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sequence types representing two distinct groups. On the ... 1995; Hocker et al., 2005; Rabodonirina et al., 2004; Singer et al., 1975). ... impacted on the surface of a rotative cup. ... round of PCR were as follows: each cycle consisted of denaturation ...... of Education, Research and Technology) and 'Eurocarinii' European.
Microbiology (2005), 151, 3117–3125

DOI 10.1099/mic.0.28059-0

Molecular and serological evidence of Pneumocystis circulation in a social organization of healthy macaques (Macaca fascicularis) Christine Demanche,1 Fane´lie Wanert,2 Mathieu Barthe´lemy,3 Je´roˆme Mathieu,4 Isabelle Durand-Joly,5 Eduardo Dei-Cas,5 Rene´ Chermette1 and Jacques Guillot1 Correspondence Jacques Guillot [email protected]

1

Equipe de Mycologie, UMR 956 INRA-AFSSA-ENVA-UPVM Biologie Mole´culaire et Immunologie Parasitaires et Fongiques, Ecole Nationale Ve´te´rinaire d’Alfort, 7 Avenue du Ge´ne´ral de Gaulle, 94704 Maisons-Alfort, France

2

Centre de Primatologie, ULP Strasbourg, Fort Foch, Niederhausbergen, France

3

Laboratoire de Parasitologie, Universite´ Pierre et Marie Curie, Paris VI, France

4

Laboratoire d’Ecologie des Sols Tropicaux, UMR 137 BioSol, IRD/Paris VI, 32 avenue Henri Varagnat, 93143 Bondy Cedex, France

5

EA3609-Parasitologie-Mycologie, Faculte´ de Me´decine et CHRU de Lille and IFR-17-Ecologie du Parasitisme, Institut Pasteur de Lille, France

Received 22 March 2005 Revised

6 June 2005

Accepted 13 June 2005

Simian populations represent valuable models for understanding the epidemiology of human pneumocystosis. The present study aims to describe the circulation of Pneumocystis organisms within a social organization of healthy crab-eating macaques (Macaca fascicularis) living in a natural setting in France. Animals were followed for up to 2 years. Deep nasal swab and blood samples were collected monthly from each animal under general anaesthesia. Environmental air was sampled for a 1 week period every month in the park where the macaques dwelt. Pneumocystis DNA was detected by nested-PCR of mitochondrial large subunit rRNA (mtLSU) gene in nasal swab and air samples. Anti-Pneumocystis IgG antibodies were detected in serum samples by indirect immuno-fluorescence assay. Pneumocystis DNA was detected in 168 of 500 swab samples examined (33?6 %). The number of macaques with detectable Pneumocystis DNA was highly variable from one month to another. Positive detection of Pneumocystis DNA was not related to the detection of serum anti-Pneumocystis antibody. During the second year of the study, Pneumocystis DNA was amplified more frequently from unweaned macaques than from adults or subadults. The mtLSU sequence showed marked polymorphism with eight Pneumocystis sequence types representing two distinct groups. On the whole, a constant and intensive circulation of Pneumocystis organisms within the community was observed. However, the implication of the various members of the colony was probably different and several levels of colonization by Pneumocystis may occur in immunocompetent macaques.

INTRODUCTION Pneumocystis organisms are considered as major opportunistic fungal pathogens that infect humans and a wide range of other mammalian host species (Cushion, 1998, 2004; DeiCas et al., 1998; Guillot et al., 2001; Morris et al., 2004). However, the basic biology of these atypical fungi and the subsequent epidemiology of Pneumocystis infections are not

Abbreviations: IFA, immuno-fluorescence assay; mtLSU, mitochondrial large subunit rRNA; SCID, severe combined immunodeficiency; TBO, toluidine blue O.

0002-8059 G 2005 SGM

Printed in Great Britain

well understood. Much of the blame for the lack of basic information about Pneumocystis organisms can be placed on their failure to grow well in culture. To date, most knowledge about Pneumocystis has been obtained from studies involving laboratory animal models such as rat, mouse, rabbit and ferret. These models have been extensively used as the main source of Pneumocystis organisms and for approaching important aspects of pneumocystosis natural history, pathophysiology or transmission (Cere´ & Polack, 1999). Studies involving laboratory animals clearly demonstrated that Pneumocystis organisms are transmitted from host to host via the air and that colonization may be 3117

