Pneumocystis oryctolagi sp. nov., an uncultured fungus causingpneumonia in rabbits atweaning: reviewof current knowledge,anddescriptionofa new taxonongenotypic, phylogenetic andphenotypic basesEduardo Dei-Cas1,2, Magali Chabe1,3, Raya Moukhlis4, Isabelle Durand-Joly1,2, El Moukhtar Aliouat1,3,James R. Stringer5, Melanie Cushion6, Christophe Noel7,8, G. Sybren de Hoog9,10, Jacques Guillot11 &Eric Viscogliosi8

1ECOPA (EA 3609), Lille Pasteur Institute, Lille, France; 2Parasitology-Mycology Service (EA 3609), Lille 2 University Hospital Centre, Lille, France;3Laboratory of Parasitology-Mycology (EA 3609), Faculty of Pharmacy of Lille, Lille, France; 4Parasitology Service, St-Antoine University Hospital Centre,

Paris, France; 5Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA;6Department of Internal Medicine, Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH, USA; 7School of Biology,

Institute for Research on Environment and Sustainability, University of Newcastle upon Tyne, Newcastle upon Tyne, UK; 8INSERM U547, IFR17, Lille

Pasteur Institute, Lille, France; 9Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; 10Institute of Biodiversity and Ecosystem Dynamics,

University of Amsterdam, Amsterdam, The Netherlands; and 11Parasitology-Mycology Service, UMR BIPAR, National School Veterinary of Alfort,

Maisons-Alfort, France

Correspondence: Eduardo Dei-Cas, Lille

Pasteur Institute, 1 rue du Professeur

Calmette, BP 245, 59019 Lille Cedex, France.

Tel.: 133 320877155; fax: 133 320877224;

e-mail: eduardo.dei-cas@pasteur-lille.fr

Received 13 March 2006; revised 18 May 2006;

accepted 25 May 2006.

First published online 17 August 2006.

DOI:10.1111/j.1574-6976.2006.00037.x

Editor: Graham Coombs

Keywords

Pneumocystis ; pneumocystosis; Pneumocystis

taxonomy; Pneumocystis phylogeny;

Pneumocystis morphology; Pneumocystis

oryctolagi .

Abstract

The genus Pneumocystis comprises noncultivable, highly diversified fungal patho-

gens dwelling in the lungs of mammals. The genus includes numerous host-

species-specific species that are able to induce severe pneumonitis, especially in

severely immunocompromised hosts. Pneumocystis organisms attach specifically

to type-1 epithelial alveolar cells, showing a high level of subtle and efficient

adaptation to the alveolar microenvironment. Pneumocystis species show little

difference at the light microscopy level but DNA sequences of Pneumocystis from

humans, other primates, rodents, rabbits, insectivores and other mammals present

a host-species-related marked divergence. Consistently, selective infectivity could

be proven by cross-infection experiments. Furthermore, phylogeny among primate

Pneumocystis species was correlated with the phylogeny of their hosts. This

observation suggested that cophylogeny could explain both the current distribu-

tion of pathogens in their hosts and the speciation. Thus, molecular, ultrastructur-

al and biological differences among organisms from different mammals strengthen

the view of multiple species existing within the genus Pneumocystis. The following

species were subsequently described: Pneumocystis jirovecii in humans, Pneumo-

cystis carinii and Pneumocystis wakefieldiae in rats, and Pneumocystis murina in

mice. The present work focuses on Pneumocystis oryctolagi sp. nov. from Old-

World rabbits. This new species has been described on the basis of both biological

and phylogenetic species concepts.

Introduction

Pneumocystis is a group of organisms assigned to the Fungal

Kingdom (Edman et al., 1988, 1989; Wakefield et al., 1992;

Calderon-Sandubete et al., 2002). The genus comprises

pathogens dwelling in the lungs of terrestrial, aerial and

aquatic mammals (Laakkonen et al., 1993, Laakkonen &

Sukura, 1997; Laakkonen, 1998, 2001; Mazars et al., 1997b;

Guillot et al., 1999, 2001; Durand-Joly et al., 2000; Demanche

et al., 2001, 2003). Occasionally they induce severe pneumo-

nitis, particularly in hosts with severe impairment of the

immune system. In such hosts, Pneumocystis species develop

progressively and may fill pulmonary alveolar cavities, a

process that leads to respiratory failure (Dei-Cas, 2000).

The highly ubiquitous occurrence and the marked patho-

genic potential of Pneumocystis species, especially of the

human-associated Pneumocystis jirovecii, has stimulated a

growing interest in these peculiar microfungi. On the

FEMS Microbiol Rev 30 (2006) 853–871 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

basis of morphological, phylogenetic and experimental

approaches we are now beginning to realize that Pneumo-

cystis constitutes a highly diversified biological group, with

numerous species that are host-specific and well adapted to

live inside the lungs of a great diversity of mammal species

(Guillot et al., 2001; Hugot et al., 2003).

Ultrastructural studies have shown that Pneumocystis

species from diverse mammals, especially the trophic forms

(formerly called ‘trophozoites’), attach specifically to type-1

epithelial alveolar cells (Dei-Cas et al., 2004). Trophic forms

emit cytoplasmic expansions or filopodia that vary in

thickness depending on the species (Dei-Cas et al., 1994,

2004; Mazars & Dei-Cas, 1998; Nielsen et al., 1998).

Filopodia may penetrate deeply into the cytoplasm of the

host cell (Dei-Cas et al., 1991). However, no disruption of

host cell membrane results from either attachment or

filopodial activity. In addition, no structural or functional

host cell alteration was found in in vitro or in vitro studies

using transmission electron-microscopy (TEM) (Dei-Cas

et al., 1991, 2004; Settnes & Nielsen, 1991; Aliouat et al.,

1993a), confocal microscopy (unpublished) or exploring the

alveolar epithelium cytophysiology (Beck et al., 1998).

Molecular genetic studies have revealed that Pneumocystis

gene sequences present a marked divergence with the host

species concerned. Numerous gene fragments were com-

pared in Pneumocystis from humans and other primates,

rodents, rabbits and insectivores. Consistently it was found

that specific sequences could be attributed to pathogens

from different host species (Banerji et al., 1995; Mazars et al.,

1995; Laakkonen, 1998; Wakefield et al., 1998; Denis et al.,

2000; Durand-Joly et al., 2000; Guillot et al., 2001). Karyo-

typic divergence was found among Pneumocystis strains

from diverse hosts (Keely et al., 2004). A multilocus enzyme

electrophoresis (MLEE) approach showed that laboratory

rats, mice and rabbits harbor dramatically different cate-

gories of Pneumocystis genotypes. The high level of linkage

disequilibrium found in this study suggested that Pneumo-

cystis genotypes from different hosts have been genetically

isolated from each other for a very long time (Mazars et al.,

1997a; Mazars & Dei-Cas, 1998). The evidence of robust

Pneumocystis genetic heterogeneity (Dei-Cas et al., 1998b)

has led to the replacement of ‘formae speciales’ by genuine

species (Redhead et al., 2006). The rabbit-associated organ-

ism until now has been referred to as Pneumocystis carinii

f.sp. oryctolagi (Anonymous, 1994).

A more recent study in primates showed that large

subunit of mitochondrial ribosomal DNA (mtLSU-rDNA)

sequence divergence among Pneumocystis species was corre-

lated with the phylogeny of their hosts (Demanche et al.,

2001). This observation, which could be extended to other

mammals (Demanche et al., 2001; Guillot et al., 2001),

suggested that cophylogeny can explain the current distribu-

tion of pathogens in their hosts. In order to test this

hypothesis, aligned DNA sequences of three genes [dehy-

dropteroate synthetase (DHPS), mtLSU-rRNA and small

subunit of mitochondrial ribosomal RNA (mtSSU-rRNA)]

from strains originating from 20 primate species were

subjected to separate phylogenetic analyses, and then com-

bined in a single data set (Hugot et al., 2003). At least 61%,

and perhaps as much as 77%, of the homologous nodes of

the cladograms of hosts and pathogens may be interpreted

as resulting from codivergence events (Hugot et al., 2003).

Coevolution of Pneumocystis species and their hosts could

explain both the remarkable adaptation of these pathogens

to the alveolar environment and the close host specificity of

Pneumocystis, which was proven by cross-infection experi-

ments (Gigliotti et al., 1993; Aliouat et al., 1993b, 1994;

Mazars &Dei-Cas, 1998; Dei-Cas et al., 1998b; Atzori et al.,

1999; Durand-Joly et al., 2002).

The high divergence among Pneumocystis species, prob-

ably resulting from a prolonged process of coevolution with

each mammal host and mostly associated with cospeciation

(Hugot et al., 2003), is consistent with the marked pheno-

typic divergence recently reported to exist among Pneumo-

cystis species from diverse mammals (e.g. the Pneumocystis

species selective infectivity). Pneumocystis species show little

difference at the light microscopy level, but host species-

related divergence was found using TEM (Dei-Cas et al.,

1994, 2004; Nielsen et al., 1998). Further differences were

found in growth rates (Aliouat et al., 1999) and in vitro

behaviour (Aliouat et al., 1993a). For instance, rat-derived

Pneumocystis seemed to have a higher capacity for attaching

in vitro to target cells than mouse-derived pathogens, and

in vitro attachment of rat Pneumocystis seemed to be more

sensitive to pentamidine or cytochalasin-B than attachment

of mouse-derived organisms (Aliouat et al., 1993a).

