Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 2.1.11.

BACTERIAL KIDNEY DISEASE
(Renibacterium salmoninarum)

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SUMMARY

Bacterial kidney disease (BKD) caused by Renibacterium salmoninarum occurs in most parts of the world where salmonid fish are cultured or occur in the wild (25, 26). Salmonids vary in their susceptibility to BKD, and Pacific salmon species of the genus Oncorhynchus are generally considered to be the most susceptible (21, 26, 49, 52). BKD can cause serious mortality in juvenile salmonids in both fresh water and seawater, and also in prespawning adults. The disease has been described throughout North America and in many countries in Europe, as well as Japan, Chile, and Iceland (2, 21, 25, 27). Most recorded outbreaks of BKD have occurred in fish culture facilities, and the spread of BKD has followed the expansion of salmonid culture (25). Clinical BKD has also been reported in feral fish (5, 36, 39, 45), including naturally spawning populations that have never been supplemented with hatchery fish (22, 51). Whereas the chronic nature of the disease has hindered accurate estimates of fish losses, particularly in feral fish populations, BKD remains one of the most important bacterial diseases affecting cultured salmonids. Losses as high as 80% in stocks of Pacific salmon and 40% in stocks of Atlantic salmon (Salmo salar) have been reported (25).
 
Renibacterium salmoninarum is a small (0.3-0.1 µm by 1.0-1.5 µm), nonmotile, nonspore-forming, nonacid-fast, Gram-positive diplobacillus (27). It typically causes a slowly progressing systemic infection, with overt disease rarely evident until fish are 6-12 months old (21). Fish with severe R. salmoninarum infections may show no obvious external signs, or may exhibit one or more of the following: lethargy; skin darkening; abdominal distension due to ascites; pale gills associated with anaemia; exophthalmos; haemorrhages around the vent; and cystic cavities in the skeletal muscle. Internal examination usually reveals the presence of focal to multifocal grayish-white nodular lesions in the kidney, and sometimes in the spleen and liver. In addition, there may be turbid fluid in the abdominal cavity, haemorrhages on the abdominal wall and in the viscera, and a diffuse white membranous layer (pseudomembrane) on one or more of the internal organs. In tissue sections of BKD lesions, R. salmoninarum is frequently observed within phagocytic cells, particularly macrophages. The bacterium appears to survive and perhaps replicate within these cells (4, 10, 31, 57).
 
The kidney disease bacterium can be transmitted both horizontally from infected fish sharing the water supply (6, 38), and vertically in association with eggs from infected parents (23, 43). As with other infectious diseases of salmonids that are difficult or impossible to treat, avoidance is recommended for the control of BKD in cultured salmonid stocks (1, 21). Because R. salmoninarum is often enzootic in wild salmonid populations (9, 22, 34), measures to control losses from BKD may be defeated by constant exposure of hatchery fish to waterborne bacteria shed into the water supply by wild fish residing upstream from the hatchery (33, 38). Salmonids reared in seawater present special problems because it is difficult to ensure adequate separation of groups of fish to prevent horizontal transmission, and because of the possibility that other marine species might serve as reservoirs for R. salmoninarum (7, 14, 50, 54).
 
To reduce the probability of vertical transmission of R. salmoninarum in cultured salmonids, brood stock segregation or culling is now used to select egg lots to retain as a source of juvenile fish for hatchery rearing (30, 43, 55) The selection process is aimed at rearing egg lots from mating pairs with undetectable or very low levels of R. salmoninarum. This requires the use of sensitive BKD detection methods for testing the prevalence and levels of R. salmoninarum in each parent fish. Elliott & Barila (16) believed that the membrane-filtration fluorescent antibody test (MF-FAT) would provide the sensitivity and quantification necessary to investigate the relationship between the levels of R. salmoninarum in the female parent and the probability of transmitting the disease to the progeny. Pascho et al. (43) later demonstrated the usefulness of brood stock segregation for controlling losses from BKD by using the MF-FAT in conjunction with the enzyme-linked immunosorbent assay to segregate egg lots from chinook salmon parents infected with either very low or very high levels of R. salmoninarum. These researchers reported that the losses from BKD among the progeny of parents with very low levels of R. salmoninarum were significantly less than those among the progeny of parents with very high infection levels. The aquaculturist must be aware, however, that brood stock segregation may not completely eliminate BKD from an affected population. Because the broodstock used for commercial fish farming should be free of the kidney disease bacterium, it may be necessary to repopulate a contaminated facility with brood fish from a BKD-free population.
 
Crucial to the success of any BKD control programme is the application of reliable diagnostic methods that can detect low levels of R. salmoninarum in a variety of sample types. For that reason, fish health specialists and researchers have long been interested in developing methods for more rapid and reliable detection of R. salmoninarum infections (29, 41, 42, 48, 56). As each new test has been developed, however, there has been a tendency to reject older techniques. Nevertheless, no single ideal diagnostic test has yet been developed for the evaluation of multiple samples for the presence of BKD.
 

