The authors would like to thank the BRF Aquatics team of the Francis Crick Institute for their technical help and critical input. This work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001999), the UK Medical Research Council (FC001999), and the Wellcome Trust (FC001999).

1. Exposure of Pre-filtration Sentinels Out of a Recirculating System
<ol>
	<li>Set a clean 8 L tank out of a recirculating system. Fill it up with water coming from the sumps. Add 2 ceramic beads or sponge cubes of bio-media from the systems to screen (Figure 1). Add 1 - 2 D. rerio/L (i.e., 12 fish). Use wild type fish of the dominant genetic background in the facility, for example AB.
	<ol>
		<li>Select at least 6 fish as follows: at least one female and one male below 6 months of age, one female and one male between 6 and 18 months, and one female and one male above 18 months of age.</li>
	</ol>
	</li>
	<li>Feed once a day. Vary the diets (e.g., dry diet, brine shrimp) to make sure the sentinels are exposed to all the diets used in the zebrafish facility. Change water on Monday, Wednesday, and Friday, i.e., three times a week for 4 months.
	<ol>
		<li>To conduct the water change, transfer sentinels and bio-media into a temporary tank. Empty completely the sentinel tank and clean it. Refill the sentinel tank with sump water only. Put the sentinels and bio-media back.</li>
	</ol>
	</li>
	<li>Expose the sentinels for 4 months to sump water. Euthanize the fish by an approved method such as immersion in an overdose solution of 2-phenoxyethanol (3 mL/L).</li>
	<li>To confirm death, wait for 10 min after cessation of opercula movement.
	<ol>
		<li>Grab the cadaver with forceps and freeze it entirely at -80 &#176;C in an identified container. This will be used for PCR12,15.</li>
		<li>Alternatively, cut the tailat the caudal peduncle, nick the abdominal wall, and set in 4% formalin. 
		CAUTION: Use gloves and fume cabinet for histopathology. Label the sample container.</li>
	</ol>
	</li>
</ol>
2. Sump Swabs
<ol>
	<li>Use a sterile dry swab with a plastic shaft. Wear gloves. Locate the surface to swab (sump wall at the surface of the water) and remove any item preventing easy access to the surface. Choose a sump surface with low flow.</li>
	<li>Unsheathe the swab by removing the outer packaging and expose the sterile cotton tip to the air. Avoid cross contamination of the swab by taking care not to touch untested surfaces.
	<ol>
		<li>Swab the sump wall over 5 - 10 cm to absorb the water and biofilm at the sump water surface level.</li>
		<li>Sheath the swab back or break the tip into a sterile centrifuge tube. Label the sample and send for PCR testing or freeze at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Detection of P. tomentosa Eggs at the Bottom of a Tank
<ol>
	<li>Sludge analysis by microscopy
	<ol>
		<li>Use a 60 mL syringe9 to aspirate the sludge at the bottom of a sump or any tank holding fish including sentinels. Divide the sample into 15 mL tubes. Close the tubes with their screw tops and label the tubes.</li>
		<li>Prepare the sugar saturated solution (specific gravity = 1.27) by mixing 227 g of granulated sugar in 177 mL of hot water with a magnetic stirrer14.</li>
		<li>Centrifuge the 15 mL tubes at 175 - 250 x g for 10 min in a centrifuge with swing buckets. Decant the tubes and keep the sediment in their tube.</li>
		<li>Fill up the tubes halfway with the sugar saturated solution. Close the tubes with their screw top and thoroughly mix the sediment with the solution.</li>
		<li>Place the tubes in the centrifuge swing buckets and fill them up with sugar saturated solution to the top. Set one cover glass gently on top of each tube and in contact with the sugar saturated solution.</li>
		<li>Centrifuge at 175 - 250 x g for 10 min. Note that some cover glass may fall and break during centrifugation hence there are 4 tubes for each 60 mL sample. Lift the cover glass and set it on a glass slide. Label the slide with a pencil or marker pen.
		<ol>
			<li>In case too many cover glass breakages occur, fill up most of the tube with the sugar saturated solution, centrifuge at 175 - 250 x g for 10 min, fill up to the top with the sugar saturated solution, then gently set the cover glass. Wait for 30 min.</li>
		</ol>
		</li>
		<li>Look for the P. tomentosa eggs with the microscope13 (Figure 2 and Video 1). Identify the bipolar plugs at magnification 400X6. 
		NOTE: The size of the eggs is 57 - 78 &#181;m long and 27 - 39 &#181;m diameter16,17. Note that one positive slide is enough for the 60 mL sample to be declared positive.</li>