C. Demanche and others

established early in life (Hendley & Weller, 1971; Soulez et al., 1991; Vargas et al., 2001). Consistently, Pneumocystis DNA was detected in air sampled from the close environment of infected or colonized animals, including rats (Bartlett et al., 1994; Olsson et al., 1996), rabbits (Guillot et al., 1999b) and non-human primates (Demanche et al., 2001). In humans, reports of pneumocystosis outbreaks among immunosuppressed patients in hospitals suggest that the main source of Pneumocystis is patients with a Pneumocystis infection (Helweg-Larsen et al., 1998; Hennequin et al., 1995; Hocker et al., 2005; Rabodonirina et al., 2004; Singer et al., 1975). The presence of Pneumocystis DNA has been reported in air samples collected from hospital rooms of patients with pneumocystosis (Bartlett et al., 1994, 1997). Detection of Pneumocystis DNA in the air is also consistent with the hypothesis that transmission occurs from person to person. Recent studies have shown that Pneumocystis DNA can also be detected in immunocompetent humans after close contact with patients with pneumocystosis (Miller et al., 2001; Vargas et al., 2000). In these cases, Pneumocystis DNA detection was transient and might have been the result of continuous inhalation of Pneumocystis cells, indicating upper respiratory surface contamination rather than an active infectious process. The actual role of transiently parasitized immunocompetent hosts as a source of infective elements has been demonstrated in the mouse model (Dumoulin et al., 2000) and warrants further investigations in humans. Recent studies on mice suggested that transmission from healthy host to healthy host, as an asymptomatic or minimally symptomatic infection, could be a way to maintain Pneumocystis organisms in the environment (Chabe´ et al., 2004; Gigliotti et al., 2003). The interest of non-human primate models relies on the phylogenetic closeness between monkeys and humans and therefore between Pneumocystis in monkeys and Pneumocystis in humans (Demanche et al., 2001; Hugot et al., 2003). In the present study, we describe for the first time the circulation of Pneumocystis organisms within a social organization of crab-eating macaques (Macaca fascicularis) living in a natural setting in France. These conditions simulate, with good fidelity, real conditions in wildlife, while reducing uncontrolled biotic and abiotic parameters. The aims of the study were to identify potential sources of infection, to assess Pneumocystis circulation within the colony and to test the hypothesis of airborne transmission. Deep nasal swab and blood samples were collected monthly from each animal. Environmental air samples were also examined every month. Since known Pneumocystis species have been analysed at the mitochondrial large subunit (mtLSU) rRNA gene (Demanche et al., 2001; Wakefield, 1998), which is considered as a sensitive and robust target for Pneumocystis PCR detection (Tsolaki et al., 1999; Wakefield et al., 1990), this locus was used for detecting Pneumocystis organisms in both macaque respiratory specimens and environmental air samples. 3118

METHODS Animal model. A social organization of crab-eating macaques

(Macaca fascicularis), living in a 500 m2 natural area at the Primatology Center of Strasbourg was examined for 2 years (from December 2000 to November 2002). Demanche et al. (2003) examined the same colony. At the beginning of the study, the colony comprised 18 macaques including nine sexually mature animals (one male and eight females), six subadults (one male and five females) and three unweaned macaques (one male and two females) born in September 2000. Unweaned monkeys were less than 1 year old and lived in close contact with their respective mothers. Subadults were more than 1 year old but had not reached sexual maturity. Eleven births occurred during the study (two in September 2001, one in January 2002, two in June 2002, one in July 2002, four in August 2002 and one in November 2002). At the end of the study, the colony comprised 29 macaques including 12 sexually mature animals (one male and 11 females), eight subadults (two males and six females) and nine unweaned macaques (five males and four females). In December 2001, the dominant reproductive male (M1 G139) was substituted by another male (M19 E447) because M1 was old and diabetic. No underlying illness was detected in the monkeys and no animal received immunosuppressive drugs or antimicrobials active against Pneumocystis. All monkeys in the colony were captured monthly. Inside the warm shelter, food and water were provided ad libitum. Samples for analysis. Deep nasal swabs and blood samples were

collected from each animal under general anaesthesia induced by the administration of 10 mg ketamine (Imalgene) kg21 by an intramuscular route. A moist sterile cotton-swab was introduced deep into each nasal cavity, left there for 5 s, rotated and withdrawn (Vargas et al., 2000). A blood sample (3–5 ml) was collected in a serum separator tube by puncturing a femoral vein. After centrifugation, serum samples were stored at 220 uC. Air samples from the environment of the colony were obtained by using the CAP (Capteur Atmosphe´rique de Poussie`res) device (Arelco) as previously described by Guillot et al. (1999b). This device sampled airborne particles with a flow rate of 10 litres min21. Particles were impacted on the surface of a rotative cup. Every month, air sampling was performed for 1 week in the centre of the park where the macaques lived. Deep nasal swab and blood samples were collected from the animals the last day of the air-sampling week. DNA extraction from nasal swab and air samples. Just after