Combining the above data, the presence of host specific

ultrastructural and biological differences among Pneumo-

cystis species strengthen the view of multiple species existing

within the genus Pneumocystis (Stringer et al., 2001). The

following species were subsequently described: Pneumocystis

carinii Frenkel, P. jirovecii Frenkel, Pneumocystis wakefieldiae

Cushion et al, and Pneumocystis murina Keely et al. (Frenkel,

1999; Cushion et al., 2004; Keely et al., 2004). As the genus

was assigned to the fungal kingdom, these species were

described according to rules of the International Code of

Botanical Nomenclature (ICBN).

The present paper focuses on a Pneumocystis species

identified in meat-, laboratory and wild rabbits (Oryctolagus

cuniculus) from the Old World. Genomic, isoenzymatic,

ultrastructural, and biological data, obtained mostly in

France during the past 15 years, made it possible to

distinguish rabbit-derived Pneumocystis from species or

formae speciales originating from other mammals. From

this work it became clear that rabbit-derived Pneumocystis

belongs to a hitherto undescribed species. Genotypic,

FEMS Microbiol Rev 30 (2006) 853–871c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

854 E. Dei-Cas et al.

phylogenetic and phenotypic bases for designating this new

species are summarized.

How to set about doing research onrabbit-associated Pneumocystis

Sources of rabbit-associated Pneumocystis

Rabbits hosts were obtained from the following sources: (a)

European suppliers of laboratory animals (Charles River,

Rouen, France; Iffa Credo, Lyon, France; BioMerieux, Lyon,

France; Harlan, Oxon, United Kingdom; Harlan, Zeist, The

Netherlands; Rabbit Pathology Unit, INRA, Nouzilly France;

Vasseur from Prouzel, Barrois from Nord-Pas de Calais,

France), (b) breeders of meat-rabbits (Deregnaucourt and

Pollet from Nord-Pas de Calais), and (c) wild rabbits

trapped in France and Spain. Most domestic animals were

California-New Zealand hybrid rabbits, but Dutch or

Chinchilla rabbits (from Harlan, UK and The Netherlands)

have also been used. In addition, rabbits from colonies

maintained under isolated conditions on farms in different

regions in France were used, especially for rabbit Pneumo-

cystis population genetics approaches. Strains and regions

were as follows: ‘Hollandais’, ‘brun marron’ and ‘Rex’ from

Alsatian farms; ‘Blanc de Bouscat’ from Verdun farms; ‘Nain

de Couleur’ and ‘Polonais aux yeux roses’ from Var farms;

‘Argente de Champagne’ from Saone et Loire; ‘California’

from Dordogne; ‘Sables des Vosges’ from Bas-Rhin, and

outbred rabbits from the cities of Boulogne (Pas de Calais)

and Rodez (Aveyron).

The challenge of Pneumocystis -free rabbits

Studies on Pneumocystis pneumonia (PcP) associated pul-

monary surfactant changes (Aliouat et al., 1998) and on

local immune response against Pneumocystis infection (Al-

laert et al., 1996, 1997; Rajagopalan-Levasseur et al., 1998)

have stimulated a growing need of Pneumocystis-free rabbits

(i.e., with repeated negative PCR results and/or no PcP

development after continuous corticosteroid administra-

tion) to be used as control. These were obtained from the

Rabbit Pathology Unit (INRA, Nouzilly, France) by combin-

ing prolonged cotrimoxazole administration with careful

microbial isolation measures (Cere et al., 1997b).

Sampling procedures

Pulmonary material to detect Pneumocystis was sampled

using noninvasive terminal broncho-alveolar lavage (BAL)

or post-mortem homogenization of lungs. Another non-

invasive method consisted of gently rinsing the nasal cavities

with sterile saline. This method allows the rabbits to be kept

alive, and was inspired by the success of noninvasive

sampling of nasopharyngeal aspirates from small children

(Nevez et al., 2001; Vargas et al., 2001). In rabbits, the nasal

cavities were rinsed with 1–2 mL of a sterile NaCl 0.9%

aqueous solution, using 16G� 200 I.V. catheters (Terumo

Europe N.V., Leuven, Belgium). Secretions were collected in

15 mL capped sterile tubes. After sampling, nasal wash fluids

were put on ice for transport to the laboratory. Then, the

samples were centrifuged at 2900 g for 10 min at 4 1C. The

supernatants were removed and the pellet stored at � 80 1C

until DNA extraction and Pneumocystis DNA detection by

nested PCR at the mtLSU-rDNA locus (see below) (Wake-

field, 1996).

Other sampling methods were performed after rabbit

euthanasia by pentobarbital irreversible anesthesia. For

terminal BAL the trachea was cannulated and the lungs were

rinsed five times with 10 mL of sterile NaCl 0.9% solution.

Fluid recovered was pooled on crushed ice and centrifuged

at 2900 g for 10 min at 4 1C to pellet cells. Pellets and

supernatants were stored separately at � 80 1C. Terminal

BAL fluid (BALF) samples were used as source of pulmon-

ary surfactant (Aliouat et al., 1998), alveolar macrophages

(Allaert et al., 1996) or of host-cell RNA in rabbit-PcP

immunology studies (Allaert et al., 1997). Finally, postmor-

tem sampling of rabbit lungs, which is described in the next

section, was used either to evaluate the kinetics of Pneumo-

cystis infection (Soulez et al., 1989; Dei-Cas et al., 1990b;

Aliouat et al., 1998, 1999) or as source of Pneumocystis

antigen for immunofluorescence (IFA) and Western-blot

assays (Soulez et al., 1988, 1989; Dei-Cas et al., 1990b).

Staining Pneumocystis organisms for lightmicroscopy

Rabbit-derived Pneumocystis organisms were usually de-

tected in lung impression smears (Dei-Cas et al., 1989,

1990a; Soulez et al., 1989), lung-homogenate air-dried

smears (Soulez et al., 1989, 1991; Rajagopalan-Levasseur

et al., 1998) or BALF samples (Aliouat et al., 1998).

Although rabbit pathogens can be identified using phase

contrast microscopy (Rajagopalan-Levasseur et al., 1998),

like other Pneumocystis species (Dei-Cas et al., 2004),

current detection was made using toluidine blue O (TBO)

(Chalvardjian & Grawe, 1963), Gomori-Grocott’s methena-

mine silver nitrate (GMS) (Grocott, 1955; Rajagopalan-

Levasseur et al., 1998), and methanol-Giemsa or Giemsa-

like stains with similar cytological affinities, such as the

RAL-555 kit (Reactifs RAL, Paris, France) (Cushion et al.,

1988; Soulez et al., 1988, 1991; Dei-Cas & Cailliez, 1996,

1998a). Additionally, Pneumocystis-specific fluorescein-la-

belled antibodies have helped to identify Pneumocystis

organisms in impression smears or lung-homogenate

air-dried smears (Soulez et al., 1988).

In order to approach lung tissue changes associated with

rabbit pneumocystosis (Dei-Cas et al., 1990b; Rajagopalan-

FEMS Microbiol Rev 30 (2006) 853–871 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

855Pneumocystis oryctolagi sp. nov. from rabbits

Levasseur et al., 1998), conventional histological methods

have also been used, as described in detail elsewhere (Creusy

et al., 1996; Dei-Cas et al., 1998a). In rabbit lung sections

Pneumocystis cystic forms were detected using TBO, GMS or

even Periodic Acid Schiff (PAS) stains (Emmons et al., 1977;

Dei-Cas et al., 1998a).

Fixing Pneumocystis organisms forultrastructural study

The effect of a large range of osmolarities of fixative and

washing solutions (190–2580 mOsm) on the structural pre-

servation of Pneumocystis cells was tested in our laboratory

(Palluault et al., 1992a). Tests were made on rabbit-derived

Pneumocystis, and revealed that high osmolarity

(850–1300 mOsm) of fixative and washing solutions was a

critical condition for obtaining well-preserved Pneumocystis

cytoplasmic structures. The following protocol was found to

be effective for fixation of Pneumocystis-infected lung sam-

ples from rabbits, and was therefore used in this work: (1)

fixation with a phosphate-buffered 2.5% glutaraldehyde

solution (0.1 M pH 7.5) adjusted to about 700 mOsm by

the addition of 0.18 M NaCl; (2) many washings with 0.1 M

phosphate buffer (same osmolarity); (3) postfixation for 1 h

in a 1%-osmium tetroxide solution in phosphate buffer,

dehydration in ethanol, and embedding in Epon (Dei-Cas

et al., 1998a). Finally, 2D images from serial ultra-thin

sections were used to reconstruct 3D images of two Pneu-

mocystis life cycle stages (trophic form and intermediary

sporocyte) (Palluault et al., 1991b, c; Dei-Cas et al., 2004).