	</ol>
	</li>
	<li>Screening imported animals in quarantine for P. tomentosa eggs
	<ol>
		<li>Set male and female D. rerio in a tank. Add to the tank a device normally used to harvest and preserve spawned eggs but use it here to collect feces (Figure 3). 
		NOTE: For example, a full 1 L breeding tank (outer tank and inner tank with grated bottom) is completely submerged in a 13 L tank, allowing free access for the fish to move in and out of the breeding device.</li>
		<li>Remove the breeding device after one week and harvest the collected sludge in the breeding device as described in step 3.1 &#34;sludge analysis by microscopy.&#34;</li>
	</ol>
	</li>
</ol>
4. PCR on Sludge Sediment
<ol>
	<li>Aspirate the sludge at the bottom of a tank or sump with a 60 mL syringe9 and transfer the sample to a 60 mL tube. Close the tube with its screw top and label the tube. Dispose of the syringe.</li>
	<li>Shake the 60 mL tube and transfer 15 mL to a 15 mL tube. Close the tube with its screw top and label the tube.</li>
	<li>Centrifuge the 15 mL tube at 175 - 250 x g for 10 min in a centrifuge with swing buckets. Decant the tubes and keep the sediment in the tube.</li>
	<li>Unsheathe a swab by removing the outer packaging and expose the sterile cotton tip to the air. Avoid cross contamination of the swab by taking care not to touch untested surfaces.
	<ol>
		<li>Swab the sediment in the tube for 15 s.</li>
		<li>Sheath the swab back or break the tip into a sterile centrifuge tube. Label the sample, freeze at -80 &#176;C and send for PCR testing. 
		NOTE: 45 mL remain in the 60 mL tube. This can be kept for detection of P. tomentosa eggs by sludge analysis by microscopy as described in step 3.1, for example, to confirm the PCR result. The PCR in the sludge can be tried for screening imported animals following step 3.2.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Limitations of the Techniques, Critical Steps, and Troubleshooting:
The age, gender, strain, and length of exposure of the sentinels are not standardized. This is shown in Table 1. There is very little screening of fish below 6 months of age, or of aged fish. There may be some pathogens that affect the young fish as there are some pathogens that are more prevalent in the older population10,18,19,20. Similarly, the gender is not considered in the selection of some sentinel groups despite some report that there is a gender bias for some pathogens21. The proposed technique tries to address these issues, although the choice of the strain could be made according to a specific pathogen to monitor. For example, TU could help with the detection of Mycobacterium spp.12,22, but there is a risk that the sentinels would then act as a reservoir or display clinical signs. Regarding the length of exposure, the approach of the Zebrafish International Resource Center10 increases the chances to detect pathogens that could be missed with an inadequate contamination period. The need for prolonged exposure implies that sentinels are not readily available. The addition of the environmental samples allows some flexibility and the multiplication of the screening events. For example, sampling can take place every other month with a 4 month interval between each screening method. This may reduce the lapse of time before a newly introduced pathogen is detected.
The environmental screening techniques rely on the detection of pathogens in the environment. The pathogens are shed by the fish and therefore diluted in the system water. The possibility of capturing the pathogens by water filtration23 was not explored. The methods we describe are only effective if pathogens are given enough time to multiply in fish and biofilm to reach a threshold of contamination allowing detection. This limitation of the techniques is minimized by a critical selection of the sampling sites: the sludge in the tank is sampled rather than the sump sludge, and the water and biofilm are sampled at the surface of the sump rather than in a tank or post-filtration. Nonetheless, all the samples from the same system are unlikely to give the same results. Positive results for P. tomentosa can be confirmed by using another assay (histopathology, PCR, or sludge analysis). Mycobacterial PCR positive results can be confirmed by culture or by another diagnostic laboratory. However, when establishing a health status, further samples are recommended to confirm negative results from any environmental screening technique.