sampling, from December 2000 to October 2001, deep nasal swabs were placed in a sterile tube containing 500 ml extraction buffer (10 mM Tris, 0?5 % SDS, 25 mM EDTA, 0?1 M NaCl). For air samples, the rotative cup from CAP apparatus was washed with 600 ml of the same extraction buffer. DNA was prepared by proteinase K digestion (Boehringer Mannheim) at a final concentration of 0?28 mg ml21, followed by phenol/chloroform extraction with a final precipitation in ethanol. From November 2001 to November 2002, deep nasal swab DNA extraction was performed by using a Qiagen kit (DNeasy tissue kit). Primers and PCR amplification. The presence of Pneumocystis DNA in swabs was assessed by nested-PCR at the mtLSU rRNA gene. We used the primer sets pAZ102-H/pAZ102-E (59-GATGGCTGTTTCCAAGCCCA-39/59-GTGTACGTTGCAAAGTACTC-39) and pAZ102-X/R1/pAZ102-Y/R1 (59-GGGAATTCGTGAAATACAAATCGGACTAGG-39/59-GGGAATTCTCACTTAATATTAATTGGGGAGC-39) (Wakefield, 1998). The thermocycling conditions for the first round of PCR were as follows: each cycle consisted of denaturation for 30 s at 94 uC, annealing for 1 min at 50 uC and extension for 2 min at 72 uC for 30 cycles. The second round of PCR was performed with 5 % (v/v) of the first round mix. The thermocycling

Microbiology 151

Pneumocystis circulation in a colony of primates conditions for the second PCR round were as follows: each cycle consisted of denaturation for 30 s at 94 uC, annealing for 1 min at 55 uC and extension for 2 min at 72 uC for 30 cycles. For both PCR reactions, the initial denaturation was performed at 94 uC for 10 min and the final extension step at 72 uC for 20 min. Negative controls were included in each experiment, in both DNA extraction and PCR amplification, to monitor for possible contamination. One negative control was tested per five experimental tubes. A laminar flow was used and PCR products were not manipulated in the 2 days preceding PCR reactions. We selected some amplification products corresponding to longcarriage periods (for animals F14 and F17) or to periods where amplifications were positive for both mothers (F5 and F7) and their respective babies (F16, F19 and F18). These amplification products were purified in a 2 % agarose gel (Tris borate EDTA buffer) and extracted with PCR purification kit (Qiagen). Amplification products were directly sequenced from both ends using sets of internal primers on an automated DNA sequencer (GenomeExpress). The mtLSU sequences were aligned with already known Pneumocystis sequences using the computer program CLUSTAL X (version 1.63b) (Thompson et al., 1997). When direct sequencing failed, positive amplification products were cloned with pGEM-T vector system II kit (Promega). Four separate colonies were selected from the transformant plates and examined for each positive sample. DNA extraction was performed by Wizard Plus minipreps DNA purification system kit (Promega). We used the primer set Sp6/T7 vector specific for the fragment size, and we used the primer set pAZ102-X/R1/pAZ102-Y/R1 (59-GGGAATTCGTGAAATACAAATCGGACTAGG-39/59-GGGAATTCTCACTTAATATTAATTGGGGAGC-39) (Wakefield, 1996) for the fragment specificity. Thermocycling conditions were as follows: each cycle consisted of denaturation for 30 s at 94 uC, annealing for 1 min at 50 uC and extension for 1 min at 72 uC for 30 cycles. Amplification products were sequenced from both ends on an automated DNA sequencer (Qiagen). Serum anti-Pneumocystis antibody detection. Anti-Pneumocystis

IgG titre was assessed on serum samples by indirect immunofluorescence assay (IFA). Antigen was a suspension of Pronase-treated rabbit-derived Pneumocystis organisms that was prepared as follows: infected rabbit lungs were cut into small pieces in Hanks’ solution without Ca2+/Mg2+ and homogenized either with a magnetic stirrer (4 uC, 90 min) or squeezed through a stainless steel mesh. The homogenate was poured through gauze and centrifuged (2900 g, 10 min, 4 uC). The pellet was resuspended in a 0?08 % Pronase solution in Hanks’ medium with Ca2+/Mg2+ and homogenized for 3 h at 37 uC under magnetic stirring. After incubation, the suspension was centrifuged and Pneumocystis organisms were washed three times with Hanks’ medium without Ca2+/Mg2+. The pellet was resuspended in a known volume of Ca2+/Mg2+-free Hanks’ medium in order to obtain a suspension of 106 Pneumocystis cysts ml21. Quantification of Pneumocystis cysts was performed on toluidine blue O (TBO) stained dry smears. Then, 10 ml of the suspension was spotted into each well of multiwell immuno-fluorescence slides (10 wells 5 mm black; ‘lames epoxy noir’, Polylabo). Slides were dried, fixed in cold acetone, wrapped individually in aluminium foil and stored at 220 uC until use (Soulez et al., 1989). Macaque serum samples were diluted (1/200, 1/400 and 1/800) in PBS pH 7?2 (bioMerieux) and dropped on the antigen-fixed immunofluorescence slides. After incubation for 30 min at 37 uC, the reaction was revealed by using fluorescent-conjugated anti-monkey immunoglobulin antibody IgG(c) (Kirkegaard and Perry Laboratories). Titres of 1/200 or higher were considered positive (Soulez et al., 1989). http://mic.sgmjournals.org