DIAGNOSTIC PROCEDURES

Bacteriological culture remains the benchmark method for determining the viability of Renibacterium salmoninarum in a sample, and may also be used to quantify the number of bacteria in samples. Immunodiagnostic procedures, however, have become the most widely used for screening large numbers of fish in aquaculture facilities or elsewhere in the field. The two principal immunodiagnostic methods are the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT). Nucleic-acid-based diagnostic tests, such as the polymerase chain reaction (PCR), are now acceptable for confirmatory identification of R. salmoninarum in bacteriological cultures and fish tissue or body fluid samples.
 
Sampling procedures: see Chapter I.1. Sections B and C.
 

1.	Standard Screening Methods for Renibacterium salmoninarum
	Screening for and diagnosis of bacterial kidney disease (BKD) should be based on the ELISA and the FAT. Confirmation of R. salmoninarum should be done by bacteriological culture on a KDM-2 medium (kidney disease medium), or by the PCR. Sample preparation and processing procedures are detailed below.
2.	Diagnostic Methods for Renibacterium salmoninarum
	2.1.	Enzyme-linked immunosorbent assay for testing tissue, plasma, and coelomic fluid
		The recommended procedure is based on the method of Pascho & Mulcahy (44) as modified by Pascho et al. (43). Other ELISA systems are also available commercially (46).
		a)	Supplies and reagents
			i)	Sample tubes
				Recommended sample tubes are sterile, 2 ml microcentrifuge tubes with screen-printed graduations, a writing space, and a gasketed screw cap.
			ii)	Test sample and control preparation diluent
				Phosphate buffered saline (PBS), pH 7.4, supplemented with 0.05% (v/v) Tween 20 (PBST), and 0.01% (w/v) thimerosal as a preservative. For 1 litre: 8.00 g NaCl, 0.20 g KH2PO4, 1.09 g Na2HPO4, 0.20 g KCl, 0.10 g thimerosal, confirm pH = 7.4, and 0.5 ml Tween 20.
			iii)	96-well microplates
				For the ELISA a 96-well microplate that is designed for use in immunoassays must be used. The performance of these plates will vary, and for a given ELISA, microplates from several manufacturers should be tested to determine which is most suitable.
			iv)	Positive control antigen
				Renibacterium salmoninarum cells (0.5% [w/v] wet packed cells) in PBS, pH 7.4, with 0.01% (w/v) thimerosal. The bacterial cells can be lyophilised for long-term storage; lyophilised bacteria normally contain a carrier compound, such as dextrose, for stability. Rehydrate lyophilised bacteria with 1.0 ml reagent-grade water and prepare necessary dilutions, 1/100, 1/500, 1/1000, and 1/5000 (v/v), in PBST and store at -70°C.
			v)	Coating buffer
				Prepare sodium carbonate or purchase a commercial coating solution.
				Sodium bicarbonate, pH 9.6: store at room temperature and discard after 30 days. For 1 litre: 1.59 g Na2CO3, 2.93 g NaHCO3, and 0.10 g thimerosal, confirm pH = 9.6.
				Commercial coating solution concentrate: store and dilute according to the manufacturer's instructions.
			vi)	Wash solution
				PBST, or a commercial wash solution that contains Tween 20. Prepare fresh wash solution for each ELISA. Either make PBST as described above, or dilute commercial wash solution concentrates according to the manufacturer's instructions.
			vii)	Conjugate diluent
				Prepare fresh for each ELISA. Use PBST as described above, or 2% (w/v) nonfat dry milk may be substituted for the Tween 20. Commercial products are also available as concentrates, often marketed as diluents or blocking solutions.
			viii)	ABTS-peroxidase-chromogen substrate
				Commercial products are available, and often are provided as a two-part system: the ABTS chromogen - 0.6 g/litre ABTS (2,2'-azino-di-[3-ethyl-benzthiazoline]-6-sulphonic acid) in a glycine buffer, and a hydrogen peroxide substrate - 0.02% hydrogen peroxide in a citric acid buffer.
			ix)	Stop solution
				5% (v/v) sodium dodecyl sulphate (SDS). Prepare in reagent-grade water immediately before use.
		b)	Coating antibody and conjugate
			i)	Coating antibody
				Affinity purified immunoglobulin to R. salmoninarum: rehydrate lyophilised coating antibody by first mixing 1.0 ml glycerol + 1.0 ml reagent-grade water that contains 0.2% (w/v; 2x) thimerosal, and then transferring 1.0 ml of the 50% glycerol solution to each product vial. Rehydrate the contents of a sufficient number vials to test the anticipated number of fish that will be sampled for a given spawning season. Pool the contents of all vials, then dispense into several cryovials and store at -70°C.