Significance of the Technique with Respect to Existing/Alternative Methods:
Mycobacterium spp. are common in the environment and their presence in the sump does not predict their pathogenicity12. Mocho9 showed that monitoring mortality rates is key to survey the developments of health issues. The use of animal samples remains essential to any veterinary investigation. Health monitoring implies detection of all prevalent pathogens in a facility and this cannot be achieved with the sole use of environmental screening techniques. Nevertheless, a lack of sensitivity of the diagnosis tools can delay or prevent an accurate description of the health status. Whilst the use of sentinels reduces the number of fish required to detect a prevalent microbe in the population, the lack of sensitivity adds weight to using a combination of methods, including environmental screening5,23. Indeed the Specific Pathogen Free status is usually defined as the absence of a species in the facility such that environmental and animal samples must test negative24,25.
The sump swab results to identify Mycobacterium spp. show that relying on fish samples may lead to a false negative health status. The 6 tested mycobacterial species are described as pathogenic or potential pathogenic in zebrafish15 and some would not be eliminated by egg surface disinfection with chlorine26 as routinely performed in quarantine. Therefore, the false negative may have some consequences for collaborators who import lines. For example, M. fortuitum was missed by the PCR on fish sample but more than half of the sump swab PCR detected it. Considering that these mycobacteria are more resistant to chlorine than others and their ability to grow in the water systems27, it is a risk for the non-contaminated importing facility. To allow the import of lines, managers need to trust and compare the health reports of the exporting facility with theirs. The ICLAS Performance Evaluation Program28 is key to that process in rodents. The RESAMA network reports the detection of M. gordonae and M. mucogenicum in French D. rerio11. These Mycobacteria are not proposed in the panels of the commercial laboratories that we use. It would be useful to extend the ICLAS program and to harmonize the diagnostic assays as well as the list of pathogenic species29.
A. hydrophila is also a pathogen that has the potential to be introduced when importing animals, although its susceptibility to chlorine30 makes its elimination more likely during routine egg surface disinfection. The sump, swab, and sludge results show that environmental screening can be used to detect this pathogen. Other bacteria like Mycobacterium spp. have been detected in the sludge by PCR23. This type of sample is particularly relevant since it allows the detection of shed pathogens. For example, another new application is the sludge analysis to screen imported fish in quarantine for P. tomentosa. The parasite is a threat to animal&#39;s health13 and neoplasia models16. Moreover, chlorine concentrations used in routine zebrafish egg surface disinfection are not efficient31. Therefore, the ability to screen the imported animals with a one-week turnover and without any fish euthanasia seems very attractive. This technique can influence the quarantine and biosecurity rules by allowing a triage of imports. A decision-making process is then designed according to the prevalent pathogens in the exporting facility, the detected pathogens in the samples from the imported fish, and the risk of compromising the health status of the importing facility10.
Future Applications or Directions after Mastering These Techniques:
Even if routine quarantine treatment is the chosen option, the efficacy of such medication32,33,34,35,36 can be assessed with the breeding device sludge analysis. More generally, the environmental screening could be used to test compounds against bacteria and parasite eradication, including in the fish biotope. Another niche application of the environmental screening is to monitor the pathogen population in the live feed37,38. Though the main application of these techniques is as a valuable addition to the diagnosis toolbox for the health monitoring in zebrafish facilities. Thanks to a more accurate, cost and time efficient definition of the health status, sump swabs and sludge analysis are complementary to the sentinel surveillance and the routine quarantine practice. Indeed, the future of these techniques is to be a routine part of any aquatic laboratory health report.