Statistical analyses. Carriage of Pneumocystis and antibodies were

analysed separately at each month. The detection of antibodies was treated by ‘presence’ (titre >1/200)2‘absence’ (titre (1/200). In all cases, hypotheses were tested by using x2 tests on 262 table of contingency (Everitt, 1977; Sokal & Rohlf, 1995). Observed probability distributions were compared with the excepted distributions corresponding to the case where the stated null hypotheses would have been true. The first error type rate was fixed at 0?05.

RESULTS Pneumocystis DNA detection in nasal samples A total number of 500 deep nasal swab samples were collected. After a single round of PCR using the primers pAZ102-H and pAZ102-E, there was no sample that amplified a Pneumocystis PCR product. Pneumocystis DNA was detected by performing nested-PCR with primers pAZ102X/R1/pAZ102-Y/R1 in 168 of 500 swab samples (33?6 %) (Table 1). Pneumocystis DNA amplification products of about 270 bp were detected. Sequencing was performed on 19 PCR products and yielded eight sequence types. These sequences formed two distinct groups already described by Guillot et al. (2004). Group 1 included three sequence types (GenBank accession nos AF362467, AY265386 and AY265389) without insertion. Group 2 comprised five sequence types (accession nos AY265384, AY265385, AY265387, AY265390 and AY265391) characterized by two successive insertions separated by a few nucleotides (Table 2). The first insertion comprised 24 to 30 bp and the second one 3 to 12 bp. The extent of genetic divergence within a sequence group was low (2?5 % in group 1 and 2?3 % in group 2). One co-infection of two sequence types was observed in unweaned macaque F17 (Mf102) in January 2002 (Table 1). Pneumocystis DNA was detected every month in several animals within the colony. The prevalence of Pneumocystis DNA was highly variable from one month to another. Higher prevalence rates were recorded in March and September 2001 when Pneumocystis DNA amplification occurred in nasal samples from 83?3 to 100 % of the animals, respectively. The lowest prevalence rate was found in February 2001, when only one macaque was positive (Table 1). Seasonal influence of climatic factors on Pneumocystis carriage was examined in detail elsewhere (Demanche et al., 2003). For each animal, the apparent duration of Pneumocystis DNA carriage varied from less than 1 month (one positive amplification) to 6 months (positive amplifications on seven consecutive sampling dates for F14 and F17). For the longest carriage periods, the same sequence type (accession no. AY265385) (for F14 from November 2001 to April 2002) or closely related sequence types (for F17 from January to June 2002) were detected. Considering only consecutive amplifications, Pneumocystis DNA carriage duration averaged 2 months. All the monkeys were positive at least once during the study. No difference was observed according to 3119

For each animal, the first line corresponds to serological results and the second line to nested-PCR results. Animal reference*

Adults M1 G139

Anti-Pneumocystis serum antibody titres and presence of Pneumocystis DNA in nasal swab samplesd§ Dec. 00

Jan. 01

Feb. 01

Mar. 01

Apr. 01

May 01

Jun. 01

Jul. 01

Aug. Sept. 01 01

Oct. 01

Nov. 01

Dec. 01

Jan. 02

Feb. 02

Mar. 02

Apr. 02

May 02

Jun. 02

Jul. 02

2 2

2 2 2 2

2 2 2 2

2 2 2 2

2 2 2 +

2 + 2 2

2 2 2 2

2 2 2 +

2 + 2 +

Subadults M2 Mf975 F11 Mf984 F12 Mf991 F13 Mf992 Mothers and unweaned monkeysD F4 D823

