Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 2.1.14.

GYRODACTYLOSIS
(Gyrodactylus salaris)

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SUMMARY

Gyrodactylus salaris (Monogenea: Gyrodactylidae) is a highly pathogenic parasite of wild and farmed Atlantic salmon parr (Salmo salar) and smolt. Several other salmonids are susceptible to G. salaris, such as rainbow trout (Oncorhynchus mykiss), Arctic char (Salvelinus alpinus), North American brook trout (S. fontinalis), grayling (Thymallus thymallus), North American lake trout (Salvelinus namaycush) and brown trout (Salmo trutta) (in declining order of susceptibility). Gyrodactylus salaris may be present in farmed salmonids for years, especially rainbow trout, without any signs of clinical disease. Single parasite infestations are common and demand a special procedure when monitoring for the occurrence of Gyrodactylus specimens. The whole surface of a fish, including gills and mouth cavity, must be examined under a dissecting microscope. Identification of G. salaris is based on morphology and morphometry of hooks and bars in the opisthaptor, or by DNA analysis.
 

INTRODUCTION

Gyrodactylosis of Atlantic salmon (Salmo salar), is caused by the viviparous freshwater monogenean Gyrodactylus salaris (4). The presence of the parasite in Swedish hatcheries has been known since the beginning of the 1950s, but it was regarded as harmless until its introduction into Norway in the mid-1970s.
 
Gyrodactylus salaris is restricted in its distribution to Europe. It has been found in farmed Atlantic salmon or farmed rainbow trout (Oncorhynchus mykiss) in several (mainly northern) European countries. The parasite has been found in wild salmonids, mainly Atlantic salmon parr, from rivers in Russia, Sweden and Norway. Gyrodactylus salaris is much more common in farmed rainbow trout than previously thought, and is likely to be present in more countries than those currently known. Great Britain and Ireland appear to be free from the parasite.
 
In experiments, Atlantic salmon from a Scottish river have shown equal susceptibility to G. salaris as Norwegian salmon, while Atlantic salmon from the River Neva in the Baltic have shown significant resistance to the parasite.
 
Gyrodactylus salaris survives and reproduces in several salmonids, such as rainbow trout (Oncorhynchus mykiss), Arctic char (Salvelinus alpinus), North American brook trout (S. fontinalis), grayling (Thymallus thymallus), North American lake trout (Salvelinus namaycush) and brown trout (Salmo trutta) (in declining order of susceptibility).
 
Gyrodactylus salaris has spread between rivers and farms mainly by the transport/restocking of live fish. Fish swimming through brackish water can also cause the parasite to be spread between rivers. Although G. salaris is a freshwater parasite, it has an almost normal reproduction at 5% salinity. The survival time at higher salinity may be significant, for example G. salaris survives for up to 240 and 42 hours at 10% and 20% salinity, respectively.
 

SAMPLING PROCEDURES

Gyrodactylus specimens may die or detach from the host if the water quality/chemistry is changed. Furthermore, Gyrodactylus specimens die rapidly if not covered with water, and often leave a host soon after it dies. When fish are sent to a laboratory for Gyrodactylus examination, the sampling procedures used must be somewhat different from those described in Chapter I.1.
 
Fish must be alive when collected and should be transported in the same water as that in which they lived. Because excretory (and other) products from the fish may change the water's quality, the number of fish/fish volume should be relatively small compared with the volume of water in the container. Transportation time should also be considered. The container should carry one fish species only because experiments and experience have shown that mixing fish species may initiate detachment of Gyrodactylus specimens from their hosts.
 
Dead fish, transported on ice, are not acceptable for Gyrodactylus examination, even if the fish are sent separately in plastic bags, etc. The parasites soon die if not covered in water, and as these parasites do not have an exoskeleton, dead parasites disintegrate as quickly as the mucus and epidermis cells of the fish. If such dead fish are rinsed in water, Gyrodactylus specimens may be found in the bottom sediment. However, if specimens are not found in the sediment, it cannot be concluded that the fish were uninfected.
 