In order to protect research and animal welfare1,2, the presence of pathogens is monitored within animal facilities. In the case of zebrafish, health monitoring3,4,5,6,7,8,9,10,11 often relies on animals analyzed post mortem by histopathology, bacteriology culture, or molecular methods. Testing only colony animals is not the recommended method due to the number of fish and related expenses that would be required to detect pathogens of low prevalence. Therefore, the preferred method is to expose a small group of animals to a higher load of contaminants. These fish are called pre-filtration sentinels. This exposure lasts for months and it involves an increase in the animal carer&#39;s workload and/or some purpose-built engineering solution. Another challenge is the screening of imported lines in quarantine where the fertile animals are to be kept alive and this is not compatible with routine assays on carcasses.
We describe here some methods to detect certain zebrafish pathogens (A. hydrophila, Mycobacterium spp., and P. tomentosa) by screening the aquatic system&#39;s environment. The aim is to reduce the number of fish used for health monitoring and to optimize turnover, cost, and sensitivity of the detection. Such methods are an alternative to the use of animals and some techniques can be applied to screening imports in quarantine. For example, Mocho9 was able to identify more pathogenic mycobacterial species by performing PCR on sump swabs rather than on zebrafish (including sentinels), and this was obtained with fewer samples. In that same study, P. tomentosa eggs were detected with more sensitivity by screening the tank sludge using flotation and microscopy rather than testing fish by PCR and histopathology.
Table 1 summarizes the various characteristics of sentinel programs3,4,5,6,7,8,9,10 used by a number of zebrafish facilities. Post-filtration sentinels receive water in the same manner as any colony fish whereas pre-filtration sentinels receive water once it has circulated through colony fish tanks first. For example, pre-filtration sentinels can be set up on the recirculating system by continuously receiving sump water. This may not be an option when there are many independent systems in one room. In this case, one tank of pre-filtration sentinels can be used to screen the whole room. The sentinels are in a static tank, out of the recirculating system, and their water is changed regularly, using only pre-filtration water i.e., sump water from all the systems in the room. This technique is described below as a baseline for comparison with the efficacy of the environmental screening. The proposed set-up is designed to control water quality issues like a decrease of pH or a nitrogen pollution.
The concept of the bacterial environmental screening relies on the hypothesis that bacteria are detectable in biofilm such as that found on the sump wall at the water surface or in the sludge at the bottom of a tank. The sump seems an ideal sampling point in a recirculating aquaculture system since it collects waste (water, feces, feed, and other organic material) from all tanks pre-filtration. The surface of the sump is often easily reachable, the swabbing is fast, and it can be performed aseptically to avoid cross-contamination of the sample (from gloves for example). The concept is used to identify prevalent pathogenic Mycobacterium spp. in zebrafish systems9,12. The technique is described below and we are also reporting detection of A. hydrophila in zebrafish sump surface swabs and sludge.
The environmental screening for parasite eggs is based on the detection by Murray et al.13 and the flotation technique is used routinely for parasitology and microscopic screening of parasite eggs in feces14. Mocho9 proposed an alternative to the sampling process and showed that the technique could be used to detect other species of the fish biotope. Infected D. rerio pass P. tomentosa eggs with their feces and the parasite eggs remain at the bottom of the tank, in the sludge. They can be collected there due to their density being greater than water. The density of the eggs is used to process the environmental sample too. A first flotation with centrifugation separates water and light debris from heavier matter. A second centrifugation relies on saturated sugar solution (with a density greater than density of P. tomentosa eggs) to allow for the parasite eggs to emerge at the surface of the tube.
The screening for bacteria in the biofilm and for P. tomentosa from the bottom of the tank can be combined by performing PCR for all these pathogens on the sludge sample sediment obtained after the first centrifugation. This optimizes the sampling time. The method is described below. We also propose to use these techniques in a quarantine context. To screen imported adult zebrafish that need to be kept alive, a breeding device is inserted to the quarantine tank. After one week, feces and other waste materials in the breeding device are collected and screened by microscopy or PCR. The technique is described below and some P. tomentosa eggs were detected by microscopy in this context.