How to separate Pneumocystis organisms fromlung tissue

Methods to separate, purify and enumerate Pneumocystis

from rabbit-lung tissue were described previously (Soulez

et al., 1991; Dei-Cas & Cailliez, 1996). Briefly, infected lungs

are cut into small pieces in Dulbecco Modified Eagle’s

Medium (DMEM) and homogenized either by squeezing

them through a stainless steel mesh or using a magnetic

stirrer (4 1C, 90 min) or a Stomacher tissue grinder. The first

two methods are usually employed in order to keep living

pathogens for infectivity or other studies (Soulez et al., 1991;

Aliouat et al., 1993a, b, 1994). A stomacher is currently used

when the aim is simply to evaluate the number of Pneumo-

cystis organisms (Soulez et al., 1991). In all cases, the

homogenate is poured through gauze and centrifuged

(2900 g 10 min 4 1C). The pellet is incubated in a buffered

hemolytic solution (9 : 1 solution of 0.15 M NH4Cl in

20 mM Tris-HCl, 10 min 4 1C), resuspended in DMEM and

filtered successively through 250 and 63mm stainless steel

meshes. The pellet is finally resuspended in DMEM. All

procedures are performed under sterile conditions.

Finally, in order to further reduce host cell debris,

pathogens are suspended in a polysucrose gradient (Histo-

paque-1077, Sigma Chemical Co., L’Isle D’Abeau Chesnes,

France). Polysucrose solution and pathogen suspension are

mixed 1 : 1 (v/v) in a 15 mL sterile tube and centrifuged at

1000 g for 15 min at 4 1C. The band accumulated at the

interface between sterile medium (DMEM or PBS) and

polysucrose solution is collected and washed twice with

sterile medium (2900 g 10 min 4 1C). This supplementary

purification step was employed, for instance, to prepare

rabbit-derived Pneumocystis samples for Western-blot assays

or for pulsed field gel electrophoretic karyotype analysis.

Viable, purified Pneumocystis samples may be used im-

mediately or can be cryopreserved by placing them in fetal

calf serum with 10% dimethyl-sulfoxyde (DMSO) at

� 80 1C in a Nalgene 1 1C Cryo Freezing Container

(Dutscher, Brumath, France) filled with isopropyl alcohol

(cooling rate = 1 1C min�1) (Dei-Cas & Cailliez, 1996).

Then, the pathogen samples are stored in liquid nitrogen.

Under these conditions, Pneumocystis samples remain in-

fectious for at least six years (Durand-Joly et al., 2002).

Counting the Pneumocystis organisms in lungsamples

For organism counts, cystic forms were counted in 2 or 5mL

air-dried smears stained with toluidin blue O (TBO). Dry

smears were fixed with methanol, stained with Giemsa or

RAL555 stains, and used to count the relative number of

trophic forms, sporocyte and cyst stages of Pneumocystis.

The total number of pathogens is calculated as follows:

Total number of pathogens ¼ W þ ðW �%UW=%WÞwhere W is the walled forms (counted on TBO smears); %W

the percentage of walled forms (intermediate sporocytes

1late sporocytes1cysts); %UW the percentage of unwalled

forms (trophic forms1early sporocytes) (Aliouat et al.,

1993b, 1995).

Amplifying and sequencing Pneumocystis DNA

Rabbit lung and nasal wash samples were treated with

proteinase K, and genomic DNA was isolated by a phenol–

chloroform extraction, or using QIAamps DNA minikit

(QIAGEN, Courtaboeuf, France) according to manufac-

turer’s recommendations. Single or nested-PCR, using oli-

gonucleotide primers described in Table 2, were carried out

from rabbit-derived Pneumocystis DNA samples to amplify

portions of the following genes: thymidylate synthase (TS),

mitochondrial large-subunit rRNA (mtLSU-rRNA), mito-

chondrial small-subunit rRNA (mtSSU-rRNA), arom locus,

manganese-dependent superoxyde dismutase (MnSOD),

dihydrofolate reductase (DHFR), dihydropteroate synthase

(DHPS), b-tubulin (b-tub), heat-shock-protein 70

FEMS Microbiol Rev 30 (2006) 853–871c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

856 E. Dei-Cas et al.

(HSP70), and internal transcribed spacer regions (ITS)

(Banerji et al., 1993; Mazars et al., 1995; Hunter & Wake-

field, 1996; Wakefield, 1996; Denis et al., 2000; Ma & Kovacs,

2001).

Amplified PCR products were purified before sequencing

either directly or after cloning. In the latter case, recombi-

nant plasmids were sequenced in both directions with a

model ABI 377 automated sequencer using the Big Dye

Terminator Cycle Sequencing kit (Perkin Elmer-Applied

Biosystems, Foster City, CA), according to the manufac-

turer’s instructions. Sequences used for comparisons were

obtained from the literature and from the GenBank database

(http://www.ncbi.nl.nih.gov/Genbank/GenbankOverview.

html).

Pneumocystis taxonomy at the specieslevel: sequence comparison andphylogenetic analysis

Sequences compared in this study

In order to distinguish rabbit-derived Pneumocystis organ-

isms from the other Pneumocystis species or formae spe-

ciales, portions of the following genes were aligned:

manganese-containing cofactored superoxide dismutase

(SODA), accession nos AF146752, AF146753, Z79785,

AF146751, AF146754; dihydropteroate synthase (DHPS),

accession nos AF322064, AF139132, M86602, AF322065,

U66283, AY070270, AF362762, AF362761, AF362760,

AF362759, AF362758, AF362757; dihydrofolate reductase

(DHFR), accession nos AF186097, AF090368, AF322061,

AF322063, AF175561, AY017418; large-subunit mitochon-

drial rRNA (mtLSU-rRNA), accession nos S42915, S42926,

U20169, U20173, S42921, AF257179, AF362455, AF362462,

AF362461, AF362458, AF362456, AF362464, AF362470,

AF362469, AF362468, AF362467, AF362466, AF362465,

AF362463, AF362460, AF362459, AF362457, AF362454,

AF362453; mtSSU-rRNA, rabbit-derived Pneumocystis,

P. jirovecii, P. carinii, P. wakefieldiae, P.carinii f.sp. mustelae,

P. murina (Hunter & Wakefield, 1996), P. carinii f.sp. macaca

from Indian Macaca rhesus (Durand-Joly et al., 2000),

accession nos AF395579, AF395580, AF395582, AF395584,

AF395578, AF395583, AF395574, AF395585, AF395576,

AF395575, AF395577, AF395581; thymidylate synthase, rab-

bit-derived Pneumocystis, P. jirovecii, P. murina (Mazars

et al., 1995), accession nos S77510; 5-enolpyruvylshiki-

mate-3-phosphate synthase (AROM), accession nos

U31054, U31055, L18918, U31056, U31053; internal tran-

scribed spacer 1 (ITS1), accession nos DQ010098,

AF013806, L27658, AY532651, AF288827; internal tran-

scribed spacer 2 (ITS2), accession nos DQ010098,

AF013821, L27658, AY53651, AF288835; Heat shock

protein 70 (HSP70), accession nos DQ435616, U80970,

U80968, U80969, AY382182; beta-tubulin (b-tub), rabbit-

derived Pneumocystis (this paper), accession nos AF170964,

X62113.

Matrix and phylogenetic tree construction

Sequences were aligned by use of the BioEdit v7.0.1 package

(http://mbio.ncsu.edu:BioEdit/bioedit.html). Introducing a

limited number of gaps optimized the alignments. Ambig-

uous regions in the alignments were not taken into account.

The DNA alignments contained 496, 798, 330, 297, 325, 410,

167 and 283 common positions for SODA, DHPS, DHFR,

TS, AROM, HSP70, mtLSU-rRNA and mtSSU-rRNA, re-

spectively. The protein alignments contained 165, 265, 110,

99, 108 and 136 common residues for SODA, DHPS, DHFR,

TS, AROM and HSP70, respectively. For the phylogenetic

analysis of 18 Pneumocystis taxa (see below), mtLSU-rRNA

and mtSSU-rRNA sequences were concatenated to form a

sequence of 281 nt long. Full-length alignments and sites

used in analyses are available upon request from the

corresponding author.

The relatedness of pairs of aligned sequences for each

individual gene was calculated using the ‘Sequence Identity

Matrix’ option provided in the BioEdit package. The values

were obtained by dividing the number of nucleotide or

residues identities by the total number of positions com-

pared and given in percentages. The pairwise distances (%)

presented in the matrix were calculated for each pair of

aligned sequences as follows: 100�% identity. The ITS and

b-tub sequences were not included in the matrix because of

the low number of available sequences (b-tub) or unam-

biguously alignable common positions (ITS).

Phylogenetic analysis of the mtLSU-rRNA and mtSSU-

rRNA concatenated sequences dataset was carried out using

MrBAYES v3_0b4 (Huelsenbeck & Ronquist, 2001). Baye-

sian analysis was performed using the GTR (general time

reversible) G (gamma distribution of rates with four rate

categories)1I (proportion of invariant sites) model of

sequence evolution, with base frequencies, proportion of

invariant sites and the shape parameter a of the G distribu-

tion estimated from the data. The model used was evaluated

using the likelihood ratio test (LRT) implemented in

MODELTEST v.3.7. (Posada & Crandall, 1998). LRTs in-

dicated that the GTR1I1G model had the best fit to the

data.

