Impact of temperature and soil type on Mycobacterium bovis survival in the environment Elodie Barbier, Murielle Dequaire Rochelet, Laurent Gal, Maria Laura Boschiroli, Alain Hartmann To cite this version: Elodie Barbier, Murielle Dequaire Rochelet, Laurent Gal, Maria Laura Boschiroli, Alain Hartmann. Impact of temperature and soil type on Mycobacterium bovis survival in the environment. PLoS ONE, Public Library of Science, 2017, 12 (4), 10.1371/journal.pone.0176315. hal-01607023 HAL Id: hal-01607023 https://hal.archives-ouvertes.fr/hal-01607023 Submitted on 26 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Distributed under a Creative Commons Attribution| 4.0 International License RESEARCH ARTICLE Impact of temperature and soil type on Mycobacterium bovis survival in the environment Elodie Barbier1, Murielle Rochelet1, Laurent Gal1, Maria Laura Boschiroli2, Alain Hartmann1* 1 AgroeÂcologie, AgroSup Dijon, INRA, Universite de Bourgogne Franche ComteÂ, Dijon Cedex, France, 2 Universite Paris-Est, Laboratoire National de ReÂfeÂrence de la Tuberculose, Unite de Zoonoses BacteÂriennes, Laboratoire de Sante Animale, ANSES, Maisons-Alfort Cedex, France a1111111111 a1111111111 * [email protected] a1111111111 a1111111111 a1111111111 Abstract Mycobacterium bovis, the causative agent of the bovine tuberculosis (bTB), mainly affects cattle, its natural reservoir, but also a wide range of domestic and wild mammals. Besides OPEN ACCESS direct transmission via contaminated aerosols, indirect transmission of the M. bovis between Citation: Barbier E, Rochelet M, Gal L, Boschiroli wildlife and livestock might occur by inhalation or ingestion of environmental substrates con- ML, Hartmann A (2017) Impact of temperature and taminated through infected animal shedding. We monitored the survival of M. bovis in two soil type on Mycobacterium bovis survival in the soil samples chosen for their contrasted physical and-chemical properties (i.e. pH, clay con- environment. PLoS ONE 12(4): e0176315. https:// tent). The population of M. bovis spiked in sterile soils was enumerated by a culture-based doi.org/10.1371/journal.pone.0176315 method after 14, 30, 60, 90, 120 and 150 days of incubation at 4ÊC and 22ÊC. A qPCR Editor: Riccardo Manganelli, University of Padova, based assay targeting the IS1561' locus was also performed to monitor M. bovis in both Medical School, ITALY sterile and biotic spiked soils. The analysis of survival profiles using culture-based method Received: January 12, 2017 showed that M. bovis survived longer at lower temperature (4ÊC versus 22ÊC) whereas the Accepted: April 7, 2017 impact of soil characteristics on M. bovis persistence was not obvious. Furthermore, qPCR- Published: April 27, 2017 based assay detected M. bovis for a longer period of time than the culture based method Copyright: © 2017 Barbier et al. This is an open with higher gene copy numbers observed in sterile soils than in biotic ones. Impact of soil access article distributed under the terms of the type on M. bovis persistence need to be deepened in order to fill the gap of knowledge con- Creative Commons Attribution License, which cerning indirect transmission of the disease. permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information file (S1 Table). Introduction Funding: Elodier Barbier had a Doctoral fellowship Mycobacterium bovis is a pathogenic mycobacteria responsible for bovine tuberculosis (bTB). financed by the Conseil ReÂgional de Bourgogne Tuberculosis caused by M. bovis is a primarily respiratory disease that also affects various Franche Comte and FEADER Grant# 2012- organs in animals [1]. Even if bTB mainly concerns cattle, a wide range of domestic and wild 9205AAO033S04910. This work was also mammals can be infected as well [2]. On the other hand, bTB a zoonotic disease impacting supported by The Conseil Departmental de la CoÃte human health [3]. Although cattle is considered to be the principal host of M. bovis, wild fauna d'Or, the Laboratoire DeÂpartemental d'Analyse de la CoÃte d'Or. The funders had no role in study design, such as the badger (Meles meles), wild boar (Sus scrofa), red deer (Cervus elaphus) and brushtail data collection and analysis, decision to publish, or possum (Trichosurus Vulpecula) have been identified as potential wildlife reservoirs [4]. preparation of the manuscript.The order of Though transmission of M. bovis among cattle usually occurs via inhalation of contaminated