2 2

2 +

2 2

2 +

2 +

1/200 1/200 + 2 ND 2 2 +(1) 2 2

2 2

2 2

ND

ND

ND

ND

2

2

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

F5 B966

2 2

2 +

2 2

2 +

M3|| Mf002

ND

ND

ND

ND

ND

+

2

ND

ND

ND

ND

ND

ND

2 2

2 +

2 2

F17|| Mf102

2 2

2 2

2 2

2 +

2 +

2 + 2 2

2 + 2 2

2 + 2 2

ND

ND

ND

ND

ND

ND

ND

ND

+

2 2 2 2

2 2 2 2

2 + 2 +

2 2 2 2

2 + 2 2

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

2 2

2 +

2 +

M7 Mf206

Microbiology 151

F16|| Mf101 F19 Mf204 F6 C962

2 +

2 +

1/200 2 2 2 2 2 2 2 2 1/200 2 2 2 2 2 2 + + 2 + + 2 + 2 2 2 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/400 1/400 2 + 2 + 2 2 2 + 2 + 2 2 2 1/200 1/400 1/800 1/800 1/400 1/400 1/400 1/400 1/400 1/400 1/400 1/200 1/200 2 + 2 2 2 2 2 2 2 + 2 2 2 2 2 2 2 1/200 1/200 2 2 2 2 2 2 2 2 + + 2 2 2 2 2 2 + 2 2 2

2 2

F14|| Mf001

Oct. 02

Nov. 02

2 2 2 2

2 2 2 2

1/400 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/400 1/800 1/800 2 2 2 + 2 2 2 + + + +

M19 E447 F3 D824

Aug. Sept. 02 02

1/200 2 1/200 2 +(2) +(2)

2 + 1/400 2 1/200 2 2 +

2 2 2 +

2 2 2 2

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ND 2 2 2 2 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 2 2 2 2 + 2 2 2 2 2 ND 2 2 1/200 1/200 1/200 1/200 1/200 1/200 ND + 2 2 2 2 2 2 2 2 ND 2 2 2 2 2 2 2 2 2 2 2 2 2 2 + 2 2 2 2 +

1/200 2 2 2 2 2 2 2 2 2 2 2 1/200 2 2 + + 2 2 2 2 2 2 2 + + 2 2 2 2 2 1/200 1/400 1/400 1/400 1/200 1/200 1/400 1/400 1/200 1/200 1/200 1/200 1/200 1/200 +(2) 2 +(2) + +(2) + + +(2) + 2 + + + +(2) + ND ND 2 2 2 2 2 2 1/200 1/200 1/200 1/200 1/400 1/400 1/400 + 2 2 + +(1, 2) + + +(2) + +(2) 2 2 2 + + ND ND 2 2 2 2 +(1) + 2 2 2 2 2 2 2 2 2 2 2 2 1/200 2 2 + + + 2 +(1) 2 2 2 2 + 2 +(2) 2 2 2 1/200 2 1/400 1/200 1/400 1/200 1/200 2 1/200 2 1/200 1/200 1/200 1/200 1/200 + 2 2 2 2 2 2 2 2 2 + 2 + 2 + ND ND 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 + 2 2 + +(2) + 2 + 2 2 + 2 2 2 2 ND 2 2 2 2 2 +(2) + 1/200 2 1/200 2 2 2 2 2 2 2 2 2 2 1/200 1/200 + + 2 2 2 2 2 2 2 + + 2 2 2 2

C. Demanche and others

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Table 1. Anti-Pneumocystis serum antibody titres and Pneumocystis DNA amplification results from Macaca fascicularis monkeys

http://mic.sgmjournals.org

Table 1. cont. Animal reference*

F15|| Mf003

Anti-Pneumocystis serum antibody titres and presence of Pneumocystis DNA in nasal swab samplesd§ Dec. 00