<table><tbody><tr><td>Aqua-Sed 250 mL</td><td>Vetark</td><td></td><td>2-phenoxyethanol</td></tr><tr><td>Tubed Sterile Dryswab Tip</td><td>mwe</td><td>MW100</td><td>Sump surface</td></tr><tr><td>BD Plastipak Disposable Syringe 50mL Eccentric</td><td>Becton&lt;br/&gt; Dickinson</td><td>300866</td><td>They are actually&lt;br/&gt; graduated to 60 ml</td></tr><tr><td>Centrifuge tube 15 mL Corning</td><td>Corning</td><td>430766</td><td></td></tr><tr><td>Centaur 2 benchtop centrifuge with 4 x 200 mL Swing&amp;ndash;Out Rotor (unsealed)</td><td>Sanyo</td><td>MSB020.CX1.5</td><td></td></tr><tr><td>Cover Glass 22 mm x22 mm</td><td>Menzel-Glaser</td><td>MNJ-350-020H</td><td></td></tr><tr><td>Plain Swab Sterile Plastic Applicator Rayon Tipped White Cap</td><td>Sterilin Ltd Thermo Fisher Scientific</td><td>F155CA</td><td>Swab sediment from sludge</td></tr><tr><td>50 mL Self-Standing Centrifuge Tube CentriStar Cap</td><td>Corning</td><td>430921</td><td></td></tr><tr><td>In-Tank Spawning Tray Set</td><td>MBK Installations Ltd</td><td></td><td></td></tr></tbody></table>