Starting trees were random; four simultaneous Markov

chains (three heated, one cold) were run twice for two

million generations, burn-in values were set at 10 000

generations (based on empirical values of stabilizing like-

lihoods), and trees were sampled every 100 generations.

Bayesian posterior probabilities were calculated using a

Markov chain Monte Carlo sampling approach (Green,

1995) implemented in MrBAYES version 3.0b4.

FEMS Microbiol Rev 30 (2006) 853–871 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

857Pneumocystis oryctolagi sp. nov. from rabbits

Pneumocystis and pneumocystosis inrabbits

Current pneumocystosis of rabbits at weaning

Extensive corticosteroid-induced PcP was reported in rab-

bits (Oryctolagus cuniculus) as early as in the 1950s by

Sheldon (1959). In 1989, however, it was reported that

without corticosteroid administration, rabbits developed

spontaneous PcP at weaning (about 1 month after birth)

(Soulez et al., 1989). This spontaneous, natural Pneumocystis

infection which has been constantly observed in weaning

rabbits of several strains (Mazars et al., 1997a), provokes

lung histopathological changes typical of PcP, often asso-

ciated with blood biochemical abnormalities (Soulez et al.,

1989; Dei-Cas et al., 1990b; Rajagopalan-Levasseur et al.,

1998). The infection evolves during 7–10 days; afterwards,

pathogen levels decrease gradually, becoming very low in 60-

day-old rabbits. Almost all animals recover within 3–4

weeks. The regularity of the pattern of this natural infection

(abrupt onset at the weaning time, extensive diffuse pul-

monary involvement evolving relatively shortly to complete,

spontaneous healing) has allowed the development of

kinetic studies of the host immune response against PcP

(Allaert et al., 1996, 1997). This research was facilitated by

the fact that PcP develops in this model without adminis-

tration of corticosteroids, as these drugs affect the immuno-

logical mechanisms. Corticosteroids also influence the

production and composition of pulmonary surfactant. For

this reason, the corticosteroid-free rabbit model was recently

used to investigate Pneumocystis-surfactant interactions

(Prevost et al., 1997, 1998; Aliouat et al., 1998).

Morphology of rabbit-associated Pneumocystisat the light microscopic level

TBO and GMS, having a good affinity for components of

the cyst wall, stain the cell wall of cystic forms

( = intermediate and late sporocytes plus mature cysts) in

reddish violet or dark brown, respectively. These techniques

are highly sensitive, allowing an easy detection of cystic

forms even at low magnifications (Fig. 1). However, trophic

forms and early sporocytes remain unidentified with these

metachromatic stains (Dei-Cas et al., 2004). Therefore, in

order to detect all the Pneumocystis life cycle stages, metha-

nol-Giemsa or Giemsa-like stains (Fig. 1) have to be

associated with TBO or GMS stains. Giemsa and other

stains with similar cytological affinities, such as Diff Quick

(Cushion et al., 1985) or RAL-555 (Dei-Cas & Cailliez,

1996), cause pinkish-purple stains on the Pneumocystis

nucleus, and blue stains on the cytoplasm. In fact, only

methanol-Giemsa and similar polychrome stains (e.g. RAL-

555 or Diff-Quick) allow the identification of the different

Pneumocystis life-cycle stages (Cushion et al., 1988; Dei-Cas

et al., 2004). These stains also allow Pneumocystis cells to be

distinguished from other organisms (Dei-Cas et al., 1998a).

These dyes do not, however, stain cystic or sporocytic thick

cell walls, which appear like a clear peripheral halo around

the fungus cell.

Trophic forms are irregular in shape and size (2–8 mm

diameter). Their cytoplasm contains a unique, homoge-

nous, well-stained nucleus. A spheroid shape and a usually

easily visible Giemsa-unstained thick cell wall characterize

the 4–7mm cystic forms (intermediate and late sporocytes

and mature cysts). The number of nuclei increased as

organisms proceed in their development from the mono-

nuclear trophic form to the mature cyst, which contains

eight well-individualized mononuclear spores. Thus, there is

one nucleus in the early sporocyte, two to eight nuclei in the

intermediate sporocyte, and eight nuclei in the late spor-

ocyte (Fig. 1) (Dei-Cas et al., 2004).

At the light microscope level, rabbit-derived Pneumocystis

cannot be distinguished unequivocally from other Pneumo-

cystis species. In methanol-Giemsa stained smears, however,

whereas rabbit-associated Pneumocystis forms are usually

well detached from each other, pathogens from rats or

primates constitute often large stacks where the different life

cycle stages are closely clustered (Table 1). Furthermore, in

rabbits with spontaneous PcP the cystic/trophic form ratio

is usually higher (about 0.10–0.15) than in immunosup-

pressed rodents with PcP (about 0.02–0.05) (Table 1).

Finally, histological differences between rabbit and rodent

or even primate pneumocystoses were reported (Creusy

et al., 1996). They involved the pathogens, their location in

the lung tissue and the pattern of the inflammatory response

(Table 1). Host immune status could surely influence the

immune and inflammatory responses to the infection, but

whether one or other specific difference is due to immuno-

depression remains unclear. First, pathogens lined the

alveolar epithelium in rabbits (Fig. 1), whereas in cortico-

steroid-treated rats, SCID mice or AIDS patients, they were

usually more numerous, closely clustered, and located in the

alveolar lumen. Second, pulmonary congestion was less

important in rabbits (Fig. 1) than in murine hosts and AIDS

patients, where it was widespread and severe. Third, no

collagen fibrosis at all was observed in rabbits, though they

had not received corticosteroids, whereas diverse degrees of

interstitial fibrosis were observed in both AIDS patients and

SCID mice. Fourth, among the inflammatory cells, eosino-

phils and plasma cells were observed in rabbits, a fact that

was confirmed using TEM (Fig. 2) (Creusy et al., 1996;

Rajagopalan-Levasseur et al., 1998), and occasionally in

humans with AIDS-related PcP (Fleury-Feith et al., 1989).

Fifth, inflammatory infiltrates were usually diffuse in rodent

and human hosts, while they appeared as clearly delimited

nodular areas scattered in the rabbit lung (Fig. 1), contain-

ing therefore eosinophils and plasma cells. Sixth, the typical

FEMS Microbiol Rev 30 (2006) 853–871c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

858 E. Dei-Cas et al.

Fig. 1. Pneumocystis oryctolagi sp.nov.: morphology and pathology at the light microscope level. (a) Cystic forms in an air-dried lung homogenate smear

stained by TBO. (b) A mature cyst containing eight ascospores is seen close to the nucleus of a host cell. Air-dried lung smear stained by methanol-Giemsa. (c)

Cystic forms (arrowheads) mostly lining an alveolar space. Histological section of the lung of a weaning rabbit stained by TBO (technique for tissue sections).

(d–f) Trophic forms (d), a mononucleate sporocyte (e) and a mature cyst (f) containing eight ascospores. Air-dried lung smears stained by methanol-Giemsa. (g)

Lung of a weaning rabbit with pneumocystosis. Alveolar septa are thickened; macrophages and other inflammatory cells infiltrate mildly the alveolar lumens.

In a severely infected area (at the top, on the right) alveoli are entirely occupied by cell infiltrates. Lung section stained by hematoxylin-eosin stain. (h) Lung of a

weaning rabbit with pneumocystosis. A nodular lesion is clearly observed. Lung section stained by hematoxylin-eosin stain. Bar = 10mm (a–f) or 100mm (g, h).

FEMS Microbiol Rev 30 (2006) 853–871 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

859Pneumocystis oryctolagi sp. nov. from rabbits

eosinophilic foamy honeycomb material, present in rodent

and human hosts, was rarely found in rabbits (Creusy et al.,

1996).

Ultrastructure of rabbit-associatedPneumocystis

Most knowledge about rabbit-derived Pneumocystis cell

structure resulted from studies made in the 1990s thanks to

both significant improvements of TEM-fixation methods

(Palluault et al., 1992a, b), and computer-aided 3D recon-

struction studies (Palluault et al., 1991a, b, c). Interestingly,

these studies revealed distinctive ultrastructural features of

rabbit Pneumocystis (Dei-Cas et al., 1994; Mazars & Dei-Cas,

1998; Nielsen et al., 1998; Durand-Joly et al., 2000). Most

features however can be extended to other Pneumocystis

species. The detailed ultrastructure of rabbit-derived Pneu-

mocystis life cycle stages was reviewed recently (Dei-Cas

et al., 2004). It will be summarized shortly here.

All known rabbit-associated Pneumocystis life cycle stages

were found in the lungs of infected rabbits, though mole-

cular methods suggested pathogens may spread to other

organs (Cere et al., 1997a). The usually accepted Pneumo-

cystis life cycle involves an amoeboid, thin-walled, mono-

nuclear trophic form, which becomes a thick-walled cystic

stage, in which multiple nuclear division leads to the

formation of eight spores (Fig. 2). These forms would be

able to leave the cyst, presumably by a pore-like zone located

at the thickest part of the cyst cell wall, as was suggested for

P. carinii (Itatani, 1994), to attach specifically to type-I

epithelial alveolar cells and to evolve into the cystic stage.

