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Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes: URMITE, CNRS UMR 6236 – IRD 198, Faculté de Médecine, IFR48, Université de la Méditerranée, Marseille, France
Correspondence
Michel Drancourt
Michel.Drancourt{at}medecine.univ-mrs.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Sporadic cases are also reported in several countries. In Algeria, plague re-emerged in 2003 in the Oran area after six decades of silence (Bertherat et al., 2007; Bitam et al., 2006). Indeed, long-term persistence in geographical areas of so-called plague foci, where plague re-emerges after decades of silence, is a characteristic feature of plague.
Plague is thought to exist indefinitely in rodent populations in so-called enzootic (maintenance) cycles that involve transmission between partially resistant rodents (enzootic or maintenance hosts) and their fleas. The disease can spread from enzootic to more susceptible animals (epizootic or amplifying hosts), causing rapidly spreading die-offs (epizootics) (Gage et al., 1995). Yersin (Yersin, 1894), Simond (Simond, 1898) and Raybaud (Gauthier & Raybaud, 1903) established that rat fleas transmit Y. pestis to humans. This scheme, later endorsed by the Indian National Advisory Committee on plague (Anonymous, 1906), was oversimplified and became the epidemiological dogma for plague (Drancourt et al., 2004). This classical scheme, however, does not explain the particular epidemiological patterns observed during large historical pandemics or the existence of plague foci that appear to persist for many decades in the absence of human cases or noticeable die-offs among local rodent populations (Drancourt et al., 2006).
The initial report by A. Yersin indicated that Y. pestis had been isolated from the soil of a house where the inhabitants had died of plague (Yersin, 1894). Y. pestis was later isolated from a burrow of plague-infected rodents (Karimi, 1963), and Mollaret (1963) reported the experimental persistence of Y. pestis in soil. Long-term persistence of virulent Y. pestis in soil could play a role in the particular epidemiology of plague. In this perspective, we assessed the long-term preservation of live, virulent Y. pestis biotype Orientalis using a non-quantitative model of artificially inoculated soil and a mouse model of infection.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Y. pestis strain 6/69M biotype Orientalis, a virulent strain (kindly provided by Professor Michel Simonet, Institut Pasteur, Lille, France), was cultured on 5 % sheep blood agar (bioMérieux). Natural sand collected in the Marseille area was steam-sterilized and further humidified by addition of 20 ml sterile distilled water per 500 g sterilized sand. Sterility was assessed by inoculating 5 g sand onto 5 % sheep blood agar and cefsulodin-irgasan-novobiocin agar (CIN, Becton Dickinson) and incubating at 30 °C under a 5 % CO2 atmosphere for 1 week. Soil was PCR-negative (see below). Sterilized soil was then thoroughly mixed with Y. pestis suspended in sterile phosphate-buffered saline (PBS) in order to achieve a homogeneous inoculum of 106 c.f.u. per g soil, and 500 g of this preparation was placed in an opaque plastic container incubated at room temperature for 40 weeks. Room temperature was monitored daily. Sterile distilled water (10 ml) was gently added twice a month to the container. The experiment was performed in duplicate. Before each sampling, soil was mixed evenly with a sterilized fork in order to ensure the homogeneity of the sample. An average 5 g aliquot of inoculated soil core was sampled 1 h post-inoculation (p.i.), and then every 24 h for 10 days, then every 2 weeks starting on day 15 for a total of 40 weeks. Aliquots of 1 g were immediately inoculated into 9 ml brain-heart broth (Difco) and gently agitated before 100 µl of the suspension was inoculated onto 5 % sheep blood agar and CIN. A second aliquot was deposited on slides with a pen nib for acridine orange staining (Chapin & Murray, 2003) and indirect immunofluorescence detection was performed using an anti-Y. pestis rabbit polyclonal antibody and FITC-conjugated goat anti-rabbit IgG (Immunotech) diluted 1 : 400 in PBS containing 3 % non-fat dry milk and 0.2 % Evans blue (bioMérieux). Slides were washed, mounted with Fluoprep (bioMérieux) and examined under an Olympus BX-51 epifluorescence microscope at x100 magnification. The remaining sample was frozen at –80 °C for further experiments.