Jan. 01

Feb. 01

Mar. 01

Apr. 01

May 01

ND

ND

ND

ND

ND

2

2

2

2 +

2 +

M4 Mf104 F2 C447

1/800 1/800 1/800 1/800 1/800 1/800 + 2 2 + 2 2

M5 Mf201 F7 C971

1/400 1/200 1/200 1/800 1/400 1/200 2 + 2 + 2 2

F18 Mf202 F8 Mf974

2 +

2 2

2 2

2 +

2 2

2 2

M6 Mf203 F9 Mf982

1/400 1/400 1/800 1/800 1/800 1/800 2 2 + + + 2

F20 Mf205 F10 Mf983

F1 F061 M8 Mf208

Jul. 01

Aug. 01

Sept. 01

Oct. 01

Nov. 01

Dec. 01

1/200 2

2 2

Jan. 02

Feb. 02

Mar. 02

Apr. 02

May 02

Jun. 02

Jul. 02

Aug. 02

Sept. 02

Oct. 02

Nov. 02

2 2 2 2 1/200 2 2 2 1/200 2 2 + 2 2 2 2 + + 2 + 2 2 2 2 2 2 2 2 2 2 2 1/200 1/200 2 2 2 2 2 + 2 2 2 2 2 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 2 2 + + + 2 2 2 + 2 2 2 + 2 2 2 2 2 ND ND ND 2 1/200 1/200 + 2 + 2 + 2 1/400 1/200 1/200 1/200 1/200 1/200 1/200 2 2 2 1/200 1/200 1/200 1/200 1/200 1/200 2 2 2 + + + + 2 2 2 2 2 2 2 + 2 2 2 +(1) + ND ND ND ND 2 2 2 2 2 + +(2) + 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 + + + + 2 2 2 2 2 2 2 2 + + + 2 2 2 ND ND 2 2 2 + ND 2 + + 1/800 1/800 1/800 1/800 1/800 1/800 1/400 1/200 1/400 1/400 1/400 1/400 1/400 1/400 1/400 1/400 1/400 1/400 2 + 2 + 2 2 2 2 2 2 2 2 + + 2 2 2 2 ND ND 2 2 2 2 2 + 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 1/800 2 2 2 + 2 2 2 2 2 2 + 2 2 2 2 2 2 2 2 2

2 2

2 +

2 +

2 2

ND

ND

ND

ND

+(1) 2 2 + 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/200 1/400 1/800 1/400 1/200 1/200 1/200 1/200 1/200 1/200 2 2 2 + + 2 2 2 + + + 2 2 2 2 2 2 2 2 + 2 2 2 2 ND

2

3121

*M, male; F, female. DUnweaned macaques were placed together with their mothers (mother F4 with unweaned macaques F14 born in September 2000, F17 born in September 2001 and M7 born in August 2002; mother F5 with unweaned macaques M3 born in September 2000, F16 born in September 2001 and F19 born in July 2002; mother F6 with unweaned macaques F15 born in September 2000 and M4 born in January 2002; mother F2 with unweaned macaques M5 born in June 2002; mother F7 with unweaned macaques F18 born in June 2002; mother F8 with unweaned macaques M6 born in July 2002; mother F9 with unweaned macaques F20 born in August 2002; mother F10 with unweaned macaques F21 born in August 2002; mother F1 with unweaned macaques M8 born in November 2002). d1/200, IgG-positive 1/200 dilution serum; 1/400, IgG-positive 1/400 dilution serum; 1/800, IgG-positive 1/800 dilution serum; 2, seronegative samples; ND, not done. §+, positive-nested-PCR; 2, negative-PCR; ND, not done; 1, sequence type from group 1 (without insertion); 2, sequence type from group 2 (with two successive insertions) (Table 2). ||Unweaned macaques F14, M3 and F15 became subadults in September 2001 when F17 and F16 were born. Unweaned macaques F16 and F17 became subadults in August 2002 when F19 and M7 were born.

Pneumocystis circulation in a colony of primates

F21 Mf207

1/800 1/800 1/800 1/800 1/800 1/800 2 2 2 + 2 2

Jun. 01

C. Demanche and others

Table 2. mtLSU sequence types of Pneumocystis isolates from Macaca fascicularis monkeys living in partial release in France Sequence types Group 1 AF362467

AY265386 AY265389

Group 2 AY265384 AY265385

AF362469 AY265387 AY265390 AY265391

Animal

Date of sampling

F14 F16 F17 F5 F21 M7 F7

(Mf 001) (Mf 101) (Mf 102) (B966) (Mf 207) (Mf 206) (C971)

March 2001 January 2002 January 2002 January 2002 August 2002 October 2002 October 2002

F6 F14 F14 F14 F14 F14 F18 F19 F17 F17 F17 F5 F6

(Mf 962) (Mf 001) (Mf 001) (Mf 001) (Mf 001) (Mf 001) (Mf 202) (Mf 204) (Mf 102) (Mf 102) (Mf 102) (B966) (Mf 962)

April 2001 September 2001 November 2001 January 2002 April 2002 October 2002 October 2002 October 2002 January 2002 April 2002 June 2002 August 2002 May 2001

the sex of the macaques (30?5 and 34?3 % positive samples from males and females, respectively). During the first year, Pneumocystis DNA was detected in 30 % of the samples from unweaned macaques, in 30?9 % of the samples from subadult macaques and 50?5 % of the samples from adults. The statistical analysis performed on adults, subadults and young showed that prevalence of Pneumocystis DNA detection tends to increase in association with the age of the animals. During the second year of the study eight births occurred, and the trend observed for the first year of the survey was found to be inverted: the proportion of animals with detectable Pneumocystis DNA was significantly higher in unweaned macaques (48?3 %) than in subadults (28?9 %) and adults (18?9 %). However, the proportion of unweaned macaques within the whole population was not correlated to the probability to carry fungal DNA within adults and subadults individuals. When Pneumocystis DNA was amplified from an unweaned macaque, parasite DNA was not systematically amplified from the corresponding mother, and when a positive amplification occurred from a mother, Pneumocystis DNA was not systematically detected in her baby. When a mother and her baby were shown to harbour Pneumocystis DNA, the Pneumocystis sequence type was not the same. In January 2002, a mtLSU sequence from group 2 was detected in the mother F5 (B966), whereas a mtLSU sequence from group 1 was observed in her baby F16 (Mf101). The same observation occurred in October 2002, in mother F7 (C971) (mtLSU sequence from group 1) and 3122

