Environmental Screening of <em>Aeromonas hydrophila</em>, <em>Mycobacterium</em> spp., and <em>Pseudocapillaria tomentosa</em> in Zebrafish Systems

Abstract

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump's bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Addition of insoluble fiber to isolation media allows for increased metabolite diversity of lab-cultivable microbes derived from zebrafish gut samples

“…32 With A. hydrophila infections being a persisting problem in zebrafish research facilities, the protective bioactivity of B. subtilis could contribute to the large representation of this species in our isolation efforts. 33 Microbes responsible for maintaining homeostasis in the gut microbiome system fall under the Actinobacteria phylum. 34 These maintenance microbes, which also reside in the colon, make up only a small portion of the gut microbiome community even though they hold a pivotal role in overall gut flora health.…”

Overview of a Disease Outbreak and Introduction of a Step-by-Step Protocol for the Eradication ofMycobacterium haemophilumin a Zebrafish System

“…Following the recommendations of two detailed methodology articles providing newly established health monitoring techniques in zebrafish facilities, we used these environmental screening techniques for our samples. 24,25…”

“…Each time the biofilm swab was taken, the collection area was concentrated at the air–water interface of the sump tank. 25…”

“…Each sludge sample swab was then prepared for PCR following routine protocols. 24,25 All 10 samples (five biofilm swab and five sludge swab) were sent to QM Diagnostics for PCR testing. Samples were sent to the company in separate tubes and then pooled by the company upon receipt.…”

Detection of the parasitic nematode, Pseudocapillaria tomentosa, in zebrafish tissues and environmental DNA in research aquaria

“…No P. tomentosa target DNA was detected in unspiked control tissue samples. (Mocho, Martin, Millington, & Saavedra Torres, 2017;Murray, Varga, & Kent, 2016;You et al, 2016). There is particular interest in the availability of non-lethal screening assays such as those using water sampling protocols and the presence of pathogens shed by in- living, yet not commonly found in municipal water systems and its presence in a facility housing fish is considered to be a clear indicator of infection.…”