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). PCRs were carried out in an Applied Biosystems 2720 thermal cycler (MJ Research). The target genes were amplified in a 50 µl reaction mixture containing 10 mM Tris/HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs (Invitrogen), 0.1 mM primer (Eurogentec) and 1 U Taq DNA polymerase (Invitrogen). Amplification of the pla gene required an initial 5 min denaturation at 95 °C and 39 cycles of 1 min denaturation at 94 °C, 30 s annealing at 60 °C, and a 1 min extension at 74 °C followed by a final 15 min extension at 75 °C; caf1 amplification used an initial 10 min denaturation at 95 °C and 40 cycles of 1 min denaturation at 94 °C, 40 s annealing at 55 °C, and a 1 min extension at 74 °C followed by 15 min final extension at 75 °C; ureD amplification used an initial 5 min denaturation at 95 °C and 39 cycles of 1 min denaturation at 94 °C, 30 s annealing at 54 °C, and 1 min extension at 74 °C followed by a 10 min final extension at 74 °C. Sterile water was used as a negative control in each PCR assay and no positive control was used. PCR products were purified using a multiscreen PCR filter plate (Millipore). Sequencing reactions were done using the d-Rhodamine Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq polymerase FS (Perkin-Elmer), and sequencing products were resolved using an ABI 3100 automated sequencer (Perkin-Elmer). Sequence analysis was performed using the ABI prism DNA sequencing analysis software package version 3.0 (Perkin-Elmer) with a Power Macintosh 7200/120. GenBank databases were searched via Internet BLAST software from the National Center for Biotechnology Information homepage (http://www.ncbi.nlm.nih.gov/BLAST/), and the sequence results were compared.
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Identification and characterization of isolates.
All isolates recovered after inoculation of agar plates and mice were identified as Y. pestis by Gram staining, oxidase test and API20E strip inoculation. Urease activity was tested in the tube (bioMérieux) after 24 h inoculation. Identification was further confirmed by PCR amplification and sequencing of the 16S rDNA (Drancourt et al., 2004), pla, ureD and caf1 genes present in single colonies.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Agar inoculation yielded >1000 colonies on blood agar over the 40-week observation period and >1000 colonies on CIN until week 25, followed by 100–1000 colonies from week 26 until week 40. Colonies were identified as Y. pestis until week 30, after which they were contaminated with Pseudomonas spp. No urease activity was observed in any Y. pestis colony. All Y. pestis-inoculated soil samples yielded positive PCR amplification of pla, ureD and caf1 genes, whereas all negative controls remained negative. The sequence of the pla amplicons yielded 93–99 % similarity with the reference (GenBank CP000310.1), the sequence of ureD amplicons yielded 85–94 % sequence similarity with the reference (GenBank AL590842.1), and the sequence of caf1 amplicons yielded 93–100 % similarity with the reference (GenBank CP000670.1). Variations in similarity values were due to variations in the sequence quality between various batches.
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). Moreover, Yersinia cells stained orange, suggesting that they were alive. Indeed, blood cultures yielded colonies identified as Y. pestis at 72 h. Likewise, cultures of lung tissues collected from dead mice yielded colonies identified as Y. pestis at 72 h, and specific pla, ureD and caf1 sequences were detected in all lung tissue specimens collected in inoculated mice, whereas negative control lungs from mock-infected mice remained negative.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Long-term persistence of Y. pestis in soil has remained a controversial issue, since several researchers failed to isolate Y. pestis from soil after Yersin's initial claim (Yersin, 1894) and most researchers believed that Y. pestis perishes quickly when outside its normal vector or hosts (Brubaker, 1991; Perry & Fetherston, 1997). Indeed, Y. pestis dies rapidly if exposed to a temperature exceeding 40 °C or when exposed to desiccation (Perry & Fetherston, 1997). Nevertheless, Mollaret (1963) reported survival of Y. pestis for 16 months in autoclaved soil and indicated that hydration of soil before experimental inoculation was necessary for Y. pestis survival (Mollaret, 1965). Furthermore, Y. pestis was isolated from the chamber of a gerbil (Meriones vinogradovi) burrow in which an animal had died from plague, but in which no animal nor dead or living fleas had been observed for 7 months (Karimi, 1963). This field observation was reproduced twice, 10 and 11 months after the closure of a contaminated burrow (Karimi, 1963). However, poorly described methodologies in these previous reports and lack of an independent confirmation prevented definite acceptance of this paradigm. Nowadays, in Madagascar, there are observations of pneumonic plague cases among persons attending ritual excavation of plague corpses in the absence of direct contact with the dead (Harilanto Razafindrazaka, personal communication). It has been previously suggested that Y. pestis urease may play a role in environmental adaptation (Sebbane et al., 2001). Indeed, unlike other Yersinia species, Y. pestis lacks urease activity due to a frame-shift mutation (Sebbane et al., 2001). We did not observe recovery of urease activity during this experiment and we did not detect reversion of the frame-shift mutation in the ureD gene, indicating that such activity is not essential for soil survival, at least under our experimental conditions.
We further demonstrated that Y. pestis strain 6/69M remained fully virulent after a 40 week incubation in humidified soil. Mice inoculated with soil samples contaminated with Y. pestis for 10–280 days developed a fatal Y. pestis septicaemia within 3 days p.i., and Y. pestis was detected in most tissues examined. Importantly, we observed lung lesions similar to those reported in a mouse model of primary plague pneumonia (Lathem et al., 2005). These data therefore indicate that Y. pestis 6/69 retained its virulence after long-term survival in soil.