			ii)	Conjugate
				Affinity purified immunoglobulin to R. salmoninarum labelled with horseradish peroxidase (HRPO): rehydrate lyophilised coating antibody by first mixing 1.0 ml glycerol + 1.0 ml reagent-grade water that contains 0.2% (w/v; 2x) thimerosal, and then transferring 1.0 ml of the 50% glycerol solution to each product vial. Rehydrate the contents of a sufficient number of vials to test the anticipated number of fish that will be sampled for a given spawning season. Pool the contents of all vials, then dispense into several cryovials and store at -70°C.
			iii)	Working concentrations of the coating antibody and conjugate
				Working concentrations for the purified goat anti-R. salmoninarum antibody (coating antibody), and the HRPO-conjugated goat anti-R. salmoninarum antibody must first be determined by checkerboard titration with the coating antibody and the HRPO-conjugate.
				NOTE: The anti-R. salmoninarum is typically applied to the microplate wells at 1 µg per ml; the dilution is made from a concentrated antibody preparation at 1 mg/ml, and the working dilution of the HRPO-conjugated antibody is normally about 1/2000 (v/v).
		c)	Sample collection and preparation
			i)	Adult fish
				Kidney tissue, 1/4 (w/v): one part tissue + three parts PBST. Collect 2-5 g total; when sampling the kidney, it is recommended that this sample consist of a pool of small tissue pieces from the anterior, mid, and posterior kidney.
				Ovarian fluid, 1/2 (v/v): one volume ovarian fluid + one volume PBST. Collect approximately 1 ml.
				Plasma, 1/5 (w/v): one volume whole blood + four volumes PBST. Collect blood in a syringe or capillary tube, then dispense the correct volume of blood into the appropriate volume of PBST, remove the cellular fraction by low-speed centrifugation, decant and then test the supernatant.
				Samples are usually taken following spawning. Care should be exercised to avoid cross-contamination between fish and contamination of tissue samples from body fluids. Keep tissue and body fluid samples on ice during collection. Store at -70°C.
			ii)	Juvenile fish
				Kidney-spleen tissue pool, recommend 1/4 (w/v) dilution, but can use 1/8 (w/v): one part tissue + seven parts PBST. Juvenile fish are often collected as whole fish and dissected on return to the laboratory. Remove the entire kidney and spleen from each fish. It is preferable to test tissues from individual fish, but a tissue pool may be made if the fish are too small. Two-fish pools are recommended when an insufficient amount of tissue is recovered from an individual fish. Store at -70°C.
				Plasma, recommend 1/5 (w/v) dilution, but can use 1/10 (w/v): one volume whole blood + nine volumes PBST. Collect blood in a syringe or capillary tube, then dispense the correct volume of blood into the appropriate volume of PBST, remove the cellular fraction by low-speed centrifugation, decant the supernatant, store fluid or heat at 100°C for 15 minutes, and test the supernatant. Store at -70°C.
		d)	Sample processing for the ELISA
			i)	Prepare fish tissue or body fluid samples for the ELISA as described above.
			ii)	Heat each sample at 100°C for 15 minutes, then centrifuge at from 8000 to 10,000 g for 10 minutes at 4°C. If the samples were prepared earlier and frozen, thaw before heating.
			iii)	Store processed and heated samples at 4°C, or freeze at -70°C for later testing.
		e)	ELISA day 1
			NOTE: Before beginning the ELISA, users should review the controls and appropriate reagent applications described in Table 1.
			i)	Dilute the concentrated goat antibody to R. salmoninarum in coating buffer: carbonate/ bicarbonate coating buffer, pH 9.6, or a commercial coating solution. When using the coating solution, make a fresh preparation for each ELISA. The carbonate/bicarbonate buffer is normally discarded after 30 days. Use water of reagent grade or equivalent.
			ii)	The conjugate control and substrate/chromogen control wells receive no coating antibody. Place 200 µl of coating buffer in each of these wells.
			iii)	The blanks, negative controls wells, positive control wells, and the wells designated for the test samples receive 200 µl of coating antibody.
			iv)	Seal each plate with an adhesive plate sealer after addition of the buffer or coating antibody. Place each plate in a humid chamber and incubate at 4°C for 16 hours.