The transition from trophic form to mature cyst occurs

through three consecutive sporocyte stages (early, inter-

mediary and late sporocyte) (Yoshida, 1989; Dei-Cas,

2000). Trophic forms and early sporocytes have a thin cell

wall 20–25 nm thick that consists of only one electron-dense

layer associated with the outer surface of the plasma

membrane (6–7 nm), which extends from the cellular body

to the filopodia or tubular expansions. Therefore, these

structures – which are frequently observed in cross, oblique

or longitudinal sections (Fig. 2) – constantly show cell wall,

plasma membrane and cytoplasm levels.

The smallest trophic forms are round to ellipsoid, appear-

ing as eukaryotic cells 1–2mm in length. Most trophic forms

are, however, larger and very irregular in size (4–8mm long)

and shape (Fig. 2), and present filopodia. Their cytoplasm

contains one nucleus (up to 1mm in diameter) that has a

typical nuclear envelope with clearly visible 55–80 nm nucle-

ar pores (Palluault et al., 1990). The chromatin generally

appears diffuse. As in small trophic forms, the perinuclear

cisterna communicates with well-developed rough (RER) or

smooth (SER) endoplasmic reticulum (Palluault et al., 1990).

The endomembranous system of rabbit-derived Pneumo-

cystis showed two types of endoplasmic structures closely

related to RER, SER, and the Golgi complex. The first, which

was named a type 1 endoplasmic saccule (ES1) (Palluault

et al., 1990), consisted of one or more coiled endoplasmic

saccules that packaged cytoplasm or mitochondria, suggest-

ing autophagic activity. ES1 could therefore be considered as

secondary lysosomes. The second type, which was named a

type 2 endoplasmic saccule (ES2), consisted of a large,

flattened, single endoplasmic saccule present in well-devel-

oped trophic forms and in intermediate sporocytes.

Table 1. Phenotypic differences between rabbit-derived Pneumocystis and other Pneumocystis species

Features Rabbit-derived Pneumocystis P. carinii and P. wakefieldiae P. murina P. jirovecii

Organisms in lung

dry smears (TBO or

Giemsa stains)

Detached from

each other

Closely clustered Clustered Closely clustered

Cystic/trophic form ratio 0.10–0.15 0.02–0.05 0.02–0.05 ND

Location Lining alveolar

epithelium

Filling alveolar

lumen

Filling alveolar

lumen

Filling alveolar

lumen (AIDS) or

lining alveolar

epithelium

(epidemic or

infantile PcP)

In vivo doubling time 1.7 days

(untreated rabbits)

4.5 days (P. carinii

in corticosteroid-

treated rats)

10.5 days (SCID mice) ND

Specific host Rabbit (Oryctolagus cuniculus) Rat (Rattus norvergicus) Mouse (Mus musculus) Man (Homo sapiens)

Intraalveolar eosinophilic

honeycomb material

Rare Present Present Present

Fibrosis Rare Frequent Frequent Frequent

ND, not determined.

FEMS Microbiol Rev 30 (2006) 853–871c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

860 E. Dei-Cas et al.

Although it seems to appear just before nuclear division, its

function remains unknown. Furthermore, the cytoplasm

contains 50–70 mm osmiophilic granules that are probably

lipoid in nature (Palluault et al., 1990).

A single mitochondrion, as shown by ultrastructural 3D

reconstruction, with budding zones occupies an important

volume in the cell (Palluault et al., 1991b). In the inter-

mediate sporocyte the mitochondrion develops active bud-

ding, becoming somewhat tree-like (Palluault et al., 1991c).

The organelle evolves apparently by budding into individual

mitochondrion of spores. Vesicles of Golgian nature (Dei-

Cas et al., 1989, 2004; Palluault et al., 1990) develop by

Fig. 2. Pneumocystis oryctolagi sp.nov.: fungus morphology and associated host cells at the ultrastructural level. (a) Trophic forms and a late sporocyte

where ascospores are being generated. Arrowheads indicate filopodia. The arrow is showing the basement membrane of the alveolar epithelium. (b, c)

An eosinophil leukocyte (b) and a plasmocyte (c), two cell types often associated with rabbit pneumocystosis. AL, alveolar lumen; SP, sporocyte; TF,

trophic form. Bar = 1 mm.

FEMS Microbiol Rev 30 (2006) 853–871 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

861Pneumocystis oryctolagi sp. nov. from rabbits

budding from either endoplasmic saccules or nuclear envel-

ope. Their number increase from about 20 in trophic forms

to about 200 in sporocytes (Palluault et al., 1990), as

organisms proceed in their development from the trophic

form to the intermediate sporocyte stage, suggesting that

this transition is associated with an increased synthesis of

cell wall compounds (glucan, chitin, glycoprotein). The key

event of early to intermediate sporocyte transition is the

development of the thick cell wall (Fig. 2). The mono-

layered cell wall of the early sporocyte becomes thickened by

the appearance of a glucan-rich electron-lucent middle layer

that results in an increase of the cell wall thickness from 40

to 100 nm (Yoshida, 1989; Dei-Cas, 2000).

The mature cyst is about 5mm in diameter, round, and

thick-walled. Its surface is rather smooth, with rare filopo-

dia. It contains a maximum of eight spores (Fig. 1). These

are mononuclear cells, which result from invaginations of

the late sporocyte cell membrane. Spores present a single

mitochondrion, a well-developed rough endoplasmic reti-

culum and an electron-dense one-layered cell wall that is

externally lined by an outer cell membrane (Fig. 2) (De

Stefano et al., 1990; Palluault et al., 1992a). Actually, like P.

carinii, rabbit-associated organisms have an outer mem-

brane (Fig. 2), a structure apparently absent from the cell

wall of other fungi, that appears as a more or less discontin-

uous osmiophilic deposit that lines the trophic-form plasma

membrane or is embedded in the electron-dense outer layer

of thick-walled stages (De Stefano et al., 1990; Palluault

et al., 1992a).

By ultrastructure, rabbit-associated Pneumocystis is not

distinguishable from primate Pneumocystis species (Frenkel,

1976; Durand-Joly et al., 2000) but can be easily distin-

guished from rodent Pneumocystis (Table 1). Most differ-

ences between rabbit and rodent Pneumocystis species

involve filopodia. These typical structures of Pneumocystis

trophic forms are markedly more numerous, thin, and tree-

like in Pneumocystis organisms from mouse than in those

from rabbit, human, or macaque (Dei-Cas et al., 1994, 2004;

Creusy et al., 1996; Nielsen et al., 1998; Durand-Joly et al.,

2000). Filopodia of rat-derived organisms were also found

to be smaller than those from rabbit-derived Pneumocystis.

Additionally, the density and diameter of membrane-limited

electron-dense cytoplasm granules were found to be respec-

tively higher and larger in mouse- than in rabbit-derived

Pneumocystis cells (Nielsen et al., 1998).

Growth rate and host specificity of rabbit-associated Pneumocystis

The growth rates of Pneumocystis species (Aliouat et al.,

1999) and their strong host specificity (stenoxenism)

(Aliouat et al., 1993b, 1994; Dei-Cas et al., 1994, 1998b)

express at best the importance of biological divergence

among Pneumocystis species. Actually, the doubling time of

Pneumocystis organisms developing in the host lung was

highly variable in terms of the host species: 1.7 days for

rabbit-derived Pneumocystis, 4.5 days for rat-derived Pneu-

mocystis and 10.5 days for P. murina growing in SCID mice

(Aliouat et al., 1999). In these experiments, the quantitation

of Pneumocystis organisms was performed microscopically,

as explained in the ‘Methods’ section. Data were plotted in a

semi-logarithmic curve, and doubling time (DT) was calcu-

lated at the exponential phase as follows: DT = ln 2 m�1,

where m represents the specific growth rate (i.e. slope of the

curve) (Aliouat et al., 1999).

The strong host specificity of Pneumocystis species was

demonstrated in cross-infection experiments that aimed to

establish to what extent host species-related genetic varia-

tion among Pneumocystis isolates entailed restricted infec-

tious power. Pneumocystis-free SCID mice (infected by nasal

route) and Nude rats (infected by tracheal route) have been

used as experimental hosts in most experiments (Gigliotti

et al., 1993; Furuta et al., 1993; Aliouat et al., 1993b, 1994;

Durand-Joly et al., 2002). Inocula were pathogens isolated

from rabbits, rats, mice, ferrets, monkeys or humans. In

these experiments, rabbit-associated Pneumocystis, like iso-

lates of the other nonrodent mammals, were not able to

develop in the mentioned deeply immunosuppressed ex-

perimental hosts. A highly sensitive PCR assay followed by

hybridization with DNA-probes specific to each Pneumo-

cystis species (Aliouat, 1995) was used but no Pneumocystis

organism was detected in nonspecific hosts (mouse to rat-

derived organisms) as soon as 3 days postinfection. Only

mouse-derived Pneumocystis developed in SCID mice, and

only rat-derived Pneumocystis developed in Nude rats

(Aliouat et al., 1993b, 1994; Furuta et al., 1993; Gigliotti

et al., 1993; Dei-Cas et al., 1998b; Wakefield et al., 1998;