A good alternative to examination of 'live' fish, is examination of formaldehyde-fixed or ethanol-preserved fish. The concentration of formaldehyde after fixation should not be lower than 4% (10% formalin). The formaldehyde concentration should be 8-10% (20-25% formalin) before adding the fish, because water is freed from the fish during fixation. The concentration of ethanol after conservation should be about 70%. If the concentration is lower, the mucus and epidermis may disintegrate and Gyrodactylus specimens, even if they are preserved, may drop off. Because water is released from the fish during preservation, the concentration of ethanol may be as high as 96% before the fish are added. Gyrodactylus specimens are more easily detected on formaldehyde-preserved compared with ethanol-preserved fish because with the former, the parasites become more whitish and easier to detect. Also, for the safety of laboratory staff, formaldehyde is a better preservative than pure ethanol with no additives. However, if examination of DNA probes is to be used for identification of Gyrodactylus specimens, preservation in pure ethanol is (at present) a prerequisite.
 
The fish should be fixed or preserved in relatively large bottles that provide excess space and fixative (5-10 fold). The opening to the bottles should be wide to avoid the possibility of scraping off Gyrodactylus specimens when the fish are taken out for examination. Bottles should be stored in a horizontal position, as should the fish, during fixation/preservation. This makes later examination of the fish easier. When fish are fixed/preserved, the bottles should be stored in a vertical position after 2-3 days.
 

DIAGNOSTIC PROCEDURES

When present on wild and farmed Atlantic salmon parr and smolt, G. salaris usually causes gyrodactylosis. Disease outbreaks may occur at any time and at any water temperature, but are most common in spring and in periods when the water temperature is 7-17°C. The fins, especially the dorsal and pectoral fins, are most commonly infested, but parasite specimens may occur all over the epidermis of the host, including the nostrils, the gills and mouth cavity. Gyrodactylus specimens are usually present in scrapings from gills, skin or fins of fish with gyrodactylosis. However, if the number of Gyrodactylus specimens is low, the chances of detecting the parasites by this method are limited.
 
Gyrodactylus salaris may be present in very low numbers on fish without any signs of clinical disease. This occurs occasionally with Atlantic salmon, but commonly with other salmonids, especially rainbow trout. Single parasite infestations are common, and the single parasite may be attached to any site on the fish surface. A special procedure is therefore required when monitoring for the occurrence of Gyrodactylus specimens on asymptomatic fish (healthy fish).
 

1.	Screening and Presumptive Diagnostic Methods for the Presence of Gyrodactylus Specimens
	Fish should be examined individually under a binocular dissecting microscope with good illumination. The fish should be placed in a box and completely covered in transport water. Living parasites are more easily detected by their movements, so disturbing light refraction on the skin of the fish should be avoided. Fish should be killed using a needle to the brain to avoid blood leaking into the water. If the dissecting microscope is illuminated from above, the bottom of the box should be black. This will increase the contrast and the parasites will be detected more easily. The whole surface of the fish, including gills and mouth cavity, must be examined. It is best to use two forceps for this process. Each fish should be examined for at least 5 minutes. The fins of relatively small, unfixed fish, which are usually less than 10 cm, can also be studied using illumination through the bottom of the box. This way, Gyrodactylus specimens on the fins can usually be easily observed.
	Fixed or preserved fish should be studied in a similar way under a dissection microscope with illumination from above. Gyrodactylus specimens turn almost white when fixed in formaldehyde, while ethanol-preserved specimens are usually only slightly opaque. Before examination, the fish should be rinsed in tap water. This can be done by placing the transport container under a tap and letting the water flow slowly (gently) through the container for a couple of hours. Before rinsing, however, the fixative/preservative, including the bottom sediment in the container, should be examined separately for the presence of Gyrodactylus specimens that may have washed off during fixation and transportation. For protection purposes, the dissecting microscope should be placed on a suction bench with a downwards outlet to avoid inhalation of evaporated fixative/preservative.
2.	Confirmatory Diagnostic Methods for the Identification of Gyrodactylus salaris
	2.1.	Morphology and morphometry of sclerites in the attachment organ
		Identification of Gyrodactylus species is based on morphology and morphometry of marginal hooks, anchors (hamuli) and bars in the opisthaptor (the attachment organ). Good preparation of specimens is a prerequisite for discrimination of species (6).
		Malmberg's ammonium picrate glycerine (APG) method for preparing whole mounts of small haptor worms (Monogenea) by means of APG (5) is superior to other methods, especially when the temporal relation is considered. According to this method, a drop of water is placed on a slide (76 x 26 mm), a worm is transferred to the water and a cover-slip (18 x 18 mm) is gently placed on top. Using a piece of filter paper, as much water as possible is absorbed at the edge of the cover-slip so that the worm is very depressed but not squashed. A small drop of APG is added to the edge of the cover-slip. The parasite will be fixed as the yellow APG solution penetrates the space between the slide and the cover-slip. The slide should be labelled with the host species, location, locality, date of collection and water temperature. If the slide is to be transported, a small drop of nail-polish or a similar substance should be added to each corner of the cover-slip for 'permanent' attachment to the slide. The worm should be kept in vivo during preparation, but a fixed/preserved worm may be used, although as the latter is harder to depress, species identification may be difficult.
		The APG solution is made by mixing one part saturated ammonium picrate solution and one part glycerine/glycerol (puriss). Identification of G. salaris should be in accordance with refs 5, 7-9.
		Morphology and morphometry in G. salaris are presented in Table 1 and Figures 1 and 2, respectively.
		Gyrodactylus salaris is morphologically very similar to G. teuchis from brown trout, Atlantic salmon, rainbow trout and North American brook trout, and to G. thymalli from grayling. The species can be differentiated on the basis of the shape of the marginal hook sickle. Gyrodactylus teuchis has a longer and more constantly curved sickle blade while G. thymalli has a small angle on the shaft of the sickle.