Plague is characterized by decades of silence in fixed geographical foci where re-emergence of human plague has been linked to a continuous, low-level circulation of Y. pestis in rodent populations. Environmental changes leading to changes in the abundance of vector and rodent populations, reduction in rodent surveillance, and increased contact with rodents may explain the re-emergence of human plague (Duplantier et al., 2005). Among factors leading to epizootics, climatic factors including variations in temperature and precipitation appear to be important in many, but perhaps not all, areas (Gage & Kosoy, 2005). Foci characterized by long inter-epizootic periods during which Y. pestis cells are not found in the host and vector populations are also a hallmark of plague, as recently illustrated in Oran, Algeria, where plague re-emerged in 1993 after more than half a century of silence (Lounici et al., 2005). In this case, epidemiological and typing data suggested the existence of a natural focus of plague rather than recent importation of infected animals. Although such observations could simply result from a lack of adequate surveillance, this phenomenon could be explained by a chronic carrier state in rodents. The latter hypothesis relies on the co-existence of relatively resistant species that act as enzootic reservoir species and relatively susceptible animals that act as epizootic hosts. Plague-resistant species of animals may develop long-term subclinical bacteraemia and, therefore, be a durable reservoir of infection (Baltazard & Mofidi, 1950). However, field observations and experiments led the authors to conclude that resistant species are not able to sustain plague in plague foci (Baltazard et al., 1963).
The experimental data reported here provide a basis on which to examine the role of soil in the epidemiology of plague (Fig. 4). Y. pestis may exist between a short and unstable parasitic phase associated with rodents and fleas and a more stable soil phase, the so-called saprophytic phase, that would allow for survival between epizootics (Baltazard, 1964; Levi, 1997). Some animals could acquire Y. pestis by burrowing in contaminated soil, and thus initiate a new rodent/rodent flea cycle of transmission. Initial experiments by the Indian Plague Commission showed that guinea pigs could be contaminated by running freely over freshly (<24 h) contaminated soil (Anonymous, 1906). Further experiments showed that rodents, including ‘plague-resistant’ Meriones species, could acquire plague by burrowing in soils experimentally infected several days earlier. A rapid course of plague and death were hallmarks of these experiments. Of 110 dead animals, 63 had lung lesions, 31 intestinal lesions, and 20 solely spleen/lymph node lesions (Anonymous, 1906). These data indicate that either inhalation or ingestion of contaminated soil, or both, could be the routes of contamination for rodents. Other animal models have indicated that susceptible animals (e.g. mice and cats) can acquire lethal plague by either inhalation or ingestion of Y. pestis (Butler et al., 1982; Welkos et al., 2002), as reported for patients after consuming raw camel meat (Bin Saeed et al., 2005).
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). The precise environmental factors contributing to its survival in soil remain to be identified. Y. pestis is highly susceptible to variations in environmental temperature and desiccation (Mollaret, 1965; Perry & Fetherston, 1997). According to Tan and co-workers, the distribution of plague foci could be closely related to calcium- and iron-enriched soils, in agreement with the known role of calcium in the regulation of Y. pestis virulence factors (Perry & Fetherston, 1997; Tan et al., 2002). The determination of these factors may be of interest from the perspective of plague control. The experimental telluric persistence reported herein should prompt field investigations in plague foci in order to better assess the role of telluric persistence in the epidemiological chain of plague. If long-term survival of Y. pestis in natural soils that have not been sterilized can be demonstrated, along with the routine recovery of plague bacteria from soils in plague-endemic areas, Y. pestis biotype Orientalis should be added to the growing list of pathogens that persist fully virulent in soil and may be responsible for zoonoses such as Mycobacterium bovis (Young et al., 2005), Bacillus anthracis (Logan et al., 2007) and Coxiella burnetii (Evstigneeva et al., 2007). A striking parallel could be made with Vibrio cholerae, a soil- and water-borne pathogen existing in well-defined cholera foci and responsible for pandemics (Zuckerman et al., 2007). Y. pestis is therefore an additional candidate for the ‘sit-and-wait’ strategy previously described for human respiratory pathogens (Walther & Ewald, 2004). This strategy should favour evolution of pathogens towards high levels of virulence. If confirmed, the hypothesis of the telluric persistence of Y. pestis, along with the recently demonstrated transmission of Y. pestis by the body louse (Houhamdi et al., 2006), should be incorporated into revised plague epidemiology schemes.
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ACKNOWLEDGEMENTS |
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Edited by: W. Liesack
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Received 7 February 2008;
revised 26 May 2008;
accepted 29 May 2008.
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