Table 1. Controls for the ELISA to detect Renibacterium salmoninarum

		Step in the ELISA
Control group	Purpose	Coating antibody	Sample addition	Conjugate	Substrate/ chromogen	Stop solution
Blank	Background absorbance levels in the absence of a test sample	Yes	PBST only	Yes	Yes	Yes
Reference or positive controls	Internal control to insure predictable absorbance by certain levels of antigen	Yes	Yes	Yes	Yes	Yes
Negative control	Absorbance produced by sample from a negative control fish	Yes	Yes	Yes	Yes	Yes
Conjugate control	To ensure that there is no nonspecific binding of the conjugate to well surfaces or to the coating antibody	Coating buffer only	PBST only	Yes	Yes	Yes
Substrate/ chromogen control	Test for nonenzymatic production of the colour reaction	Coating buffer only	PBST only	Diluent only	Yes	Yes

 

		f)	ELISA day 2
			i)	Prepare PBST wash solution or dilute a commercial wash solution concentrate in reagent-grade water, and store overnight at 4°C. The wash buffer should remain at room temperature during the ELISA.
			ii)	Remove the unbound coating antibody by washing each plate five times, with a 30- second soak each time the wells are refilled. Shake excess wash buffer out of each plate after the five washes are completed. Wash plates in numerical order.
			iii)	Place aliquots of controls and test samples into microplate wells. The following control wells receive 200 µl of the test sample diluent (PBS, pH 7.4, supplemented with 0.05% [v/v] Tween 20): Blank, conjugate control, substrate/chromogen control. Control tissue wells receive the appropriate tissue or body fluid.
			iv)	Place 200 µl aliquots of each positive control in the appropriate wells.
			v)	Place 200 µl aliquots of each test sample in the appropriate wells.
			vi)	Cover each plate with an adhesive plate sealer and incubate for 3 hours at 25°C in a humid chamber. Wash each microplate five times as described previously.
			vii)	Apply 200 µl of the diluted conjugate to the appropriate wells. Substrate/chromogen control wells receive an equivalent amount of diluent without conjugated antibody.
			viii)	Seal each plate with an adhesive plate sealer and incubate in a humid chamber for 2 hours at 25°C.
			ix)	Wash microplates five times as described previously.
			x)	Substrate/chromogen reaction.
				The timing of the substrate/chromogen reaction is critical. The reaction must be stopped after exactly 20 minutes. The volume of stop solution is 50 µl to insure that the wells are not over-filled. The SDS stop solution is prepared from a 5% (v/v) concentrate as follows: four parts concentrate + one part water. Apply 200 µl of substrate/chromogen solution to each well. Fill all plates in numerical order. Immediately after all the wells have received the substrate/chromogen solution, seal the plate and put it in the humid chamber at 37°C.
			xi)	Stop the substrate/chromogen reaction and measure the absorbance. Begin to apply the stop solution immediately after the incubation period is complete. Wells should receive 50 µl of stop solution in the exact sequence used to apply the substrate/chromogen solution. Wipe any condensation off the bottom of the plate and immediately measure the absorbances at 405 nm.
	2.2.	Standard indirect and direct fluorescent antibody technique for testing tissue samples and bacteriological isolates
		In general, there are two types of staining procedures that use fluorescein isothiocyanate (FITC)-labelled antibodies: the indirect and direct fluorescent antibody techniques (IFAT and DFAT, respectively). The principle and techniques are similar, but the indirect uses a second antibody that is often biotinylated for increased sensitivity. The direct FAT is used more commonly for bacterial corroboration testing and staining of R. salmoninarum.
		There are three basic steps for DFAT: preparing and fixing bacterial cultures or kidney tissue on multiple-well glass slides; staining the slides with antibody reagents; and reading and interpreting the slides.
		a)	Preparing the slides
			i)	Pure bacterial cultures (corroboration testing of pure isolates R. salmoninarum): pure isolates of bacteria are diluted in sterile PBS and applied to a glass microscope slide. Air-dry and fix in absolute methanol for 5-10 minutes.
			ii)	Kidney smears from fish - DFAT for R. salmoninarum: after the tissue has completely dried, slides are fixed in methanol for approximately 5-10 minutes.
		b)	Staining
			i)	Positive and negative controls: slides containing both a non-cross-reacting bacterial species and a known positive control can be prepared in quantity and stored refrigerated for use as controls for DFAT staining. Positive controls are always used in corroboration testing to correctly identify morphology and fluorescence of the bacteria in question. The negative control is important in determining overall staining technique, background debris, and nonspecific fluorescence.
			ii)	Place slides in dark, humidified chamber, and place one drop of specific FITC-conjugated antibody on each slide.
			iii)	Incubate for 60 minutes at room temperature.
			iv)	Gently rinse the slides with PBS, pH 7.1 (see Section 2.3.).
			v)	Counterstain in Evan's blue for 3-4 minutes for R. salmoninarum. (A counterstain is not necessary for corroboration testing of pure bacterial cultures, although Rhodamine can be used if desired.)
			vi)	Rinse with PBS, pH 7.1.
			vii)	Air-dry completely, add mounting medium and a cover-slip.
		c)	Reading and rating
			Slides are read at x1000 magnification on a compound fluorescence microscope (refer to the microscope manufacturer for correct wavelength and filters required for FITC epifluoresence microscopy). The positive and negative control slides are read first. This has two purposes: (1) quality assurance for the staining process (positive control should have myriad numbers of fluorescing bacteria, the negative control should have no fluorescence); and (2) to familiarise the reader with the correct bacterial size and shape, and the magnitude of the bacteria's fluorescent halo in the positive control. The reader can refer back to the positive control as a reference, if needed, to confirm suspect bacteria in the sample wells.
			i)	Bacterial corroboration testing: positive bacterial isolates will fluoresce strongly and have the same morphology and size as the positive control.
			ii)	Kidney smears tested for R. salmoninarum: bacterium will have a distinctly apple-green fluorescent cell wall; be the appropriate size (1 µm long by 0.5 µm); and be the proper shape (bean shaped or pear shaped). Compare any suspect bacteria with the control slide to be sure all three of the above criteria are met for R. salmoninarum.