in her unweaned macaque F18 (Mf202) (mtLSU sequence from group 2) (Tables 1 and 2). The two babies, F14 (Mf001) and F17 (Mf102), of mother F4 (D823), harboured closely related Pneumocystis sequence types (from group 2) during the study, except in January 2002 when F17 was co-infected with Pneumocystis isolates corresponding to two distinct mtLSU sequence types from groups 1 and 2 (Table 2). Pneumocystis DNA detection in air samples During the study, a total number of 11 air samples were collected. Using nested-PCR with primers pAZ102-H/ pAZ102-E (first round) and pAZ102-X/R1/pAZ102-Y/R1 (second round), Pneumocystis DNA was not detected in these environmental samples. Serum anti-Pneumocystis antibody assessment A total number of 468 blood samples were collected. AntiPneumocystis antibody was detected in 238 samples (50?8 %). Titres remained quite stable throughout the study (Table 1). Seven monkeys were found seropositive (M1, F1, F2, F9, F10, F11 and F16). Two of them showed a high and constant rate of anti-Pneumocystis antibody (1/800) (F2 and F10). The remaining animals either were seronegative or had low levels of anti-Pneumocystis antibody (1/200) from time-to-time. Anti-Pneumocystis antibody was detected in monkeys younger than 1 year (F17, F16, M4 and M5). Seroconversion occurred in 1-year-old macaques F14 and M3 and in the 14-month-old macaque F15 (Table 1). Relationships between the presence of antibodies and the likelihood to carry Pneumocystis DNA Throughout the study, anti-Pneumocystis IgG titres were less variable than results of Pneumocystis DNA amplification. There was no statistical difference in PCR amplification results between samples from seropositive and seronegative animals (Fig. 1). Similarly, no statistical difference was observed when the analysis was made independently on the following three categories: seronegative macaques (n=8), animals in which a seroconversion was detected during the study (n=13), and seropositive animals from the beginning to the end of the study (n=7). For instance, Pneumocystis DNA was detected in 10 nasal swab samples from the adult female F3 and F5, and in nine samples from adult females F8 (Table 1), although these animals remained seronegative. Conversely, Pneumocystis DNA was detected in only three nasal samples from the seropositive female F10.

DISCUSSION The rationale for undertaking the present study was to examine the potential of a normal animal population to serve as the reservoir for Pneumocystis by exploring the animal-to-animal circulation of the organisms. The originality of the study resides in both the use of a simian colony Microbiology 151

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mice were able to transmit the infection to SCID mice (Chabe´ et al., 2004; Gigliotti et al., 2003).

Fig. 1. Evolution of the probability of Pneumocystis DNA detection in nasal swab samples from seropositive or seronegative monkeys during this study.

instead of rodents, and the very long follow-up. Three parameters were used to assess the circulation of Pneumocystis organisms within the colony: the detection and characterization of Pneumocystis DNA from individual nasal swab samples, the detection of anti-Pneumocytis antibody from individual serum samples and the detection of Pneumocystis DNA in air samples. We first demonstrated a relatively high prevalence of Pneumocystis DNA in the upper respiratory tract of healthy macaques (33?6 % of PCR-positive nasal swabs). However, marked variations were observed in the number of animals with detectable Pneumocystis DNA during the experiment. Also, each month, a positive amplification occurred systematically from nasal samples from at least one animal in the colony. The mean duration for Pneumocystis DNA carriage in PCR-positive monkeys was relatively short (2 months). By using immunohistochemical methods to detect the presence of Pneumocystis in fixed lung tissues, Vogel et al. (1993) reported that latent Pneumocystis infection was uncommon in rhesus macaque. To account for the apparent discrepancy between our results and those from Vogel et al. (1993), it may be hypothesized that PCRpositive results occurred in transient carriers, which were not colonized by Pneumocystis organisms. Essentially, it was not clearly established if the finding of Pneumocystis DNA in nasal samples provided information about the actual presence of cysts or trophic forms in lung alveoli or whether these parasite stages were viable or infective if present. Recent experiments, however, showed that healthy BALB/c mice transiently colonized with Pneumocystis parasites were able to transmit them by an airborne route either to severe combined immunodeficiency (SCID) (Dumoulin et al., 2000) or to BALB/c mice, which showed seroconversion (Chabe´ et al., 2004; Gigliotti et al., 2003). In addition, parasites were detected histologically in the lungs of healthy BALB/c mice that contracted their infection from SCID mice with pneumocystosis (Chabe´ et al., 2004). What is more, in these experiments, healthy mice infected by the aerial way by co-housing with transiently infected healthy http://mic.sgmjournals.org