Durand-Joly et al., 2002). These observations attested that

Pneumocystis host specificity is an all-or-none event. In

short, until now, cross-infection experiments have shown

that Pneumocystis organisms from a given mammal cannot

infect hosts of another species. Host-species restriction, as

established further by molecular identification studies on

Pneumocystis species in domestic or wild mammals (Wake-

field et al., 1992, 1997; Peters et al., 1994a, b; Banerji et al.,

1995; Mazars et al., 1995, 1997b; Bishop et al., 1997; Guillot

et al., 1999, 2001; Denis et al., 2000; Durand-Joly et al., 2000;

Demanche et al., 2001; English et al., 2001; Hugot et al.,

2003) seems therefore to be a universal feature of Pneumo-

cystis species. One of the few exceptions was the discovery of

macaque-derived Pneumocystis strains, likely to be different

species (as mean mtLSU-rDNA sequence divergence was

4.3� 1.4%), recovered from both Macaca mulatta (rhesus

macaque) and M. fascicularis (cynomolgus macaque) (Guil-

lot et al., 2004). However, this apparent transgression of the

species barrier might indicate that rhesus and cynomolgus

FEMS Microbiol Rev 30 (2006) 853–871c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

862 E. Dei-Cas et al.

macaques are closely related and may belong to a single

species. Actually, rhesus and cynomolgus monkeys might

have had a large overlapping geographic distribution in the

past (Fooden, 1980). Consistently, hybrids between rhesus

and cynomolgus monkeys have been reported in Thailand

(Fooden, 1964) and in captivity, and are known to be fertile

(Bernstein et al., 1980). There is also some phylogenetic

evidence of hybridization between M. fascicularis and M.

mulatta (Tosi et al., 2000), though mitochondrial DNA

analyses indicate these two species are separated (Hayasaka

et al., 1996; Morales & Melnick, 1998).

Species-specific gene sequences in rabbit-associated Pneumocystis

All studied DNA sequences of rabbit-associated Pneumocys-

tis were found to be significantly different from the homo-

logous fragments of the other known Pneumocystis species

and formae speciales. Targeted genes are shown in Table 2,

with PCR primers used, and Table 3, showing the pairwise

distances (%) of eight Pneumocystis genes among the

Pneumocystis species and formae speciales. ITS1 and ITS2

loci and b-tub genes were not included in the matrix

because the number of sequences available was too small

(b-tub) or because the variability of sequences was too high

(ITS). Considering the compared gene portions, the extent

of genetic divergence at the mtLSU-rDNA locus ranged

from 18% (between rabbit-derived Pneumocystis and P.

jirovecii) to 26.4% (between rabbit-derived Pneumocystis

and P. carinii) (Table 3). Distances reported previously were

comparable (Wakefield, 1998; Durand-Joly et al., 2000). In a

more recent study, divergences at the same locus were 18.1%

between rabbit-derived Pneumocystis and P. jirovecii, and

29.1% between rabbit-derived Pneumocystis and P. carinii

(E. Dei-Cas & A.E. Wakefield, unpublished). At the arom

locus 25% divergence was found between rabbit-derived

Pneumocystis and P. carinii at the deduced amino acid

sequence level (present study and Banerji et al., 1995).

Table 2. List of explored rabbit-derived Pneumocystis genes

Genes Primers Accession number References

TS Sense: 50-ATTTATGGGTTTCAATGG-30 Unpublished Mazars et al. (1995)

Antisense: 50-TGCAATATTAAAGGGAAC-3 0

mtLSU-rDNA First round PCR: S42915 Wakefield (1996)

Sense: H 50-GTGTACGTTGCAAAGTACTC -30

Antisense: E 50-GATGGCTGTTTCCAAGCCCA -30

Second round PCR:

Sense: X 50-GTGAAATACAAATCGGACTAGG-30

Antisense: Y 50-TCACTTAATATTAATTGGGGAGC-3 0

mtSSU-rDNA Sense: pAZ112-10 F 50-TAGACGGTCACAGAGATCAG-30 Unpublished Hunter & Wakefield (1996)

Antisense: pAZ112-10 R 50-GAACGATTACTAGCAATTCC-30

DHFR Sense: 50-ATGAATCAGCAAAAGTCTTTAACATTGATTGTT-30 AF186097 Ma et al. (2001)

Antisense: 50-TTATAAATCTCTTGTCCACATTTCGAATTC-30

DHPS Sense: 50-GTTAATCCTGGTATTAAACCAGTTTTGCCATT-30 AF322064 Ma et al. (2001)

Antisense: 50-TCTTGAAACTTTATACATTTCATAAACA-30

Arom locus First round PCR: U31054 Banerji et al. (1993)

Sense: 50-(T,C)TNGGNAA(T,C)GCNGGNACNGC-3 0

Antisense: 50-(G,C)(A,T)(T,C)TTICCIGCI(G,C)CIC(G,T)CAT-30

Second round PCR:

Sense: 50-GGGAATTCATATGGA(G,A)(T,C)CAATGACNGAT(G,A)C-3 0

Antisense: 50-GGGAATTCATCCCACCAN(T,C)(A,C)NGGCCA-30

HSP70 Sense: 50-GATGAAAGAATTAGCAGAAACTAA-3 0 DQ435616 Chabe et al. (2004)

Antisense: 50-CTTCTCCTCCTAAATGTGTATC-3 0

Sense: 50- TTGAGAAAGCAATTGGTATT-30

Antisense: 50- CTGCTGCAGTAGGCTCATTG-30

Mn SOD Sense: 50-TGTTTTACCAAGTCTTCCTTATGATTATCA-30 AF146752 Denis et al. (2000)

Antisense: 50-TGATTCATAACTTTCCAATT-30

ITS First round PCR: Unpublished Present work

Sense: 50-AGTTGATCAAATTTGGTCATTTAGAG-30

Antisense: 50-CTCGGACGAGGATCCTCGCC-30

Second round PCR:

Sense: 50-TTCCGTAGGTGAACCTGCG-3 0

Antisense: 50-CTGATTTGAGATTAAAATTCTTG-3 0

b-tub Sense: 50-TGGGCAAAAGGGCATTATAC-3 0 Unpublished Present work

Antisense: 50-GTAATACCACTCATTACTGC-30

FEMS Microbiol Rev 30 (2006) 853–871 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

863Pneumocystis oryctolagi sp. nov. from rabbits

Divergence between rabbit-derived Pneumocystis and P.

jirovecii was similar at the deduced amino acid sequence

level of DHFR locus (25%), and higher (38–39%), at the

same locus, between rabbit-derived Pneumocystis and P.

carinii (present study and Ma et al., 2001). Over 99 deduced

amino acids of a fragment of the TS locus, divergence was

lower: 14.2% between rabbit-derived Pneumocystis and P.

carinii, and 9.1% between rabbit-derived Pneumocystis and

P. jirovecii (present study and Mazars et al., 1995). In all the

cases, levels of divergence are indicative of species-level

variation (Stringer, 1996).

No gene flow between rabbit- and rodent-associated Pneumocystis species

In an early study investigating the genetic diversity of

Pneumocystis organisms isolated from rabbits, mice and rats,

multilocus enzyme electrophoresis (MEE) was used to

analyse five Pneumocystis enzyme systems: malate dehydro-

genase (MDH), glucose phosphate isomerase (GPI), leucine

aminopeptidase (LAP), malic enzyme (ME) and 6-phos-

phogluconate dehydrogenase (6PGDH) (Mazars et al.,

1997a). This study revealed high genetic divergence among

the Pneumocystis isolates from the three targeted host species

(22 weaning rabbits, 30 corticosteroid-treated rats and 17

corticosteroid-treated mice). Isolates from different host

species exhibited clearly distinct isoenzyme patterns (near

maximum genetic distance possible) (Mazars et al., 1997a).

Within a given host species, Pneumocystis isolates from mice

and rabbits showed very little or no genetic diversity.

Especially, the 22 Pneumocystis isolates from rabbits of

diverse strain or geographic origin did not show genetic

diversity. Rat-derived pathogens diverged by the MDH

system that showed three distinct profiles, which were

however closely related to each other. However, the most

important outcome was that this work enabled us to

evaluate the degree of genetic isolation between Pneumocys-

tis genotypes. All linkage disequilibrium tests showed con-

siderable departures from panmictic expectation, and

strongly suggested that Pneumocystis genotypes from differ-

ent hosts species have been genetically isolated from each

other for a very long time, representing dramatically distinct

gene pools (Mazars et al., 1997a).