PLOS ONE | https://doi.org/10.1371/journal.pone.0176315 April 27, 2017 1 / 12 Survival of Mycobacterium bovis in soil reference cited has been corrected, some numbers aerosols released by infected animals during close contacts between animals [1, 5], indirect carried over during successive amendments were transmission either by inhalation of environmental bioaerosols or ingestion of infected envi- removed. ronmental matrices may be alternative potential routes of animal infection [5±7]. Indirect Competing interests: The authors declare that no transmission through feed or water has already been demonstrated between deers, deers and competing interests exist. cattle [7, 8] and badgers and cattle [9] in laboratory experiments. Investigating this type of indirect transmission is challenging because it results at least from the combination of three essential factors i.e., i) the environmental contamination by shedding from infected animals, ii) the persistence of the bacteria under a viable state in environmental matrices and finally iii) the interaction between a new susceptible host with the contaminated matrices. The shedding of M. bovis has already been demonstrated in many species via oro- nasal mucus, sputum, urine, feces and wound discharges, depending on the species [7, 10, 11]. Previous experimental studies using various environmental substrates showed that M. bovis was able to survive for a long-time period outside of its host [12±16]. For example, using a cul- ture-based method, Ghodbane et al. [16] recovered viable M. bovis from sterile soils incubated in controlled laboratory conditions 12 months after their inoculation whereas an incubation under natural weather conditions of Michigan, USA, allowed the persistence of bacteria in sterile soils for 88 days [15]. Moreover, numerous studies have identified several climatic fac- tors such as a low temperature, an adequate moisture and a protection against solar radiation (ultra violet) as positive factors for the persistence of M. bovis in environmental matrices like feces [12, 17], food supply such as corn, hay, apples [15, 18], water [9, 15] and soil [13, 15, 17, 19]. When Fine et al. [15] compared the persistence of M. bovis in four substrates (corn, hay, water and soil), the longest survival was found in cool and moist soil (88 days in winter/spring) suggesting that this substrate probably ensured better conditions of persistence for M. bovis. Until now, environmental detection of M. bovis has been mainly achieved in soil, especially in badger sett soil or in pasture soil [14, 20±23]. To the best of our knowledge, no accurate data on the influence of soil characteristics on M. bovis persistence are available. A low pH and a high iron content are often described as major factors driving mycobacterial survival in soils, especially for Mycobacterium avium ssp. paratu- berculosis [24, 25]. Concerning M. bovis, one study based on a large enquiry in cattle farms established a relationship between the occurrence of bTB in animals and soil parameters thus showing that a 1% increase in the sandy content of soil led to an increase in odds of bTB infec- tion by 4% [26], suggesting that soil type may promote or hinder M. bovis environmental persistence. Culture-based method is the gold standard for direct diagnosis of M. bovis in animals. How- ever this method is unsuitable for analysing environmental samples due to the abundance and diversity of soil microorganisms and to the slow growth rate of M. bovis. Moreover, pre-treat- ments applied to prevent competition of fast growing organisms, such as a harsh decontamina- tion, strongly reduce M. bovis viability [14, 15]. That is why molecular detection tools such as semi-quantitative PCR or real-time PCR (qPCR) have been preferentially used to detect M. bovis environmental samples [14, 20, 21, 23]. The objective of this study was to further assess the role of the soil characteristics on persis- tence of M.bovis. Thus, the effects of both temperature and physicochemical characteristics of the soil on the survival of M. bovis were studied under controlled laboratory conditions. For this purpose, two different soils selected for their contrasted properties either sterile or natural (biotic) were spiked with known concentrations of M. bovis and incubated at two different temperatures to mimic seasonal temperature variation. The persistence of M. bovis over the time was monitored using a culture-based