		d)	Fluorescent antibody technique rating guide
			(Based on those described in Fish Pathology Section Laboratory Manual, Meyers, T.R., ed. Special Publication 11, Alaska Dept Fish and Game, P.O. Box 25526, Juneau, AK 99802 USA.) Standard rating criteria for interpretation of the FAT on tissue smears are based on a minimum of 60 fields examined at x1000 magnification.
			i)	Negative (-): no organisms seen in thirty fields examined.
			ii)	Plus/minus (±): organisms observed with questionable fluorescence or morphology not typical of the target organisms. Total of one typical organism observed but suspected of not originating from the sample examined, i.e. wash over from a high-level positive sample.
			iii)	One plus (1+): one to five organisms observed. If only one organism is found, examination of up to 100 fields continues in an attempt to find a second organism that would confirm the 1+ status. If no other organism is detected the final ± or 1+ interpretation is at the discretion of the individual reader.
			iv)	Two plus (2+): 6 to 50 organisms observed in which some fields will be negative and some will typically contain several organisms.
			v)	Three plus (3+): 51 to 150 organisms observed with a typical field containing a dozen or more organisms.
			vi)	Four plus (4+): Greater than 150 organisms with no more than 200 organisms in an average field.
			vii)	Clinical (C5+): Greater than 200 organisms in an average field. Gross lesions are likely to be observed in the sampled kidneys from this category.
	2.3.	Membrane-filtration FAT for testing coelomic fluid
		This procedure is based on the method of Elliott & Barila (16) as modified by Elliott & McKibben (17).
		a)	Reagents
			i)	0.01 M PBS, pH 7.1
				8.50 g NaCl, 1.07 g Na2HPO4 (anhydrous), 0.34 g Na2H2PO4H2O (monohydrate), and distilled water to 1 litre. Preserve the solution by adding 0.01% (w/v) thimerosal.
			ii)	PBS/Triton
				5.0 ml Triton X-100 (Bio-Rad), and PBS, pH 7.1, to 1 litre. Filter through a 0.2 µm bottle filter.
			iii)	Trypsin solution
				1.0 g Trypsin powder (Difco) 1/250, and DH2O to 1 litre. Mix trypsin with water at 4°C. Clarify the solution by filtration through Whatman No. 1 filter paper (or by centrifugation at 4000 g, followed by filtration through a 0.2 µm filter). Dispense in small aliquots and freeze at -20°C or colder. (Trypsin will retain activity after storage at -20°C for 1 or 2 months; longer storage should be at -70°C.) Thaw new aliquots of trypsin each day as needed; do not re-freeze.
			iv)	Eriochrome black T counterstain
				1/2000 stock suspension
				0.25 g Eriochrome black T, and PBS, pH 7.1, to 500 ml. Filter through Whatman No. 1, then Whatman No. 42 filter papers (the latter is optional) to remove large chunks of stain. Store the counterstain in a dark or foil-covered bottle.
				NOTE: There are variations in dye content among lots of the compound. Eriochrome black T dilutions (w/v in PBS) ranging from 1/2000 to 1/20,000 may be required for appropriate staining, depending on the lot of stain used.
				Working suspension
				Check the stain quality on test filters. The prepared counterstain suspension should be a medium purple colour, and the filters should show a definite purple colour after counterstaining, but should not be so heavily counterstained that they are nearly black.
			v)	Glycerol mounting medium
				0.01 M PBS, pH 7.4
				3.36 ml solution A: 0.5 M KH2PO4, (anhydrous, 68.04 g/litre), 16.0 ml solution B: 0.5 M K2HPO4, (anhydrous, 87.09 g/litre), 8.5 g NaCl, and distilled water to 1 litre.
				Mounting medium
				90 ml glycerol, 2.5 g 1,4-diazobicyclo-(2,2,2)-octane (DABCO), and 10 ml 0.01 M PBS, pH 7.4. Add the DABCO to the glycerol and dissolve by heating the mixture gently in a water bath. Then, add the PBS. Adjust to pH 8.6-9.0 by adding 0.1 N HCl or 0.1 N NaOH as necessary. Store at room temperature in a dark or foil-covered bottle.
		b)	Sample preparation
			i)	Mix 0.5 ml of ovarian (coelomic) fluid with 0.5 ml of PBS/Triton and 0.5 ml of trypsin solution in a small centrifuge tube. Mix vigorously for 20-30 seconds.
			ii)	Heat the mixture at 50°C for 10 minutes.
			iii)	Withdraw the sample from the centrifuge tube with a 3 ml syringe equipped with a needle (22 g x 1.5 inch or similar). Triturate the sample a few times with a syringe to break up any remaining clumps of material.
		c)	Membrane filtration
			i)	Attach the syringe containing the sample to a Swinney-type filter holder or a disposable pop-top holder. The filter holder should contain a 13 mm diameter, 0.2 µm pore size polycarbonate filter (Whatman Nuclepore, Cambridge, Massachusetts, USA) and a supporting 13 mm diameter, 5.0 µm (or larger) pore size nylon membrane filter (Osmonics, Minnetonka, Minnesota, USA).
				NOTE: The polycarbonate filter should be placed shiny side up (toward the syringe) in the filter holder. Polycarbonate filters are thin, and frequently develop 'ridges' identical to the ridges on the support screens of the filter holders. Placing a thicker nylon filter between the polycarbonate filter and the support screen helps to reduce the formation of these ridges and therefore makes the filter surface flatter for easier observation and counting of bacteria.
			ii)	Force the sample through the filter.
			iii)	Rinse each filter with 3 ml of PBS/Triton (force through the filter with a syringe; use a separate syringe for each sample).
			iv)	Leave the filter in the holder, and drop on 100 µl of FITC-conjugated anti-R. salmoninarum serum at the optimum working dilution. Cover the top of the filter holders with paraffin film or foil (alternatively, place them in a humid chamber), and incubate upright in the dark at room temperature for 1 hour.
			v)	After incubation, rinse each filter with 3 ml of PBS/Triton by forcing the rinse through the filter with a syringe.
			vi)	Counterstain by forcing 1 ml of Eriochrome black T suspension through the filter with a syringe.
			vii)	Remove the polycarbonate filters from the holders and place them on microscope slides to air-dry. Discard the support filters. When the filters are dry, place a drop or two of glycerol mounting medium, pH 9, on the centre of each filter, and mount with cover-slips. Examine at x1000 magnification with a microscope equipped for FITC epifluorescence.
			viii)	Filter counts of the number of R. salmoninarum in a given number of microscope fields can be converted to cells/ml of the original ovarian fluid sample according to the formula:

