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The prevalence of undiscovered genotypes of cryptosporidium in fish has been a big mystery, such knowledge would potentially impact aquaculture industries stock losses, pathogenicity and endangered species as well as raising the public health issue given the potential pollution of coastal and estuary waterways with cryptosporidium-infected faeces. This study investigates the prevalence of cryptosporidium in 51 aquarium fish by PCR amplification at the 18S RNA and actin locus. 1 positive was successfully sequenced and is to be aligned with other cryptosporidium clades in the taxa.
Cryptosporidium is a protozoan enteric parasite which causes illness in the gastrointestinal tract of mammalian species. Their oocysts are resilient to disinfectants when outside their hosts and transmission is done through faecal-oral route  or waterborne  . Although there have been extensive studies of C. parvum  and C.hominis  in regards to their epidemiology, taxonomy, pathology and host specificity, there is less information on cryptosporidium species infecting piscine hosts. A study in 2002  and 2004  identified 2 species in fish: C. molnari found in gilthead sea breams (Sparus aurata L.) and European sea basses (Dicentrarchus labrax L.) and C. scophthalmi (piscine genotype 1  ). A study in 2010  sequenced piscine genotypes of ornamental fish at the 18S rRNA locus and conducted a phylogenetic analysis and discovered that they formed basal clades to all other cryptosporidium species, suggesting that piscine genotypes could be the most primitive form of the species ancestry  . Finding novel genotypes of piscine-infecting species can help us isolate and study their properties such as their zoonotic significance.
2. Materials and methods
2.1 Sample collection
A total of 51 fishes were collected. 35 of them were ornamental fishes collected from a random aquarium in WA (Vebas Aquariums  ), 16 marine mullets (Mugilidae) from a random bait shop in WA. Among the fresh-water aquarium fishes, 26 were dead and 9 were alive. Live fishes were euthanized by putting them in ice. Epithelial cell scrapes of the fishes' intestines and stomachs were taken using a surgical scalpel blade and stored 1.5 ml Eppendorf tubes. Faecal matter was also present in the intestines of the 16 mullet sample scrapes and was collected as well.
2.2 DNA extraction and amplification
The 35 ornamental fish was extracted using Qiagen DNeasy blood/tissue extraaction kit. The protocol was conducted exactly as stated in the kit with the exception of the final elution step; purified DNA was eluted in 60 Âµl of AE buffer instead of 100 Âµl to concentrate the DNA. One of the tubes with roughly 250 mg of intestinal and stomach tissue was found to be mostly undigested with protease K. Additional protease K was added and incubated overnight to digest. Clogged filters were observed in 3 spin columns with sample during purification and additional centrifugation at 10,000 rpm was applied; as advised in the Qiagen troubleshooting section. 16 marine mullet fish sample scrapes were given 5 cycles of freeze-thawing and incubated in 100Â°C for 10 minutes. The samples are then extracted with PowerSoil kit protocol with the exception of the final elution being 50 Âµl of C6 elution buffer. Eluted samples are then measured for DNA concentrations by NanoDrop UV-vis spectrometry (260nm).
The target sequence is the conserved region of the cryptosporidium 18S ribosomal subunit locus and was amplified with nested PCR (double PCR) as described in (Ryan et al., 2003.)  (table 1) Fish Samples that have positive PCR products were also amplified at the actin gene locus using a hemi-nested PCR (using a common reverse primer for both primary and secondary PCR steps with different forward primers).
Table 1: Working concentrations of reagents used in the 18S rRNA locus PCR (nested). 10X Kapa
Taq buffer with dye (with 1.5mM Mg at 1X), Kapa Biosystems 5U/ul Kapa Taq polymerase. And Promega
100mM dNTP. Reactions were topped up with hospital-grade injection water to a final volume of 25Âµl.
2.4 DNA sequencing and phylogenetic analysis
Amplified PCR products were then separated and screened via gel electrophoresis. The desired PCR products (587bp) were cut out with a scalpel blade and purified by loading them into filtered 100ul pipette tips and placing them into 1.5 ml Eppendorff tubes and microcentrifuge at max speed for 30 seconds to 1 minute to filter out the DNA. 1ul of filtered DNA from gel electrophoresis was then added to a sequencing reaction using the protocol from the ABI PrismTM Dye Terminator cycle sequencing kit with alterations made to the annealing temperatures: 58Â°C and 48Â°C for 18S locus and actin locus respectively. Reverse primers were used to sequence the 18S rRNA locus and forward primers for the actin locus. Nucleotide sequences generated were analysed with chromas lite version 2.0  and FinchTV software and aligned with established genotypes from GenBank using Mega 5.05 alignment software.
3.1 PCR amplification and gel electrophoresis
The first attempt at amplifying the first 12 fish samples showed smears and primer dimmers in all samples including cryptosporidium positive controls troubleshooting experiments were then conducted to test the primers, a fresh stock concentration was prepared from the master stock and was used in tandem with the old primer stock to test the primer quality, the new primer stock showed clear bands for known positives and validates the primer quality of the older primer stock.
ladder12 fish samples nested 18sc 17 jan 2013 new primers.Tif
Figure 1: Electrophoresis 18S rRNA amplification in 1.5% agarose gel of 12 fish samples with Promega 100bp DNA ladder and one control positive. Single confirmed positive band (~580bp) in fish, other samples show smears and/or primers dimmers
All fish samples were diluted the same concentration of 100 ng/Âµl. A new primer stock was prepared and used to screen the 12 samples again and 1 positive was found in Fish 2 (Figure 1), a juvenile green chromis (Chromis viridis). The remaining samples were screened as well and 2 faint bands were found in fish 44 and 45 during gel electrophoresis as well as one undefined band in fish 51 (fish 44,45 and 51 are marine mullets/ Mugilidae). Fish 2, 44, 45 and 51 were also amplified at the actin locus with known positive controls. Positive bands were found only in fish 2 and 45 when amplified with actin locus primers, the bands along with the positive control also appear faint.
3.2 DNA sequencing
The initial 18S rRNA chromatograms generated for fish 2 and 44 had failed, showing multiple peaks and excessive backgrounds noise, which made it difficult to name bases in the sequence. Both reaction products were diluted, resequenced and a resolute chromatogram for fish 2 was generated, fish 44 also had better resolved peaks (figure 2) but had dye blob artefacts at 72-78 bases and 110-120 bases as well as multiple peaks within the sequence. From 120 bases onwards in fish 44, the terminator dye signals become greatly reduced.
The first sequencing results of the actin locus of fish 2 and 45 also showed unrecognisable signals and the reaction was resuspend and diluted. The reactions still appeared to be unrecognisable with multiple peaks (sequencing failure) except at a later sequence at 45 at 150bp onwards with a few multiple peaks with background noise (figure 3).
Figure 2: DNA sequencing results of fish 44 via ABI PrismTM Dye Terminator cycle sequencing kit showing dye blobs at 70bp and 110bp.
Figure 3: Sequencing results of fish sample 45 with unclear signals up till 150bp where there is a better signal ratio with noisy backgroun data.
18S rRNA sequence of fish 2 had been analysed via BLAST and was found to closely match a mullet genotype 18S ribosomal RNA gene partial sequence by 97%. Sequence of fish 44 was confirmed a non piscine genotype. Fish 45 actin sequence was mostly unsuitable for analysis, except at 150bp onwards, and was found to match molnari actin-like gene by 92%.
Failure to amplify the first 12 samples in the first attempt was possibly due to the poor primer quality of the old stock concentration as a result of freezing and thawing when removed from -20Â°C freezers. Another possible reason for having only secondary products (smears) and primer dimmers is that the original yield of DNA concentration was too high which could have been suboptimal for amplifying the 18S locus. Articles of PCR inhibition may have been present in the fish samples. A spike analysis was conducted: 0.5 Âµl of fish 2 positives were used as a positive control and added to other test sample reactions to find presence of PCR inhibitors, the results showed solid spiked bands from fish 44 and 51, but 41 showed no bands, this indicates that fish sample 45 has inhibition; most likely protein products that were not digested by proteases during extraction hindered PCR. The notion of fish 45 having inhibitors is conflicted by another observation: fish 45 had a positive band when amplified at the actin locus. One possible suggestion is the different primers used in each locus had different efficiency levels to the same amount of inhibition in the DNA template; Longer PCR templates increase the incidence of secondary structures which interferes with the annealing and elongation steps, making the PCR less efficient and easily aggravated by inhibitors. Primer sets of the 18S locus in this study amplify ~800bp and 587bp DNA products (external and internal respectively) whereas the primers used for the actin locus amplified 392bp and 278bp products. To reduce the level of inhibition, organic additives like bovine serum albumin (BSA) can be added to bind to protein inhibitors to improve PCR reactions  . Dimethy sulphoxide (DMSO) can also be added to the reaction to reduce the melting temperatures of GC bonds to destabilise secondary structures, however the drawback to adding consolvents like DMSO is that it also compromises the annealing of primers to the templates  . Another alternative is to dilute the DNA template to reduce the amount of inhibitors also present in the final reaction volume. BSA was added to samples 44, 45 and 51 to see if PCR would work, however all bands including non-template controls showed false positive bands, indicating contamination of PCR reagents. A troubleshooting experiment was conducted to test sterility of PCR reagents used. Hospital-grade injection water was replaced, as well as two types of reactions were prepared in tandem: one with and without 3Âµl BSA per reaction tube which were put through the same under the same thermal cyclic conditions. The gel electrophoresis results revealed positive bands in all tubes with BSA including non-templates control whereas their non-BSA counterparts showed expected bands for known positives and negative results for non-template controls and control negatives.
Having no recognizable sequence in the initial screen sample sequences (18S rRNA and actin locus) was possibly due to too much DNA product after the reaction which creates excessively strong signals. Making dilutions helped resolve the signals in the second screen. The remaining abnormalities in the chromatograms were the dye blobs in Fish 44. This occurs when there are free dye terminators that remain in the pallet after the ethanol purification step, a result of poor post-sequencing preparation. A solution for this is to scrutinize the ethanol dilutions of each purification step; the 100% ethanol solution absorbs water vapour when expose to air and may have become 95% or lower, which affects the precipitation. Partial sequence analysis of sample 45 matched C. molnari actin-like gene by 92% and further suggest that it is cryptosporidium positive and should be amplifiable in the 18S rRNA locus. The reason for having poor resolution of dye signals is possibly due to multiple templates made by the secondary PCR products. More steps can be taken to optimize the semi-nested PCR of the actin locus, such as adding a gel purification step after a primary PCR to obtain a purer template for the secondary PCR with less unwanted PCR products.
Zanguee et al. (2010) conducted a test for cryptosporidium in fish with 200 ornamental fish and found 21 positives when amplified with the 18S rRNA locus. Among the fish samples, 13 of them were green chromis (Figure 4), 2 of which tested positive. This current study concurs with that finding with 1 positive in green chromis in 51 fish samples. Several studies have also observed the incidence of cryptosporidium infections being more prevalent in juvenile piscine hosts  ,  .
C:\Users\Aspire\Desktop\notes\Summer scholarship\pics\Fish 2.JPG
Figure 4: Fish 2, a juvenile green chromis (Chromis viridis)  , a marine tropical damselfish that grows up to 8 cm.
Once molecular and phylogenetic analysis has been concluded, the seeing the percentage difference in the sequences of the isolate compared to other established piscine-derived cryptosporidium species (e.g. C. molnari) would fulfil one criteria to support species status; Genetic distance between well-known cryptosporidium species such as C. parvum and C. hominis differ by 0.6%, by that molecular analogy, comparing novel genotypes to their closest relatives within the cryptosporidium taxonomy would serve as a confident gauge to weather the novel genotypes could be considered a species of their own  . However to declare species status, morphological characterisation must be established as well. The pathogenesis of the fish specimens was unknown as they were deceased for variable lengths of time before collection.
Among 51 fish samples, 1 isolate from a juvenile green chromis has been sequenced and analysed, a phylogenetic alignment is needed to compare the evolutionary relationship with other established cryptosporidium clades. Sample 45 is a confirmed cryptosporidium positive with PCR inhibitors. Actin gene locus requires PCR optimization to obtain a cleaner template prior to sequencing reactions.