In the present work, the detection of anti-Pneumocystis antibody may help the interpretation of PCR results. Throughout the study, two groups of macaques could be distinguished according to serological results. A first group included animals (n=7), which either were seronegative or showed fluctuating but low anti-Pneumocystis antibody titres, suggesting transient colonization or intermittent contact with an infective source. Monkeys of the second group (n=8) showed a constant level of anti-Pneumocystis antibody, which might indicate a durable colonization. However, the prevalence of Pneumocystis DNA detection was not higher in seropositive animals than in seronegative ones. This apparent divergence between PCR and serological results may indicate that high levels of anti-Pneumocystis antibody do not necessarily reveal colonization. An alternative explanation would be that the deep nasal sampling method used in this work was not invasive enough for the detection of Pneumocystis carriage. In human communities, infants could constitute a major infectious reservoir for Pneumocystis organisms. Pneumocystis DNA has been frequently detected in nasopharyngeal aspirates from immunocompetent infants (Nevez et al., 2001; Vargas et al., 2001), suggesting that colonization may occur at higher rates in healthy children than in healthy adults. Similar observations were made in different animal species. In wild rabbits, positive amplification systematically occurred with samples collected from 1-month-old or younger animals (Guillot et al., 1999a). A large retrospective study concerning Pneumocystis infection in pigs indicated that animals from herds where adult and young pigs shared the same air space were more heavily infected than those from herds in which adults and weaners were reared separately (Kondo et al., 2000). In the present study, the proportion of animals with detectable Pneumocystis DNA was significantly higher in young macaques but only during the second year when eight births occurred. The detection of antibodies to Pneumocystis in the serum of the unweaned macaque suggested that seroconversion took place very early in life. However, when positive amplification occurred from unweaned macaque samples, Pneumocystis DNA was not systematically detected in the mother samples. When mothers and their unweaned monkeys were PCR-positive their respective strains did not correspond to the same sequence type, excluding a possible transmission from the young macaques to their mothers and conversely. Many observations suggested that infection from undefined environmental sources of Pneumocystis organisms was possible (Hughes, 1982). In humans, the risk of pneumocystosis has been linked to the degree of soil exposure (Navin et al., 2000). Wakefield (1996) was able to detect DNA from rat- and human-derived Pneumocystis in air samples from rural locations in the UK. This result suggested that Pneumocystis organisms might be current components of the air spora. In the present study, we found a constant circulation 3123

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of Pneumocystis organisms within the members of the colony but Pneumocystis DNA could not be detected in air. Reasons for this could be that the time sampling was probably too short (168 h vs 240 h in the study from Wakefield, 1996) or that the device for air sampling was not used at convenient places. In conclusion, we used an original model for the study of Pneumocystis transmission in a group of nonimmunocompromised animals. Throughout the study, the presence of Pneumocystis DNA was frequently detected from nasal swab samples. Anti-Pneumocystis antibodies were also detected from sera but serological titres could not be clearly correlated with the detection of Pneumocystis DNA in the upper respiratory tract. These results indicated a constant and intensive circulation of Pneumocystis organisms within the community. However, both occurrence of Pneumocystis carriage and colonization level varied significantly throughout the study in the members of this colony of apparently immunocompetent monkeys. DNA sequencing demonstrated that closely related animals (unweaned monkeys and their mothers) frequently harboured different Pneumocystis strains. This result does not contradict the hypothesis that young animals may represent a source of infection in a community of immunocompetent individuals. However, other infection sources could operate during the follow-up of the colony. Further studies including quantification of Pneumocystis DNA, and a more discriminative genotyping of isolates from monkeys should clarify this complex picture and help to elucidate both routes of transmission and carriage of Pneumocystis in non-human primates.

ACKNOWLEDGEMENTS This study was developed in the framework of both ‘Pneumocystis’ PRFMMIP network (Programme de Recherche Fondamentale en Microbiologie et Maladies infectieuses et Parasitaires, French Ministery of Education, Research and Technology) and ‘Eurocarinii’ European network (QLK2-CT2000, 01369). Financial support was provided by the ENVA Parasitology Unit. We would like to acknowledge Jacques Cabaret (INRA Nouzilly) for skilled assistance in statistical analysis, and Christophe Gaertner (Centre de Primatologie de Strasbourg) and Manju Deville (Ecole Nationale Ve´te´rinaire d’Alfort) for technical support. This work is dedicated to late Professor Ann E. Wakefield and Francine Touati.

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