environmental screening of aeromonas hydrophila, mycobacterium spp., and pseudocapillaria tomentosa in zebrafish systems

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&#39;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.

The benefits of the sump swabs to identify prevalent Mycobacterium spp. compared to fish samples are supported by the results in Figure 4. Of 115 fish tested, M. chelonae and M. haemophilum were detected in 5% and 3% of the samples, respectively. No other pathogenic mycobacterial species was identified. From the same systems, 49 sump swabs revealed the presence of 5 mycobacterial species. Odds ratio are calculated with the hypothesis that M. chelonae and M. fortuitum are detected more frequently by PCR in the surface sump swabs than in the fish sample. This is statistically significant with respective odds ratio of 11 (95% CI: 4 to 29; p &#60;0.0001) and 306 (95% CI: 18 to 5208; p = 0.0001). The results show that the surface sump swab technique is a valuable alternative to the sole use of sentinels to screen the zebrafish facility for Mycobacterium spp. The environmental samples were also used to screen for A. hydrophila. These bacteria were detected in fish, sludge, and surface samples (Figure 4). This also supports the ability of the proposed techniques to screen the fish biotope.
Regarding the sludge analysis to detect P. tomentosa eggs, Mocho9 detected the parasite in 27% of the fish samples by PCR and histopathology whereas the eggs were detected in 93% of analyzed sludge from the same system. Here, the technique was challenged to reproduce quarantine screening. In this context, imported animals cannot be sampled, and assessing their health status in a timely manner helps with the biosecurity management. Fish of unknown health status from a P. tomentosa positive facility were set in 8 tanks with breeding devices: maximum of 16 fish/13 L tank, 7 transgenic and 1 wild type line, mixed gender, aged 4 - 24 months. The sludge was harvested from the devices after one week and analyzed by microscopy. P. tomentosa eggs were seen in 7 (88%) samples. Finally, the PCR detection of the parasite was trialed on sump swabs and sludge sediments. 4 out of 6 sludge samples were PCR positive and all results were negative for the surface swabs. This is not surprising since the sludge screening relies on the ability of the eggs to fall to the bottom of the tank. This shows that the sludge analysis techniques can be used to screen D. rerio aquaria for P. tomentosa infestation and that the methods can be adapted for the screening of imported animals.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55306/55306fig1.jpg" /> 
Figure 1: Pre-filtration sentinel tank out of the recirculating system. An 8 L tank is filled up with water and bio-media from the sumps of the systems to screen. The two white ceramic bio-media beads sit at the bottom of the tank (in the middle of the picture). 12 fish are selected according to their age, strain, and gender, and they are added to the sentinel tank. <a href="https://cloudflare.jove.com/files/ftp_upload/55306/55306fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55306/55306fig2.jpg" /> 
Figure 2: P. tomentosa egg detected during sludge analysis by microscopy. Magnification used was 400X. Arrows indicate the bipolar plugs. Scale bar = 50 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/55306/55306fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55306/55306fig3.jpg" /> 
Figure 3: Breeding device submerged in a fish holding tank to collect sludge for analysis. This tank is set on a bench for the purpose of the picture; it is set otherwise in the recirculating system. <a href="https://cloudflare.jove.com/files/ftp_upload/55306/55306fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55306/55306fig4.jpg" /> 
Figure 4: Percentage of identification of Mycobacterium spp., A. hydrophila, and P. tomentosa by PCR on fish, surface sump swabs, and sump sludge. Percentage is obtained by dividing the number of positive results for each pathogen species by the number of tested samples. The percentage of positive results is given by sample type as FISH, SURFACE, or SLUDGE, and indicated after the bacteria&#39;s name. 115 fish and 49 surface sump swabs were tested by PCR for identification of Mycobacterium spp. The data are compiled with Mocho&#39;s9 results since it is an extension of this study. The fish are mainly pre-filtration sentinels as per protocol section 1. Rarely, when sentinels were not available, escapees and old colony fish (&#62;18 months) were sampled. All tested systems for Mycobacterium spp. were tested on fish and sump swabs. Odds ratio are calculated with the hypothesis that M. chelonae and M. fortuitum are detected more frequently by PCR in the surface sump swabs than in the fish sample. This is statistically significant with respective odds ratio of 11 (95% CI: 4 to 29; p &#60;0.0001) and 306 (95% CI: 18 to 5208; p = 0.0001). Mycobacterium marinum PCR was negative for all the samples and it is therefore deemed absent from these facilities and not included in the analysis. 12 fish, 14 surface sump swabs, and 6 sump sludge were tested by PCR for the presence of A. hydrophila. 6 sumps were tested for the presence of P. tomentosa on the surface and in the sludge. PCR = Polymerase Chain Reaction. CI = Confidence Interval. * and ** indicate statistical significance; other comparisons were not statistically significant. <a href="https://cloudflare.jove.com/files/ftp_upload/55306/55306fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/55306/55306vid1.jpg" /> 
Video 1: Scanning for an egg of P. tomentosa with the microscope. The slide is obtained from sludge analysis as described in step 3.1. The field is scanned to detect an egg of P. tomentosa. Once a structure is recognized, it is zoomed in to confirm the identification at higher resolution. <a href="http://cloudflare.jove.com/files/ftp_upload/55306/video_scan_for_egg.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Authors</td>
			<td>Age at the start of exposure</td>
			<td>Length of exposure</td>
			<td>Sampling age</td>
			<td>Pre or Post 
			filtration</td>
			<td>Gender</td>
			<td>Strain(s)</td>
		</tr>
		<tr>
			<td>Barton et al.3</td>
			<td>4 months</td>
			<td>6 months</td>
			<td>10 months</td>
			<td>Pre-filtration</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Borges et al.4</td>
			<td>6 months</td>
			<td>6 months</td>
			<td>12 months</td>
			<td>Pre-filtration</td>
			<td>N/A</td>
			<td>AB wild type</td>
		</tr>
		<tr>
			<td>Collymore et al.5</td>
			<td>As young as possible</td>
			<td>3 months</td>
			<td>&#60;6 months</td>
			<td>Pre-filtration</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Geisler et al.6</td>
			<td>4 months</td>
			<td>4 months</td>
			<td>8 months</td>
			<td>Pre and post 
			filtration</td>
			<td>N/A</td>
			<td>AB wild type</td>
		</tr>
		<tr>
			<td>Liu et al.7</td>
			<td>3 months</td>
			<td>1-6 months</td>
			<td>4-9 months</td>
			<td>Pre and post 
			filtration</td>
			<td>N/A</td>
			<td>Wild type</td>
		</tr>
		<tr>
			<td>Martins et al.8</td>
			<td>3 months</td>
			<td>3 months</td>
			<td>6 months</td>
			<td>Pre-filtration</td>
			<td>N/A</td>
			<td>Wild type</td>
		</tr>
		<tr>
			<td>Mocho9</td>
			<td>&#60;6 months, 
			6 - 12 months, 
			&#62;18 months</td>
			<td>4 months</td>
			<td>7 - 24 months</td>
			<td>Pre-filtration</td>
			<td>1 female and 
			1 male of 
			each age group</td>
			<td>AB wild type</td>
		</tr>
		<tr>
			<td>Murray et al.10</td>
			<td>3-4 months</td>
			<td>3 months, 6 months, 1 year</td>
			<td>7, 10, and 16 
			months</td>
			<td>Pre and post 
			filtration</td>
			<td>N/A</td>
			<td>AB wild type</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of sentinel settings in zebrafish facilities. The sentinel fish may be selected according to their age, gender, or strain. They are exposed for a defined period and they receive pre- or post-filtration system water. The data are compiled from the 2016 special issue on health of the Zebrafish journal3,4,5,6,7,8,9,10.