Cells/ml =	(conversion factor) (dilution factor) (total number of cells counted)
	Total number of fields examined

 

				Where the conversion factor is the filtering surface area divided by the area of a single field at the magnification used. One can calculate the theoretical sensitivity of the technique for any desired number of fields to be examined by entering '1' in the equation for the total number of bacteria counted. In general, 50 or more fields are examined per filter.
			ix)	To confirm MF-FAT results, a procedure such as a nested PCR for R. salmoninarum can be used (11, 41).
	2.4.	Bacteriological culture
		a)	Sampling
			Tissue samples for diagnosis and identification of R. salmoninarum should be taken aseptically from kidney lesions or from lesions in other organs. When no lesions are present, the kidney is the preferred organ for sampling. In mature females, the coelomic fluid may also represent convenient material.
			For routine controls for detecting infected individuals in a population, a sufficient number of fish must be sampled (see Chapter I.1., Section B.1.).
		b)	Isolation
			Renibacterium salmoninarum is a fastidiously growing organism that requires prolonged incubation (2-3 weeks, sometimes more, at 15°C) to produce colonies. L-Cysteine and serum or serum substitutes are requisite factors, and different media or ingredients have been proposed to improve its growth or reduce the development of associated microorganisms. The following two special media are currently used:
			.	Kidney disease medium enriched with serum (KDM-2) or charcoal (KDM-C) (15, 19): 0.1 g l-cysteine (chlorhydrate), 1 g tryptone, 0.05 g yeast extract, 1.5 g agar and 100 ml distilled water. Adjust the pH to 6.5-6.8 with NaOH, distribute into flasks or tubes and autoclave for 20 minutes at 120°C. Can be stored for 1 month at 4°C. Regenerate prior to use and add 5-10% fetal calf serum (FCS), or 0.1% activated charcoal.
			.	Selective kidney disease medium (SKDM-2) (3): 0.1 g l-cysteine (chlorhydrate), 0.005 g cycloheximide, 1 g tryptone, 0.05 g yeast extract, 1 g agar and 100 ml distilled water. Adjust the pH to 6.8 with NaOH and autoclave for 20 minutes at 120°C. Cool to approximately 48°C, and add 10% FCS and the following components, previously filter sterilised (0.22 µm): 0.00125 g D-cycloserine, 0.00025 g oxolinic acid and 0.0025 g polymyxin B sulfate (all final concentrations). Dishes with isolation medium are dried at room temperature for 24-48 hours, inoculated by streaking a 0.1-0.2 ml drop of infectious material across the agar surface, and incubated at 15°C in plastic bags or humid chambers when the absorption is complete. The effect of storage of SKDM-2 plates on the effectiveness of the antimicrobial supplements has not been reported. It is recommended that the SKDM-2 dishes before stored at 4°C and used shortly after preparation
			It seems that serum and charcoal are more likely to act as detoxifying agents than as sources of essential nutrients, and their efficacy has been proved comparable (15), and even optional (53). Supplementing KDM-2 medium with antibiotics may reduce the problem of fast-growing organisms (bacteria and fungi), but there is some evidence that they may also inhibit R. salmoninarum itself (40). Another possibility is to inspect the dishes regularly at intervals of 2-3 days, and to remove aseptically the colonies produced by fast-growing organisms. In order to maintain the viability of the R. salmoninarum, Evelyn (20) recommends the preparation of tissue suspensions in isotonic saline enriched with peptone (0.1 % [w/v]).
			When a stock culture of R. salmoninarum is already available, it is possible to take advantage of the 'satellitism' phenomenon described by Evelyn et al. (24) for accelerating the growth of the isolates. A heavy suspension of the laboratory feeding strain is dropped on to the centre of the plate, and the samples to be tested are inoculated in the periphery. The growth rate and the colony size of the isolates are noticeably increased. Growth enhancement may also be achieved by adding 1.5% (v/v) sterile spent KDM-2 broth to the medium (24).
		c)	Characteristics (28, 35)
			After a sufficiently long incubation period on KDM-2, KDM-C or SKDM-2, R. salmoninarum produces white or creamy, shiny, smooth, round, raised, entire colonies that are pinpoint to 2 mm in size. Bacteria from diseased fish will produce visible colonies after 2-3 weeks on average, however up to 8 weeks have been reported for initial growth on KDM-2, and 12 weeks on the selective medium SKDM-2. Old cultures may achieve a granular or crystalline appearance. Transverse sections through such colonies will reveal the presence of Gram-positive rods in a crystalline matrix. The crystalline material is thought to be cysteine precipitated from the medium. Growth does not occur on blood agar medium without cysteine supplement or on trypticase-yeast agar. For some strains a uniformly turbid growth occurs in broth, but for others a sediment may develop. Renibacterium salmoninarum appears as small (0.3-1.5 x 0.1-1 µm) Gram-positive, PAS-positive, asporogenous, nonmotile, nonacid-fast rods, frequently in pairs, short chains or pleomorphic forms as 'Chinese letters', especially in fish tissue.
			Renibacterium salmoninarum is catalase positive and oxidase negative. Its phenotypic characteristics have been established using API-Zym systems and conventional tests (Table 2). The API-Zym profile for R. salmoninarum is: - + - + - + - - + - + + - - - + - - +/- - (Austin & Austin 1999). However, the slow growth of the organism does not render such tests very useful in practice, and serological methods are more usually employed to confirm the identity of the isolated strains.

Table 2. Characteristics of Renibacterium salmoninarum (18, 28)

Characteristic	Response	Characteristic	Response
Production of:		Degradation of (continued):
Acid phosphatase	+	DNA	-
Alkaline phosphatase	+	Elastin	-
Butyrate esterase	-	Gelatin	-
Caprylate esterase	+	Guanine	-
Catalase	+	Hyaluronic acid	-
Chymothrypsinase	-	Hypoxanthine	-
Cystine arylamidase	-	Lecithin	-
alpha-fucosidase	-	RNA	-
alpha-galactosidase	-	Starch	-
beta-galactosidase	-	Testosterone	-
beta-glucosaminidase	-	Tributyrin	+
alpha-glucosidase	+	Tween 40	+
beta-glucosidase	-	Tween 60	+
beta-glucoronidase	-	Tween 80	-
Leucine arylamidase	+	Tyrosine	-
alpha-mannosidase	+	Xanthine	-
Myristate esterase	-	Acid production from sugars	-
Oxidase	-	Growth on/at:
Trypsinase	+	pH 7.8	+
Valine arylamidase	-	Bile salts, 0.025%, (w/v)	-
Nitrate reduction	-	Crystal violet, 0.0001% (w/v)	+
Degradation of:		Methylene blue, 0.001% (w/v)	-
Adenine	-	Nile blue, 0.00001% (w/v)	+
Aesculin	-	Phenol, 0.025% (w/v)	-
Arbutin	-	Potassium thiocyanate, 1% (w/v)	-
Casein	+	Sodium chloride, 1%( w/v)	+ (poor)
Chitin	-	Sodium selenite, 0.01% (w/v)	-
Chondroitin	-	Thallous acetate, 0.001% (w/v)	-

Table 2 (cont.). Characteristics of Renibacterium salmoninarum (18, 28)

Characteristic	Response	Characteristic	Response
Use of::		Use of::
4-umbelliferyl (4MU)-acetate	+	4PU-heptanoate	+
4MU-butyrate	+	4PU-laurate	+
4MU-beta-D-cellobiopyranoside monohydrate	-	4PU-nonanoate	+
4MU-elaidate	-	4MU-oleate	+
4MU-alpha-L-arabinopyranoside	-	4MU-palmitate	-
4MU-2-acetamido-2-deoxy-beta-D-galactopyranoside	-	4MU-propionate	+
4MU-beta-L-fucopyranoside	-

 

	2.5.	Nested polymerase chain reaction for testing tissue samples, whole blood, and coelomic fluid
		This procedure is based on the nested PCR method described by Chase & Pascho (11), and Pascho et al. (41). The nested design must only be used with rigorous controls to ensure the accuracy of each diagnosis, and to avoid cross-contamination between samples. Other designs may prove desirable when this can be difficult to achieve, as false-positive results may have serious consequences such as the unnecessary culling of an entire broodstock. Other molecular detection methods have also been reported for diagnosing R. salmoninarum infections (8, 13, 32, 37, 47).
		a)	General considerations
			i)	DNA extractions should be done in an area that is free from amplified PCR product to reduce the risk of contamination.
			ii)	To prevent false-positive results from carry-over of amplified DNA sequences, first round reaction mixtures should also be set up in a separate area, or under a UV hood.
			iii)	Each work area should include a separate set of supplies, reagents and pipetting devices.
			iv)	For each PCR analysis include multiple reagent controls without DNA to test for contamination, and select a weak positive control that will be indicative of PCR performance.
		b)	Primer design
			i)	Two pairs of oligonucleotide primers are used in the nested PCR protocol. The primers were designed from the published sequence of the p57 protein of R. salmoninarum (12).
			ii)	The primers used in the first round were: forward 75-93 (5'-AGC-TTC-GCA-AGG-TGA-AGG-G-3'; P3) and reverse 438-458 (5'-GCA-ACA-GGT-TTA-TTT-GCC-GGG-3; M21). Primer location number corresponds to the nucleotide sequence of the open reading frame.
			iii)	The primers used in the second round of amplification reaction were: forward 95-119 (5'-ATT-CTT-CCA-CTT-CAA-CAG-TAC-AAG-G-3', P4) and reverse 394-415 (5'-CAT-TAT-CGT-TAC-ACC-CGA-AAC-C-3'; M38). Primer location number corresponds to the nucleotide sequence of the open reading frame.
		c)	DNA extraction and purification
			To purify nucleic acids from tissue use a DNA recovery kit and follow the manufacturer's instruction for DNA recovery from tissue. DNA extraction protocols typically do not account for the rigid cell wall of a Gram-positive bacterium, and the bacteria could remain intact during the lysis steps. Treatment of tissue and body fluid samples with lysozyme, however, has been reported to be effective and essential for recovery of high-quality bacterial DNA and rRNA (11, 47). After the initial cell lysis, add 50 µl of 4x lysozyme buffer and incubate at 37°C for 1 hour. Consult the manufacturer for specific instructions regarding this lysis step.
			Manufacturers of DNA recovery kits from tissue include: DNeasy Tissue Kit from Qiagen, Chatsworth, California (CA), USA; Nucleospin Tissue Kit from Clonetech Palo Alto, CA, USA, or Clinipure Genomic DNA Kit from GeneMate, Klaysville, Utah, USA.
			i)	Kidney tissue: cut 25-50 mg of tissue into small pieces and place in a 1.5 ml microfuge tube. Add tissue lysis buffer and incubate according to the manufacturer's instructions. After the initial tissue lysis, add 50 µl of 4x lysozyme buffer (80 mg/ml lysozyme, 80 mM Tris/HCl, pH 8.0, 8 mM EDTA [ethylene diamine tetra-acetic acid]; 4.8% Triton) and incubate for 1 hour at 37°C. Continue with DNA purification as instructed.
			ii)	Ovarian fluid: pippette 50 µl of ovarian fluid into a 1.5 ml microfuge tube, add tissue lysis buffer and incubate according to the manufacturer's instructions. After the initial tissue lysis, add 50 µl of 4x lysozyme buffer and incubate for 1 hour at 37°C. Continue with DNA purification as instructed.
			iii)	Whole blood: pippette 50 µl of whole blood into a 1.5 ml microfuge tube, add tissue lysis buffer and incubate according to the manufacturer's instructions. After the initial tissue lysis, add 50 µl of 4x lysozyme buffer and incubate for 1 hour at 37°C. Continue with DNA purification as instructed.
			iv)	Renibacterium salmoninarum cells: Pellet bacteria solution by centrifugation at 7000 g for 15 minutes. Pour off supernatant, resuspend pellet in lysis buffer and incubate according to the manufacturer's instructions. After the initial tissue lysis, add 50 µl of 4x lysozyme buffer and incubate for 1 hour at 37°C. Continue with DNA purification as instructed.
		d)	Determination of yield and purity of nucleic acid samples
			On the basis of their absorbance value at 260 nm, adjust the DNA samples with molecular biology-quality water to a concentration between 0.01 and 0.1 ng/µl. Determine the purity of each DNA sample by calculating the ratio of the readings at 260 nm and 280 nm. Pure DNA samples have an A260/A280 ratio of 1.8-2.0.
		e)	First round PCR protocol
			i)	Preparation of first round PCR reaction mixture.
				Total volume of the first round PCR is 50 µl: 10 µl of the nucleic acid sample and 40 µl of the reaction mixture. To prepare the reaction mixture, combine 0.2 mM of each nucleotide, 50 mM KCl, 10 mM Tris/HCl, pH 8.3, 1.5 mM MgCl2, 0.2 mM each of primers P3 and M21 and 2 U of Taq polymease.
			ii)	Add the PCR reagents except the template DNA into the 'Master Mix' tube.
			iii)	In the PCR tubes, aliquot 40 µl of Master Mix and overlay samples with one drop of mineral oil. Close the cap tightly after each addition.
			iv)	Add 10 µl of extracted DNA to the PCR tubes.
			v)	In each reaction well of the thermal cycler, add two drops of mineral oil. Load the samples into the thermal cycler.
			vi)	Programme the thermal cycler for 30 cycles as follows: denaturing at 94°C for 30 seconds; annealing at 60°C for 30 seconds; and extending at 72°C for 1 minute.
		f)	Second round PCR protocol
			i)	Preparation of the second round PCR reaction mixture.
				Total volume of the reaction mixture is 50 µl; this will include 1 µl of amplified DNA from the first round as template DNA. Use the same reaction mixture as described for the first round except that primers P4 and M38 are used.
			ii)	Add the PCR reagents except the template DNA into the Master Mix tube.
			iii)	In nested PCR tubes, aliquot 49 µl of Master Mix and overlay the samples with one drop of mineral oil. Close the cap tightly after addition.
			iv)	Add 1 µl of first round PCR product to the nested PCR tubes.
			v)	In each reaction well of the thermal cycler, add two drops of mineral oil. Load the samples into the thermal cycler.
			vi)	Programme the thermal cycler for 30 cycles as follows: denaturing at 94°C for 30 seconds; annealing at 60°C for 30 seconds; and extending at 72°C for 1 minute.
		g)	Visualisation of amplified DNA
			i)	Use 10 µl of the second round PCR product for gel electrophoresis on a 2% agarose gel. Each electrophoresis gel should include a 1 kb DNA ladder.
			ii)	Stain gels for 30 minutes in a solution of 5 µg/ml ethidium bromide, 0.02 M hydroxymethyl aminomethane, 0.02 M glacial acetic acid, and 0.5 mM EDTA.
			iii)	Examine the gels under UV transillumination. Samples are considered positive for R. salmoninarum if the anticipated 320 base pair product is observed.

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