Table 1. Total range of variation in 14 characters measured on marginal hooks, anchors, and ventral bars of Gyrodactylus salaris on Atlantic salmon parr and rainbow trout. The water temperature at the time of measuring varied between 0.0°C and 20.0°C. All measurements are in µm (n = number of specimens measured)

Character measured	n	Range of variation
Total length of marginal hook	783	33.0-46.6
Length of marginal hook handle	807	26.0-38.5
Length of marginal hook sickle	849	7.0-9.5
Total length of anchor	872	58.0-85.0
Length of anchor shaft	872	42.8-63.9
Length of anchor point	878	27.8-44.2
Length of anchor root	875	15.0-32.1
Maximal distance between processes of ventral bar	700	18.5-33.2
Length of ventral bar	747	19.5-32.0
Total basal width of ventral bar	715	20.3-36.5
Basal width of ventral bar	759	7.1-18.7
Total median width of ventral bar	720	17.0-35.5
Median width of ventral bar	778	5.0v15.5
Length of ventral bar membrane	736	12.5-23.0

Figure 1. Morphological variability/variation in opisthaptoral hand parts of Gyrodactylus salaris.
a) Marginal hooks; b) Anchors; c) Ventral bar. Scale bars: 40 µm, 50 µm and 30 µm, respectively.

Figure 2. Fourteen characters measured on marginal hooks, anchors and ventral bars
of Gyrodactylus salaris.

lmh = total length of marginal hook	lvb = length of ventral bar
lh = length of marginal hook handle	tbwvb = total basal width of ventral bar
lsi = length of marginal hook sickle	bwvb = basal width of ventral bar
la = total length of anchor	tmwvb = total median width of ventral bar
las = length of anchor shaft	mwvb = median width of ventral bar
lap = length of anchor point	lvbm = length of ventral bar membrane
lar = length of anchor root	(tmwvb = mwvb + lvbm)
mdpvd = maximum distance between processes of ventral bar

 

	2.2.	DNA analysis of Gyrodactylus specimens (1-4)
		a)	Preparation of samples
			Individual specimens should be removed from ethanol, blotted to remove excess ethanol and placed in individual 0.5 ml microfuge tubes containing 7.5 µl lysis solution (0.45% NP40, 0.45% Tween 20, 60 µg/ml Proteinase K). Tubes should be incubated at 65°C for 20 minutes and then at 95°C for 10 minutes. This lysate is used as the DNA template in the polymerase chain reaction (PCR), without further purification.
		b)	Examination of the small subunit ribosomal RNA (srRNA) gene V4 region
			.	PCR amplification of the V4 region
			Microfuge tubes should be prepared containing all PCR reagents except template and Taq polymerase. These reagents are: 1 x Buffer, 1.5 mM MgCl2, 200 µM of each dNTP, 1 µM of each primer (5'-CTA-TTG-GAG-GGC-AGT-CT-3' and 5'-CTT-TTC-AGG-CAA-CAA-GG-3') and dH2O. Reagents should be overlaid with mineral oil. An aliquot of 2.5 µl lysate is added to the reaction mixture and the tubes are incubated at 95°C for 5 minutes. Then 1 unit Taq polymerase is added and the tubes are subjected to 28 cycles of 94°C for 1 minute, 50°C for 30 seconds and 72°C for 30 seconds.
			A 4-µl aliquot is examined by agarose gel electrophoresis and the presence of a 358 bp PCR product should be confirmed. Any remaining PCR solution is decanted into a sterile microfuge tube and stored at 4°C. If this PCR product is to be used in hybridisation with DNA probes, it should be applied to membranes within 2 days of PCR amplification.
		c)	DNA probe detection of Gyrodactylus salaris
			Probe labelling and detection are carried out according to the protocols in the DIG System Users' Guide using DIG labelling and detection kits.
			i)	Control PCR products amplified from G. salaris, G. derjavini and G. truttae should be included on all membranes. Denature the DNA before applying to the membranes. Denaturation is achieved by heating 10-µl aliquots of each PCR reaction in 0.5 ml microfuge tubes in a boiling water bath or a dry block-heater at 99°C for 10 minutes, and placing on ice at once.
				Immediately spot 1 µl of each solution on to each of three positively charged nylon membranes already marked to allow identification of samples. Bake membranes at 120°C for 30 minutes and store at room temperature until hybridisation is carried out. The remainder of the PCR solution should be stored at 4°C until diagnosis has been made.
			ii)	Label probes GsV4B (5'-GTG-AAT-TGA-TTT-CGG-A-3'), GdV4 (5'-GGG-TTT-CGG-CCT-TGT-3') and GtV4 (5'-GTC-TAC-ACT-TTC-GGA-3') with dioxigenin-11-ddUTP using the reagents and protocol supplied with the DIG Oligonucleotide 3'-end Labelling Kit. Estimate the yield of labelled oligonucleotide by comparing the signal intensities with those produced by labelled control DNA.
			iii)	Place replicate membranes in separate hybridisation bottles and incubate in 20 ml hybridisation buffer for 1 hour at 30°C in a hybridisation oven. Dilute labelled oligonucleotides in hybridisation buffer to a concentration of 10 pmol/ml. Discard prehybridisation solution and replace with 6 ml of probe solution (10 pmol/ml). Each membrane is incubated with a different probe at 30°C for 3.5 hours. Following hybridisation, decant the probe solutions and store at -20°C for re-use. Wash membranes at room temperature twice for 5 minutes each in 2 x SSC (standard saline citrate), 0.1% (w/v) SDS (sodium dodecyl sulfate), and twice for 15 minutes each in 0.1 x SSC, 0.1% (w/v) SDS at 30°C.
			iv)	Chemiluminescent detection of bound probe is carried out using reagents and the protocol given in DIG Chemiluminescent Detection Kit and Wash and Block Buffer Set.
			v)	Species are identified by comparing the signals from diagnostic samples with signals from control samples of G. salaris, G. derjavini and G. truttae. Gyrodactylus salaris, G. teuchis and G. thymalli will all react positively with the probe GsV4B and thus cannot be distinguished by this method.
		d)	srRNA gene V4 region sequencing
			Amplified V4 DNA prepared as in Section 2.2.b above may be sequenced and this sequence is compared with those of G. salaris, G. derjavini, G. truttae and G. teuchis EMBL nucleotide database accession numbers Z26942, Z35128, Z35129 and AJ249349, respectively.
		e)	Examination of the ribosomal RNA gene internal transcribed spacer region
			i)	PCR amplification of the internal transcribed spacer (ITS)
				Microfuge tubes should be prepared containing all PCR reagents except template and Taq polymerase. These reagents are: 1 x Buffer, 1.5 mM MgCl2, 200 µM of each dNTP, 1 µM of each primer (5'-TTT-CCG-TAG-GTG-AAC-CT-3' and 5'-TCC-TCC-GCT-TAG-TGA-TA-3') and dH2O. Reagents should be overlaid with mineral oil. An aliquot of 2.5 µl lysate is added to the reaction mixture and the tubes are incubated at 95°C for 5 minutes. Then 1 unit Taq polymerase is added and the tubes are subjected to 30 cycles of 94°C for 1 minute, 50°C for 1 minute and 72°C for 1 minute.
				A 4-µl aliquot is examined by agarose gel electrophoresis and the presence of a 1300 bp PCR product should be confirmed.
			ii)	Restriction enzyme digestion of ITS
				An aliquot of 7-15 µl PCR product (approximately 100-400 ng) is digested with approximately 2 units HaeIII enzyme. Tubes containing DNA, enzyme, and appropriate buffer are incubated at 37°C for at least 1.5 hours. The digested DNA is then subjected to agarose gel electrophoresis in a 1.5% (w/v) gel alongside controls of HaeIII-digested ITS from G. salaris, G. derjavini, G. truttae and G. teuchis. ITS from G. salaris produces HaeIII fragments of 513, 399, 234 and 154 bp.
		f)	ITS region sequencing
			Amplified ITS prepared as in Section 2.2.e.i. above may be sequenced and this sequence compared with those of G. salaris, G. derjavini, G. truttae and G. teuchis EMBL nucleotide database accession numbers Z72477, AJ132259, AJ132260 and AJ249350, respectively.

REFERENCES

1.	Cunningham C.O. (1997). Species variation within the internal transcribed spacer (ITS) region of Gyrodactylus (Monogenea; Gyrodactylidae) ribosomal RNA genes. J. Parasitol., 83, 215-219.
2.	Cunningham C.O., McGillivray D.M., MacKenzie K. & Melvin W.T. (1995). Identification of Gyrodactylus (Monogenea) species parasitizing salmonid fish using DNA probes. J. Fish Dis., 18, 539-544.
3.	Cunningham C.O., McGillivray D.M., MacKenzie K. & Melvin W.T. (1995). Discrimination between Gyrodactylus salaris, G. derjavini and G. truttae (Platyhelminthes: Monogenea) using restriction fragment length polymorphisms and an oligonuleotide probe within the small subunit ribosomal RNA gene. Parasitology, 111, 87-94.
4.	Cunningham C.O., Mo T.A., Collins C.M., Buchmann K., Thiery R., Blanc G. & Lautraite A. (2001). Redescription of Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel & Vigneulle, 1999 (Monogenea: Gyrodactylidae), a species identified by ribosomal RNA sequence. Syst. Parasitol., 48, 141-150.
5.	Malmberg G. (1957). Om förekomsten av Gyrodactylus på svenska fiskar. Skr. söd. Sver. FiskFör., (Årsskr.) 1956, 19-76. (In Swedish, species descriptions and summary in English).
6.	Malmberg G. (1970). The excretory systems and the marginal hooks as a basis for the systematics of Gyrodactylus (Trematoda, Monogenea). Ark. Zool. Ser. 2, 23, 1-235.
7.	Mo T.A. (1991). Seasonal variations of opisthaptoral hard parts of Gyrodactylus salaris Malmberg, 1957 (Monogenea: Gyrodactylidae) on parr of Atlantic salmon Salmo salar L. in the River Batnfjordselva, Norway. Syst. Parasitol., 19, 231-240.
8.	Mo T.A. (1991). Variations of opisthaptoral hard parts of Gyrodactylus salaris Malmberg, 1957 (Monogenea: Gyrodactylidae) on rainbow trout Oncorhynchus mykiss (Walbaum, 1792) in a fish farm, with comments on the spreading of the parasite in south-eastern Norway. Syst. Parasitol., 20, 1-9.
9.	Mo T.A. (1991). Variations of opisthaptoral hard parts of Gyrodactylus salaris Malmberg, 1957 (Monogenea: Gyrodactylidae) on parr of Atlantic salmon Salmo salar L. in laboratory experiments. Syst. Parasitol., 20, 11-19.

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