Watch this Scientific Journal Video about Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems at JoVE.com

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, ...

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Research

JoVE Journal

Biology

9.7K Views.  The Francis Crick Institute. This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

Video: Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Environment

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Immunology and Infection

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social defects.

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, ...

Behavior

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is important to standardize protocols to ensure proper analyses and interpretations. In our previous studies, we have measured boldness by monitoring a series of behaviors (time near surface, latency in transitions, number of transitions, and darts) in a 1.5 L trapezoidal tank. Likewise, we quantified aggression by observing bites, lateral displays, darts, and time near an inclined mirror in a rectangular 19 L tank. By dividing a 76 L tank into thirds, we also examined shoaling preferences. The shoaling assay is a highly customizable assay and can be tailored for specific hypotheses. However, protocols for this assay also must be standardized, yet flexible enough for customization. In previous studies, end chambers were either empty, contained 5 or 10 zebrafish, or 5 pearl danios (D. albolineatus). In the following manuscript, we present a detailed protocol and representative data that accompany successful applications of the protocol, which will allow for replication of behavioral syndrome experiments.

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is ...

Medicine

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

Neuroscience

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, therapeutic drug discovery, and other biomedical research applications. The presented protocol aims to provide a clear guide for drug screening and toxicity testing in early-stage zebrafish larvae (aged 3-7 days post fertilization, dpf). Several methodologies describing toxicity testing in zebrafish larvae have been published, albeit lacking a standardized protocol for exposure durations, rate of media changes, and morphology scoring, amongst others. We experimentally determined the ideal larvae ages for conducting the experiments, the plate sizes that work best for those ages, the rate of media changes that provide reliable results while limiting wastage&#160;of test compound, and a morphology scoring system that is easily quantifiable in a statistical manner. The manuscript describes in detail a protocol for toxicity testing in zebrafish larvae aged between&#160;3 to 7 dpf, aiming to target the gap in drug toxicity testing and taking the first step towards a standardized method similar to the Fish Embryo Acute Toxicity test.

This protocol describes a drug screening procedure in zebrafish larvae 3-7 days post fertilization followed by morphological assessment.

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, ...

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

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Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

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Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae