZ Burrow � �92 SJZ Burrow � �93 SJZ Perturbed soil � �94 SJZ Burrow � �95 SJZ Burrow þþ þþ Coccidioides spp.

96 SJZ Burrow � þþ Coccidioides spp.

97 SJZ Small burrow � �98 SJZ Soil þþ �99 SJZ Soil � �100 SJZ Large mammal den � �

VPD Valle de las Palmas, E Ensenada, SJZ San Jose de la Zorra.

þþ clear band; þ faint band; � smear; � no amplification.

a Sample location can be seen in Fig 6.


Flora and fauna of VDP

VDP vegetation was dominated by Artemisia californica

(California Sagebrush), Baccharis sarathroides (Desert Broom),

Haplopappus juarezensis (Goldenbush) and Viguiera laciniata


(San Diego Sunflower). Other species found in the area were

Cylindropuntia californica (Snake Cholla), Eriogonum fasciculatum

var. fasciculatum (Coastal California Buckwheat, Fig 7G), Feroc-

actus viridescens (Coast Barrel Cactus), Lycium californicum (Cal-

ifornia Desert Thorn), Mirabilis laevis (Desert Wishbone Bush),


Fig 3 e Climatology of Baja California from 2007 to 2010. Monthly mean precipitation (pp), maximum temperature (Tmax) and

minimum temperature (Tmin) were plotted. Themean temperature (Tm) was estimated. Arrows indicate themonths inwhich

we sampled in VDP.


Prosopis glandulosa var. glandulosa (Honey Mesquite, Fig 7H) and

Simmondsia chinensis (Jojoba) (Roberts 1989).

The two species of heteromyids trapped in VDP were

identified as Dipodomys simulans (Dulzura Kangaroo Rat,

Fig 7E) and Chaetodipus fallax (San Diego Pocket Mouse,

Fig 7F).

Fig 4 e Coccidioidomycosis incidence inBajaCalifornia andclimat

coccidioidomycosis in Baja California for the period from 1988 to 1

and temperature, both for the period from1971 to 1995. Reported i

the National Meteorological System. Coccidioidomycosis cases fo

Epidemiological Surveillance.


Edaphology of the sampling sites

The edaphic characteristics of VDP were eutric fluvisols,

which contain non-consolidated material, a light horizon A

with scarce organic carbon, and a thin and hard stratus when

dry. Saturation grade was estimated to be 50 % or more in the

ologyofValle de lasPalmas.Monthlymean incidence ratesof

994were plotted alongwithmonthlymean precipitation (pp)

nValle de las Palmas. Climate data reported for 1971e1995by

r 1988e1994 available at the National Service of


Fig 5 e Gaussen ombrothermic analysis for Valle de las Palmas (Baja California, Mexico). The mean monthly precipitation

(pp) and twice the mean temperature (2Tm) are plotted against time (months). In the dry period, the line of precipitation is

under the 2Tm line. The area between the two lines shows the intensity of drought in the place. Climate data reported by

National Meteorological Service 1971e1995.


top 20e50 cm of soil, without significant calcium carbonate

in the aggregates. The original material was geologically

recent and originated by pluvial deposition. Different char-

acteristics were found in SJZ, with eutric regosols of medium

texture.

Discussion

We designed a relatively fast and reliable method to directly

detect presence of CoccidioidesDNA in soil samples. Of the three

sites analyzed, VDP, a previously reported endemic area (Cairns

et al. 2000), presented a considerably higher number of samples

positive to Coccidioides spp. (38 %). In contrast, at the SJZ site we

found only 25 % of the samples positive to Coccidioides spp. This

higher percentage of positive samples found at VDP could be

the result of the more lengthy and intensive nature of the

sampling. An equally comprehensive sampling would be

necessary to fairly compare both sites. No Coccidioides spp. were

detected in Ensenada. However, this could only be corroborated

after sequencing, as the diagnostic PCR resulted positive for

two of the 10 samples, which corresponded to Aphanoascus spp.

Thus, nested PCR cannot be used as a diagnostic method by

itself as it should be supplemented with sequencing or a high

resolution gel such as PAGE because the length of the ITS2

sequences belonging to Coccidioides spp. and Aphanoascus spp.

differ in approximately eight nucleotides. Nevertheless, diag-

nostic nested PCR without sequencing can be used as an

environmental screening tool for soils potentially inhabited by

Coccidioides spp.

The primers ITS2F and ITS2R proved to be extremely sensi-

tive and specific for Coccidioides spp. when used in a real-time

PCR assay to detect Coccidioides spp. directly from clinical


specimens (Binnicker et al. 2007). However, for soil samples, the

primerswere not sensitive enough to use directly in a diagnosis

PCR. Therefore, it was necessary to first run a nested PCR, sug-

gesting that probably there isnot enoughquantityofCoccidioides

spp. DNA in the total extracted DNA. Recently, using other

primers designed to amplify a region of the 28S rDNA, a PCRand

semi-nested PCR approach has been reported to efficiently

identify Coccidioides spp. in 24 soil samples from Brazil

(De Macedo et al. 2011). Although in that study 100 % recovery

efficiency was reported, no sequencing results were shown.

Given thehigh identity of the rDNAsequence inCoccidioides spp.

and closely related species of the Onygenaceae family (Millar

et al. 2003), it would be ultimately desirable to have

sequencing confirmatory results to avoid false identification.

MostVDP, SJZ andE soil sampleshad less genomicDNA than

that reported previously for environments with higher mois-

ture content (Miller et al. 1999; Fierer et al. 2007).We believe that

this could provide a significant clue about the quantity of

microorganisms adapted to extreme habitats.

Our BLAST analysis with the obtained sequences identified

with similar score values C. immitis and C. posadasii. Previous

biogeographical evidence regarding the spatial distribution of

Coccidioides spp. in America, has shown that C. immitis, formerly

called Californian-strain, is exclusively found in San Joaquin

Valley in southern California, and eastern Arizona, whereas

C. posadasii is distributed in the rest of arid lands in the

southwest United States, northern Mexico and endemic areas

in SouthAmerica (Fisher et al. 2001; Barker et al. 2007). An earlier

study reported isolation of C. immitis from soil samples from

VDP (Cairns et al. 2000). However, only that species of Cocci-

dioides was known at that time. The ITS2 sequenced region is

useful for basic taxonomic identification, but not to differen-

tiate between species of the same genus. To distinguish one


Fig 6 e Satellite images showing the sampling sites in Valle de las Palmas and San Jos�e de la Zorra. (A) Landsat image with

general overview of VDP (courtesy of Landsat image archive, glovis.usgs.gov). (B) High resolution 2008 satellite image with

polygon enclosing the sites sampled for Coccidioides (image courtesy of Digital Globe Image Library, accessed through Image

Connect. www.digitalglobe.com). (C) Landsat image with general overview of SJZ (courtesy of Landsat image archive, glovis.

usgs.gov). (D) High resolution 2008 satellite image with polygon enclosing the sites sampled for Coccidioides (image courtesy

of Digital Globe Image Library, accessed through Image Connect. www.digitalglobe.com).


species from the other and to conduct a biogeographical anal-

ysis would require analyzing defined microsatellite-containing

loci and mutations in genes encoding fungal enzymes.

The spatial distribution of Coccidioides spp. in VDP is very

similar to that observed in highly endemic areas of the San

Joaquin Valley basin in southern California and the Santa Cruz

basin in southern Arizona, which suggests a probable prefer-

ential spatial distribution of Coccidioides in vegetation corridors

along intermittent or dry water courses. We did not find Larrea

tridentata, whose presence near positive isolation points has

been historically linked to this fungal disease (Maddy &

Crecelius 1967; Swatek 1970).

It is widely accepted that the soil is the natural habitat for

Coccidioides spp. (Barker et al. 2007; Fisher et al. 2007). None-

theless, since Coccidioideswas once described as a soil saprobe,


many hypotheses have been postulated to explain the spatial

distribution of the fungus apparently limited to arid lands of

the America continent. Some authors, based on fungal

isolations from six different heteromyids’ species, accepted

the correlation between this fungal pathogen and small desert

mammals (Stewart & Meyer, 1932; Emmons 1942; Swatek

1970). More recent evidence suggested that soil enrichment

with animal organic material supports the development of

different fungal pathogens (Hawkins 1996). Accordingly,

organic debris such as animal carcasses could well support

the life cycle of Coccidioides. Previous analyses confirmed that

spherules and endospores exposed to biological fluids survive

a long time in extreme environmental conditions. However,

this theory has neither been confirmed nor refuted because of

the scarce available information and low rates of isolation of


http://glovis.usgs.gov

http://www.digitalglobe.com



http://www.digitalglobe.com

Fig 7 e Landscape, heteromyids and vegetation of the sampling area in Valle de las Palmas. (A, B) The characteristic

landscape of VDP in Jan. 2009 and Jun. 2010, respectively. (C) Example of an active burrow sampled. (D) Example of a larger

den sampled. (E) Dipodomys simulans. (F) Chaetodipus fallax. (G) Eriogonium fasciculatum (Coastal California Buckwheat).

(H) Prosopis glandulosa (Honey Mesquite).


Coccidioides in desert environments after extensive sampling

(Maddy & Crecelius 1967; Swatek et al. 1967; Lacy & Swatek

1974; Greene et al. 2000). Historical evidence suggests that

the highest isolation rates are correlated with heteromyids

burrows (Egeberg & Ely 1956). It is well known that the

microenvironmental conditions in desert burrows provide the

ideal conditions for fungal development (Reichman et al. 1985;

Hawkins 1996). We found no correlation between sample type

(burrow, large den, soil or letrine) and presence or absence of

Coccidioides spp. in VDP and SJZ. However, both positive loca-

tions had abundant active burrows, indicative of high rodent

presence, which was confirmed by several capturing studies

conducted later (data not shown).

Among Coccidioides animal infection, wild rodents are

probably themost reported in the literature. Previous research


found positive isolation in several species of mice and rats. Of

these, higher prevalence was found in kangaroo rat

(Dipodomys merriami with 17 %) and desert pocket mice

(Chaetodipus penicillatus, previously known as Perognathus

penicillatuswith 15 % prevalence). Other species reported were

Citellus baileyi, Citellus intermedius, Citellus formosus, as well as

Citellus leucurus (ground squirrel) and grasshopper mouse

(Onychomys torridus) (Emmons 1942; Emmons & Ashburn 1942;

Emmons 1943; Ajello 1967). The species described in this

study, D. simulans and C. fallax are taxonomically closely

related to these species (http://pir.uniprot.org/taxonomy/).

Dipodomys spp. constitute 80 % of the desert mammal biota

in the Sonora desert ecosystem. Heteromyids communities’

research documented a population succession to Perognathus

spp. during the rainy season and desert blossoms (Mares


http://pir.uniprot.org/taxonomy/


1981). The biomass increase and the subsequent integration to

the ecosystem by action of detritivores and decomposers,

enhance the availability of C and N sources in soil hotspots.

This could explain the correlation between the incidence of

Coccidioides and weather conditions in highly endemic regions

described in previous bioclimatic analyses (Kolivras & Comrie

2004; Park et al. 2005; Zender & Talamantes 2006). Presumably,

soil moisture increase and, therefore, the water availability in

the microenvironment of the endemic areas could be one of

the important consequences of weather changes contributing

to Coccidioides spp. growth. An alternating sequence of wetting

and drying of soils leads to fungal growth and spore formation

(Comrie & Glueck 2007). Recent reviews suggest that warm

and dry summers in combination with heavy wintertime

precipitation in desert-mediterranean ecotone provide

optimal conditions for infectious fungal spore propagation

(Greer et al. 2008). We found Coccidioides spp. in soil samples of

all the sampled years, including the winter sampling in Jan.

2009 in VDP. The ecological and meteorological changes

related to seasonal climate variabilitymay affect the structure

of soil microbial populations, so that better environmentally

fitted fungal pathogens survive and propagate.

Conclusion

Positive environmental samples belonged to a transition biome

between Mediterranean and desert with dry ombrothype and

high soil evapotranspiration. This study supports the link

between Coccidioides spp. ecological niche and a rain-dry

seasonal pattern and suggests an important correlation

between regional climate and the epidemiology of this fungal

disease. Previous reports linked coccidioidomycosis spread to

increased rain associated to El Ni~no/Southern Oscillation

(ENSO) conditions, with drought conditions preceded by flood-

ing (Colwell & Patz 1998). In our study period only the winters

2006e2007 and 2009e2010 were characterized by El Ni~no

conditions; as can be seen in Fig 3, non-El Ni~no winter like

2008e2009, can be also characterized by high rainfall in the

study area, and therefore may favour the incidence of the

fungus. Moreover, under global warming more extreme events

may becomemore frequent (Meehl et al. 2007), and the arid and

semi-arid regions of the subtropics may become more arid

(Seager et al. 2007); therefore, these scenarios of extreme

conditions may increase the risk of hazards such as coccidioi-

domycosis (e.g., Bernard et al. 2001; Gamble et al. 2008). Regional

warming, rain season changes and other meteorological

phenomena can be expected to modify the spatial distribution,

timing and natural hosts for this fungal diseases (Kolivras &

Comrie 2004; English et al. 2009). This is a major concern to

public health, because the Western North American region is

extremely vulnerable to the impacts resulting from global

climate changes, which include an increase in the frequency of

extreme weather events with high temperatures, longer

droughtperiodsandan increase inextremeprecipitationevents

associatedwithmonsoon (Patz&Kovats 2002; Diffenbaugh et al.

2008). Altogether, these changes provide a scenario with highly

favourable conditions for a spatial redistribution of the known

endemic areas for coccidioidomycosis and subsequent changes

in the incidence rates of the disease.


Acknowledgements

This research was supported by a grant from CONACyT-

SEMARNAT 2006-1 23479. We would like to thank M. Orbach

and B. Barker from the University of Arizona, Tucson,

D. Pappagianis from the University of California Davis, and

G. Gonzalez from the School of Medicine at the Autonomous

University of Nuevo Le�on, M�exico, for helpful discussions and

unpublished information. We are grateful to A. Guillen

Gonz�alez and E. Flores Rojas, for technical field assistance.

Supplementary material

Supplementary material related to this article can be found

online at doi:10.1016/j.funeco.2011.08.004.

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