Exhaust Air Particle PCR Detects Helicobacter hepaticus Infections at Low Prevalence

Miller, Manuel; Ritter, Bärbel; Zorn, Julia; Brielmeier, Markus

doi:10.4172/2157-7579.1000343

Cited by 7 publications

(16 citation statements)

References 4 publications

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“…All 8 opportunistic microbes recovered were overall and individually more readily PCR-detectable in IVC exhaust airways than in murine specimens. These findings extend prior reports regarding the utility of PCR testing of IVC exhaust airways as a component of murine health surveillance 2,12,24,31,39,44,45 and are especially applicable to improved health monitoring of immunodeficient mice, in which such agents serve as opportunistic pathogens. 1,5,15,20,56,58,59,74 Although sanitization by mechanized washing reduced the PCR evidence of these microbes, most species (that is, 5 of the 8 obtained) remained PCR-detectable.…”

Section: Discussionsupporting

confidence: 84%

“…12,44 Consequently, PCR testing of the interiors of IVC exhaust airways has increasingly become a component of murine health monitoring. 2,12,24,31,40,44,45,64 As immunodeficient and genetically engineered murine inventories continue to grow and as the complexities of coevolving host-microbiota homeostasis are further defined, 9,10,26 additional challenges to murine biosecurity, data integrity, and study replication likely will emerge. 63 In immunodeficient murine settings, members of the microbiota may function as opportunistic pathogens, 15,74 and commensal symbionts may function together as pathobionts.…”

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confidence: 99%

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PCR Prevalence of Murine Opportunistic Microbes and their Mitigation by Using Vaporized Hydrogen Peroxide

Ragland

Miedel

Engelman

2019

j am assoc lab anim sci

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Exposing immunodeficient mice to opportunistic microbes introduces risks of data variability, morbidity, mortality, and the invalidation of studies involving unique human reagents, including the loss of primary human hematopoietic cells, patient-derived xenografts, and experimental therapeutics. The prevalence of 15 opportunistic microbes in a murine research facility was determined by yearlong PCR-based murine and IVC equipment surveillance comprising 1738 specimens. Of the 8 microbes detected, 3 organisms-Staphylococcus xylosus, Proteus mirabilis, and Pasteurella pneumotropica biotype Heyl-were most prevalent in both murine and IVC exhaust plenum specimens. Overall, the 8 detectable microbes were more readily PCR-detectable in IVC exhaust airways than in murine specimens, supporting the utility of PCR testing of IVC exhaust airways as a component of immunodeficient murine health surveillance. Vaporized hydrogen peroxide (VHP) exposure of IVC equipment left unassembled (that is, in a 'static-open' configuration) did not eliminate PCR detectable evidence of microbes. In contrast, VHP exposure of IVC equipment assembled 'active-closed' eliminated PCR-detectable evidence of all microbes. Ensuring data integrity and maintaining a topographically complex immunodeficient murine research environment is facilitated by knowing the prevalent opportunistic microbes to be monitored and by implementing a PCR-validated method of facility decontamination that mitigates opportunistic microbes and the risk of invalidation of studies involving immunodeficient mice.Abbreviations: AHU, air handling unit; BI, biological indicator; CI, chemical indicator; VHP, vaporized hydrogen peroxide

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Section: Discussionsupporting

confidence: 84%

mentioning

confidence: 99%

PCR Prevalence of Murine Opportunistic Microbes and their Mitigation by Using Vaporized Hydrogen Peroxide

Ragland

Miedel

Engelman

2019

j am assoc lab anim sci

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“…This method requires minimal additional work, while guaranteeing dependable and reproducible results that have already been shown to work with other viruses and bacteria. 13,15,16…”

Section: Discussionmentioning

confidence: 99%

“…The animals were housed in groups (sex-matched, 2-5 animals) in a room with a 12 h light/12 h dark cycle under specific pathogen-free conditions (see supplement Table 1 online at http://lan.sagepub.com) [AQ1] in IVCs (Greenline GM500; Tecniplast, Hohenpeißenberg, Germany) with 60 air changes per hour and a positive pressure mode (15)(16)(17)(18)(19)(20)(21)(22). The mice were kept on wood fiber bedding (Lignocel 3/4-S; JRS, Rosenberg, Germany) with weekly cage changes, and received sterile-filtered water along with a standard rodent diet (Altromin 1314; Altromin Spezialfutter, Lage, Germany) ad libitum.…”

