Combining fish and environmental PCR for diagnostics of diseased laboratory zebrafish in recirculating systems

Abstract

Precise knowledge of the health status of experimental fish is crucial to obtain high scientific and ethical standards in biomedical research. In addition to the use of sentinel fish, the examination of diseased fish is a fundamental part of all health monitoring concepts. PCR assays offer excellent sensitivity and the ability to test a broad variety of pathogenic agents in different sample types. Recently, it was shown that analysis of environmental samples such as water, sludge or detritus from static tanks can complement PCR analysis of fish and is actually more reliable for certain pathogens. In our study, we investigated whether the analysis of filtered water mixed with detritus of tanks including fish showing clinical signs of illness is suitable to complement health monitoring programs in recirculating systems. The obtained data indicate that pathogens such as Pseudoloma neurophilia or Myxidium streisingeri were exclusively or mainly found in fish, while mycobacteria were predominantly present in environmental samples. A combination of both sample types seems to be required for the detection of a broad range of infectious agents in zebrafish colonies using real-time PCR technology.

Multi-state occupancy model estimates probability of detection of an aquatic parasite using environmental DNA: Pseudoloma neurophilia in zebrafish aquaria

“…Previous studies have reported that environmental samples are not adequate for the detection of the microsporidium in zebrafish facilities (Crim et al 2017;Miller et al 2019).…”

“…Previous studies have reported that environmental samples are not adequate for the detection of the microsporidium in zebrafish facilities (Crim et al 2017; Miller et al 2019). However, this conclusion was based on the sampling of flowing water from aquaria and a sample processing method that did not incorporate a sonication step to disrupt spores as previously recommended (Sanders and Kent 2011).…”

“…The assay for P. tomentosa was shown to effectively detect the pathogens in feces, detritus, and water samples (Norris et al 2020). In contrast, P. neurophilia is not easily detectable in water with current assays (Sanders et al 2013;Crim et al 2017;Miller et al 2019).…”

Reviewing Pseudoloma neurophilia infections in the popular zebrafish model

“…The search for early detection methods lay on molecular techniques that detect the pathogen even prior the infection. Recently, and to complement PCR analysis of fish, Miller et al (2019) tested the detection of P. neurophilia by using real-time PCR taking environmental samples such as water, sludge or detritus from the tanks. However, this approach was not sensitive enough for P. neurophilia detection, contrary to what happened with mycobacteria and Myxidium streisingeri, which were mainly identified in environmental samples (Miller et al 2019).…”

“…et al (2011), Peterson et al (2011), Sanders et al (2013), Sanders et al (2014), Spagnoli et al screen large numbers of fish. Require very little tissue, it can be adapted to screen water sources, and detect all life stages of the pathogen As assay is specific to only a single pathogen, it is used as a follow-up to histology Whipps and Kent (2006), Murray et al (2011), Spagnoli et al (2015b),Miller et al (2019) of the fish in which the parasite has been found, tissues/organs in which it was present, further comments, reliability scale and references are shown. Reliability scale based on the fulfilment of the following requirements by the method: (A) useful in all live stages of the fish; (B) reliability in detecting mature spores; (C) reliability in detecting presporogonic stages of the parasite; (D) none or small amount of tissue sample/ environmental (i.e.…”