Pneumocystis strains from Old-World rabbitsrepresent a common gene pool

Comparing Pneumocystis genotypes from different hosts

with classical biological species (Mayr, 1963; Taylor et al.,

2000), it was shown that Pneumocystis isolates derived from

Table 3. Pairwise distances (%) of eight Pneumocystis genes

SODA TS HSP70 DHPS DHFR AROM mtrRNA

Nt AA Nt AA Nt AA Nt AA Nt AA Nt AA LSU SSU

P. oryctolagi/P. jirovecii 15.0 14.6 12.5 9.1 34.4 33.1 15.0 16.3 24.0 25.5 17.3 16.7 18.0 17.0

P. oryctolagi/P. carinii 24.4 29.7 16.5 14.2 35.7 35.3 17.6 18.9 33.4 39.1 21.9 25.0 26.4 17.0

P. oryctolagi/P. wakefieldiae NA NA NA NA 16.4 36.1 NA NA NA NA NA NA 22.2 18.1

P. oryctolagi/Pc f.sp. mustelae NA NA NA NA NA NA 17.8 23.1 32.8 32.8 18.8 26.0 18.0 17.0

P. oryctolagi/P. murina 24.0 29.1 15.9 15.2 23.5 16.2 17.2 19.3 33.7 35.5 21.0 25.0 21.6 16.0

P. oryctolagi/Pc f.sp. macaca 15.6 13.4 NA NA NA NA 15.2 17.4 25.2 28.2 NA NA 19.8 14.5

P. jirovecii/P. carinii 23.0 25.5 16.5 11.2 17.1 14.0 16.5 17.4 33.7 40.0 18.2 20.4 24.0 19.5

P. jirovecii/P. wakefieldiae NA NA NA NA 19.1 14.0 NA NA NA NA NA NA 23.4 20.5

P. jirovecii/Pc f.sp. mustelae NA NA NA NA NA NA 18.5 22.3 18.2 28.2 17.3 19.5 19.8 18.1

P. jirovecii/P. murina 21.8 24.3 16.2 13.2 33.5 30.2 15.1 17.4 32.5 33.7 17.3 19.5 21.6 18.4

P. jirovecii/Pc f.sp. macaca 7.1 1.9 NA NA NA NA 9.9 11.7 18.8 21.9 NA NA 12.5 10.3

P. carinii/P. wakefieldiae NA NA NA NA 14.9 10.3 NA NA NA NA NA NA 9.6 8.9

P. carinii/Pc f.sp. mustelae NA NA NA NA NA NA 18.0 23.4 28.8 33.7 18.5 23.2 21.6 16.0

P. carinii/P. murina 5.1 3.7 14.8 3.1 31.5 31.7 5.9 6.5 16.1 17.3 6.8 7.5 9.0 7.5

P. carinii/Pc f.sp. macaca 22.6 25.5 NA NA NA NA 15.5 18.5 34.3 40.0 NA NA 28.3 14.9

P. wakefieldiae/Pc f.sp. mustelae NA NA NA NA NA NA NA NA NA NA NA NA 19.2 15.2

P. wakefieldiae/P. murina NA NA NA NA 33.0 33.1 NA NA NA NA NA NA 9.6 7.8

P. wakefieldiae/Pc f.sp. macaca NA NA NA NA NA NA NA NA NA NA NA NA 26.4 17.4

Pc f.sp. mustelae/P. murina NA NA NA NA NA NA 16.8 21.9 27.3 26.4 17.6 21.3 18.6 15.6

Pc f.sp. mustelae/Pc f.sp. macaca NA NA NA NA NA NA 18.3 23.8 30.0 31.9 NA NA 23.7 14.9

P. murina/Pc f.sp. macaca 21.6 24.3 NA NA NA NA 15.3 18.5 31.3 34.6 NA NA 27.0 13.5

The pairwise distances (%) presented in the matrix were calculated for each pair of aligned sequences as follows: 100�% identity.

Common positions of the DNA alignments and common residues of the protein alignments:

SODA: nt: 496 AA: 165. TS: nt: 297 AA: 99. HSP70: nt: 410 AA: 136. DHPS: nt:798 AA: 265. DHFR: nt: 330 AA: 110. AROM: nt:325 AA: 108.

mtLSU-rRNA: 167. mtSSU-rRNA: 283. NA, nonaligned.

FEMS Microbiol Rev 30 (2006) 853–871c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

864 E. Dei-Cas et al.

a given host species exchange genes freely and represent

therefore a common gene pool. This is clearly the case of the

rabbit-associated Pneumocystis. Actually, studies performed

between 1992 and 2005 revealed no significant genetic

polymorphism in rabbit-derived Pneumocystis isolates from

both domestic and wild Old-World rabbit populations. In

these studies, in addition to the MLEE-based work discussed

above (Mazars et al., 1997a), numerous gene sequence

comparisons have been performed. Targeted loci were TS

(Mazars et al., 1994, 1995), mtLSU-rRNA (Wakefield et al.,

1992; Peters et al., 1994a; Guillot et al., 1999, 2001; Durand-

Joly et al., 2000), mtSSU-rRNA (Durand-Joly et al., 2000;

Guillot et al., 2001), arom (Banerji et al., 1995), SODA

(Denis et al., 2000), DHFR and DHPS (Ma et al., 2001).

Within some Pneumocystis species, such as P. carinii, P.

jirovecii, ferret-derived Pneumocystis- (Wakefield, 1998) and

shrew-derived Pneumocystis (Peters et al., 1994a; Laakkonen

& Sukura, 1997), intraspecies polymorphism was shown.

Regarding rabbit-derived Pneumocystis, no difference was

found among isolates from diverse domestic rabbit strains

and/or different geographic origins (Mazars et al., 1994,

1995, 1997a; Dei-Cas et al., 1998b). Some divergence at the

mitochondrial ribosomal RNA loci was found between

isolates from wild rabbits of diverse European geographic

regions on the one hand, and strains of domestic rabbits on

the other (E. Dei-Cas & A.E. Wakefield, unpublished data),

but the divergence levels were low, indicative of class II

(strain-level divergence but not species-level variation)

according to the Stringer criteria (Stringer, 1996). Data on

the same locus in Pneumocystis from European wild rabbits

published previously by other authors were consistent with

these observations (Guillot et al., 1999), though the number

of molecular studies on Pneumocystis species in wild mam-

mals is still limited (Peters et al., 1994a; Bishop et al., 1997;

Mazars et al., 1997b; Guillot et al., 1999).

Phylogeny of rabbit-associated Pneumocystis :concordance of gene genealogies

Both simple gene sequence comparison studies (mtLSU-

rDNA, TS and arom gene) (Wakefield et al., 1992; Peters

et al., 1994a, b; Mazars et al., 1995) and Pneumocystis

phylogenetic trees constructed on the basis of sequence

divergence at many loci (mtLSU-rDNA, mtSSU-rDNA,

SODA, DHPS or DHFR genes), either individually (Wake-

field, 1998; Guillot et al., 1999; Denis et al., 2000; Durand-

Joly et al., 2000; Ma et al., 2001) or concatenated (this paper;

Guillot et al., 2001; Keely et al., 2004), provided concordant

results on the relationships of rabbit-associated with other

Pneumocystis species, and on its place in the phylogeny of

Pneumocystis. In sequence comparison studies, rabbit Pneu-

mocystis gene sequences positioned constantly closer to

P. jirovecii ones than to those of rodent Pneumocystis species

(Fig. 3). Consistently, in phylogenetic trees rabbit-associated

isolates emerge regularly as a monophyletic clade placed

close to primate-derived Pneumocystis species, and relatively

far from rodent-associated Pneumocystis strains (Fig. 3).

Thus, the requirement of genealogical concordance (Taylor

et al., 2000), comprehensively discussed by Keely et al.

(2004) in their recent description of P. murina, is clearly

fulfilled for the rabbit-associated Pneumocystis species.

Rabbit-associated Pneumocystis : a newPneumocysti s species

The rabbit-associated Pneumocystis species presents a high

level of genetic and phenotypic divergence from existing

Pneumocystis species or formae speciales. Results summar-

ized in this paper demonstrate that genetic divergence from

Pneumocystis from hosts other than rabbits occurs through-

out the genome as shown by the detailed analysis of eight

independent loci, and five isoenzyme systems (Mazars et al.,

1997a). High concordance between gene trees associated

with the results of the population genetics approach suggests

that the entity has been genetically isolated from the other

Pneumocystis species or formae speciales for a very long

time, and that it has undergone, therefore, a prolonged

genetic and functional adaptation to the rabbit host (Or-

yctolagus cuniculus). Consistently, in cross-infection experi-

ments, the entity was found to be a stenoxenous species

(Aliouat et al., 1993b, 1994; Dei-Cas et al., 1994, 1998b),

similar to other studied Pneumocystis species associated to

specific animal hosts (Furuta et al., 1987; Gigliotti et al.,

1993; Durand-Joly et al., 2002). Other phenotypic differ-

ences examined in this paper (ultrastructure, growth rate)

illustrate the divergence between rabbit-associated and

other Pneumocystis species or formae speciales.

According to the biological concept (Mayr, 1963), which

hold a prominent place in mycology (Taylor et al., 2000),

species are ‘groups of actually or potentially interbreeding

natural populations, which are reproductively isolated from

other such groups’. Though mating tests are impossible to

apply in Pneumocystis due to their limited in vitro growth,

present data indicate that rabbit-derived Pneumocystis nat-

ural populations share the same gene pool and do not

exchange genes with other Pneumocystis populations. Con-

sequently the rabbit-associated entity is a biological species.

Furthermore, the concordance of topologies of multiple

gene trees shows that this entity, which emerges as a clear

monophyletic clade, is consistent with the concept of

phylogenetic species, i.e. an evolutionary lineage that has a

unique combination of DNA orthologue sequences (Taylor

et al., 2000). Therefore, the entity satisfies the criteria, at the

operational level, of both Biological Species Recognition

(BSR), and Phylogenetic Species Recognition (PSR), in the

sense of Taylor (Taylor et al., 2000).