Section: Animalsmentioning

confidence: 99%

“…This is particularly important for pathogens known to be non transmissible via soiled bedding, and has been successful for fur mites, murine hepatitis virus (MHV), Helicobacter spp, Pasteurella pneumotropica and Sendai virus. [13][14][15][16] In the present study, exhaust air prefilter quantitative reverse transcription PCR (RT-qPCR) was compared with serological monitoring of soiled bedding sentinels for the detection of murine norovirus (MNV) in a proof of principle field study with unknown prevalence, and also in an IVC rack with known MNV prevalence in order to determine detection limits. MNV was chosen because of its high prevalence worldwide [17][18][19][20][21] and as the most commonly detected viral agent in laboratory mice.…”

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Murine norovirus detection in the exhaust air of IVCs is more sensitive than serological analysis of soiled bedding sentinels

et al. 2016

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One limitation to housing rodents in individually ventilated cages (IVCs) is the ineffectiveness of traditional health monitoring programs that test soiled bedding sentinels every quarter. Aerogen transmission does not occur with this method. Moreover, the transmission of numerous pathogens in bedding is uncertain, and sentinel susceptibility to various pathogens varies. A novel method using particle collection from samples of exhaust air was developed in this study which was also systematically compared with routine health monitoring using soiled bedding sentinels. We used our method to screen these samples for the presence of murine norovirus (MNV), a mouse pathogen highly prevalent in laboratory animal facilities. Exhaust air particles from prefilters of IVC racks with known MNV prevalence were tested by quantitative reverse transcription polymerase chain reaction (RT-qPCR). MNV was detected in exhaust air as early as one week with one MNV-positive cage per rack, while sentinels discharged MNV RNA without seroconverting. MNV was reliably and repeatedly detected in particles collected from samples of exhaust air in all seven of the three-month sampling rounds, with increasing MNV prevalence, while sentinels only seroconverted in one round. Under field conditions, routine soiled bedding sentinel health monitoring in our animal facility failed to identify 67% ( n = 85) of positive samples by RT-qPCR of exhaust air particles. Thus, this method proved to be highly sensitive and superior to soiled bedding sentinels in the reliable detection of MNV. These results represent a major breakthrough in hygiene monitoring of rodent IVC systems and contribute to the 3R principles by reducing the number of animals used and by improving experimental conditions.

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Environmental samples make soiled bedding sentinels dispensable for hygienic monitoring of IVC-reared mouse colonies

Miller

Brielmeier

2017

Lab Anim

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Accurate knowledge of the health status of experimental animals is pivotal to high scientific and ethical standards in biomedical research. Individually ventilated cages (IVCs) are becoming the predominant system for housing laboratory mice, as they prevent cage-to-cage infections. However, this feature constitutes a major drawback for hygienic monitoring of mouse colonies, as traditional screening programs build on reliable transmission of infectious agents from experimental animals to sentinel mice commonly tested as representatives for the mouse colonies. In recent years, the laboratory animal community has realized that sentinels are ineffectual for screening mouse colonies in IVC systems because infections are often not transmitted to sentinels and therefore remain undetected. Furthermore, sentinel monitoring results in high numbers of used animals. In contrast, environmental monitoring provides a more reliable approach to identify and exclude pathogens in rodent colonies. In recent studies we provided evidence that polymerase chain reaction analysis of exhaust air particles is superior to soiled bedding sentinels for different agents. In this study, we show that testing pooled environmental samples generates more meaningful information compared to soiled bedding sentinels during routine hygienic monitoring in different barriers.

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Exhaust Air Particle PCR Detects Helicobacter hepaticus Infections at Low Prevalence

Cited by 7 publications

References 4 publications

PCR Prevalence of Murine Opportunistic Microbes and their Mitigation by Using Vaporized Hydrogen Peroxide

PCR Prevalence of Murine Opportunistic Microbes and their Mitigation by Using Vaporized Hydrogen Peroxide

Murine norovirus detection in the exhaust air of IVCs is more sensitive than serological analysis of soiled bedding sentinels

Environmental samples make soiled bedding sentinels dispensable for hygienic monitoring of IVC-reared mouse colonies

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