FEMS Microbiol Rev 30 (2006) 853–871 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

865Pneumocystis oryctolagi sp. nov. from rabbits

Fig. 3. Maximum likelihood phylogeny of 18 Pneumocystis taxa inferred from mtLSU-rRNA and mtSSU-rRNA concatenated sequences. Bayesian

posterior probabilities are given as percentages near the individual nodes. Nodes with values of o 50% are not shown. Scale bar = 0.1 substitutions

(corrected) per base pair.

FEMS Microbiol Rev 30 (2006) 853–871c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

866 E. Dei-Cas et al.

Pneumocystis oryctolagi Dei-Cas, Chabe¤ ,Moukhlis, Durand-Joly, Aliouat, Stringer,Cushion, Noe« l, de Hoog, Guillot &Viscogliosi, sp. nov. MycoBank MB 500511.Figs 1 and 2

Pneumocystis oryctolagi (de Oryctolaguscuniculus , in quo fungus reperitur), quae antePneumocystis carinii f.sp. oryctolagi noscebatur(Anonymous, 1994)

Non filiosi extracellularesque fungi qui in Oryctolagus

cuniculus alveolis pulmoneis inhabitant. Ad Typum Primum

epithelii alveolas in alveolare epithelio sparsas haesi sunt.

Quod trophicae sporocyticae cysticaeque formae congrega-

tae sunt, quod quidem in Pneumocystis speciebus, quae e

mordacibus primatibusque oriuntur, frequenter evenit, con-

tra in Pneumocystis oryctolagi rarius est. Hujus speciei enim

fungi alveolare lumen petunt in solis corporibus quae

permulti parasitis inhabitant. Trophicae formae sunt

1–4mm statura, uninucleatae, irregulares, tenuitunicatae

plasmaticisque cum membranis in duobus lateribus. Asci

(cysti) autem, 4–6 mm statura, crassitunicati, globosi duabus

cum plasmaticis membranis sunt, in quibus octo rotundi ad

ovatiles ascopores sunt, quisque 1–2 mm statura. Vacui,

falciformi aut irregulares videntur. Pneumocystis oryctolagi

morphologia non distinguitur ab aliis Pneumocystis specie-

bus, facili microscopio uso, sed ultrastructuralibus studiis

patet Pneumocystis oryctolagi filipodia crassiora valdeque

minus copiosa quam soricinis Pneumocystis (Dei-Cas et al.,

1994; Nielsen et al., 1998). Quin etiam cysticae trophicaeque

formae crebriores in Pneumocystis oryctolagi (0.10–0.15)

quam in mordacum Pneumocystis speciebus (0.02–0.05). In

vivo autem ad duplicandum celerior Pneumocystis oryctolagi

est (1.7 dies) quam Pneumocystis carinii (4.5 dies) Pneumo-

cystis murina (10.5 dies) (Aliouat et al., 1999). Ad alias

externas dissimilitudines cognoscendas tabula prima con-

sulenda est.

Pneumocystis oryctolagi dissimilior DNA ordine est quam

aliae Pneumocystis species. Cujus mtLSU-rDNA loco genera

inter 18.1% (a Pneumocystis jirovecii) et 29.1% (a Pneumo-

cystis carinii) differunt. DNA ordinibus regionalia genera

Pneumocystis oryctolagi SODA ordine differunt a Pneumo-

cystis carinii 29.6% atque a Pneumocystis jirovecii 5.5%;

DHPS ordine a quibusque 18% et 15%, DHFR ordine a

quibusque 31% et 23% (Ma et al., 2001). Aminis acidis 97

conjectis e TS loci parte Pneumocystis oryctolagi differebat a

Pneumocystis carinii 14.4% vel a Pneumocystis jirovecii 9.2%

(Mazars et al., 1995). AROM loco Pneumocystis oryctolagi

differt 25% sine amini acidi ordine a Pneumocystis carinii

(Banerji et al., 1995). Cujus HSP70 inter 16.2% (a Pneumo-

cystis murina) et 36.1% (a Pneumocystis wakefieldiae) differ-

unt. DNA ordinibus regionalia genera Pneumocystis

oryctolagi mtSSU-rRNA ordine differunt a Pneumocystis

f.sp. macaca 14.5% atque a Pneumocystis wakefieldiae 18.1%.

Holotypus IPL-3609, e menstrui cuniculi pulmonibus

(Officina Charles River, Rouen, Francia). Cryoservata ex-

empla et electronae traditionis microscopii imagines

primae servata sunt in Pastore Instituto apud Lillam (Lilla,

Francia). Isotypus in Centraalbureau voor Schimmelcul-

tures depositur.

Description of Pneumocystis oryctolagisp. nov. Dei-Cas, Chabe¤ , Moukhlis, Durand-Joly, Aliouat, Stringer, Cushion, Noe« l, deHoog, Guillot & Viscogliosi, sp. nov.MycoBank MB 500511. Figs 1 and 2

Pneumocystis oryctolagi (L. adj. oryctolagi , ofthe rabbit, after the host in which the organismis found, Oryctolagus cuniculus )

Formerly known as Pneumocystis carinii f.sp. oryctolagi

(Anonymous, 1994).

Nonmycelian extracellular fungal organisms resident in

the pulmonary alveoli of Oryctolagus cuniculus. They attach

to Type 1 epithelial alveolar cells lining the alveolar epithe-

lium. Clustering of trophic, sporocytic and cystic forms,

which occurs frequently in rodent- and primate-derived

Pneumocystis species, is rather rare in P. oryctolagi. In this

species, organisms extend into the alveolar lumen only in

extensively parasitized hosts. The trophic forms, measuring

1–4mm, are uninucleate, of irregular shape, thin-walled and

with inner and outer plasma membranes. Asci (cysts),

measuring 4–6mm, are thick-walled, spheroid, with two

plasma membranes and contain eight round to ovoid

ascospores, each 1–2mm. When empty, asci appear falciform

or irregular. Pneumocystis oryctolagi is morphologically in-

distinguishable at the light microscopic level from other

Pneumocystis species, but ultrastructural studies show the

filopodia of P. oryctolagi to be thicker and clearly less

abundant than those of Pneumocystis from mice (Dei-Cas

et al., 1994; Nielsen et al., 1998). In addition, cystic-trophic

form ratio is higher in P. oryctolagi (0.10–0.15) than in

rodent Pneumocystis species (0.02–0.05). Likewise, in vivo

doubling time of P. oryctolagi is shorter (1.7 days) than in

P. carinii (4.5 days) or P. murina (10.5 days) (Aliouat

et al., 1999). Other phenotypic differences are shown in the

Table 1.

Pneumocystis oryctolagi is very different at the DNA

sequence level from other Pneumocystis species. Genetic

divergence at the mtLSU-rDNA locus ranged from 18.1%

(between P. oryctolagi and P. jirovecii) to 29.1% (between

P. oryctolagi and P. carinii). DNA sequences from regions in

the genes of the P. oryctolagi SODA diverged from those of

P. carinii by 29.6%, and P. jirovecii by 5.5%; DHPS by 18%

FEMS Microbiol Rev 30 (2006) 853–871 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

867Pneumocystis oryctolagi sp. nov. from rabbits

and 15%; DHFR by 31% and 23% (Ma et al., 2001). Over 99

deduced amino acids of a fragment of the TS locus,

divergence was 14.4% between P. oryctolagi and P. carinii,

and 9.2% between P. oryctolagi and P. jirovecii (this paper,

(Mazars et al., 1995). At the arom locus P. oryctolagi diverged

by 25% (deduced amino acid sequence level) from P. carinii

(this paper, Banerji et al., 1995). Divergence was similar in

HSP70 (16.2–36.1%) and in the DNA sequence of the

mtSSU-rRNA gene (14.5–18.1%).

The type strain is IPL-3609. Extracted from lungs of 1-

month-old rabbits (Charles-River, Rouen, France). Cryo-

preserved samples and original transmission electron micro-

graphies are stored at the Lille Pasteur Institute and at the

Centraalbureau voor Schimmelcultures (Utrecht, The Neth-

erlands).

Acknowledgements

We would like to thank Professors Walter Gams (CBS,

Fungal Biodiversity Centre, Utrecht, The Netherlands) and

Scott Redhead (Agriculture and Food Ottawa, Canada) for

helpful advice in the field of fungal nomenclature and

taxonomy, as well as Professors Maria-Lucia Taylor (UNAM,

Mexico DF, Mexico), Maria-Jose Mendez Giannini (UNESP,

Sao Paulo, Brazil), Rosely M. Zancope-Oliveira (FIOCRUZ,

Rio de Janeiro, Brazil), and Francois Delaporte (‘Jules

Vernes’ University, Amiens, France) for providing valuable

papers on Pneumocystis taxonomy published in the early

20th century. We thank Miss Nausicaa Gantois for technical

assistance. The French Ministry of High Education and

Research (EA3609 Lille 2 University), Lille Pasteur Institute,

Spanish Ministry of Research and Technology (SAF2003-

06061, 2003–2006), and European Commission (‘Eurocar-

inii’ FP-5-QLK2-CT-2000-01369) have supported this work.

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871Pneumocystis oryctolagi sp. nov. from rabbits