Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 2.1.13.

PISCIRICKETTSIOSIS
(Piscirickettsia salmonis)

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SUMMARY

Piscirickettsiosis is a disease of salmonids caused by Piscirickettsia salmonis that was first reported in farmed coho salmon (Oncorhynchus kisutch). The disease was initially described in 1989 from fish in Chile. Piscirickettsia salmonis is a Gram-negative, highly fastidious, intracellular bacterial pathogen of fish. It is distantly related to the genera Coxiella and Francisella and is grouped with the gamma subdivision of the Proteobacteria.
 
The identification of P. salmonis is based on isolation of the causative agent with subsequent testing for characteristics of this intracellular pathogen. Piscirickettsia salmonis occurs in cytoplasmic vacuoles in the host cell. The organism may be distinguished from Chlamydia as it does not possess the characteristic chlamydial developmental cycle, and does not contain the group-specific lipopolysaccharide chlamydial antigen. The identity is confirmed by means of serological tests or polymerase chain reaction (PCR).
 
For a rapid result, the identity of P. salmonis isolated in cell culture or observed in smears from diseased tissue may be confirmed by means of the fluorescent antibody test or immunohistochemical methods with polyclonal or monoclonal antibodies, or by PCR with P.-salmonis-specific primers.
 
The implementation of hygienic measures and management policy are the only control methods currently available. Antibiotics have been used, but their value is questionable. Intensive efforts are underway by various groups to develop an effective vaccine.
 

INTRODUCTION

Piscirickettsiosis is a septicaemic condition of salmonids. The causative agent of the disease is Piscirickettsia salmonis (ATCC VR-1361), and the type strain is LF-89^T (13, 15). Thus far the disease has been described from Chile (5, 12), Ireland (22, 23), Norway (21), and both the West (6, 11) and East (16) coasts of Canada.
 
Piscirickettsia salmonis has been detected in coho salmon (Oncorhynchus kisutch), chinook salmon (O. tshawytscha), sakura salmon (O. masou), rainbow trout (O. mykiss), pink salmon (O. gorbuscha) and Atlantic salmon (Salmo salar). Coho salmon are believed to be most susceptible (13). Mortality in seawater net pens was reported to be 30-90% among coho salmon reared in Chile (5). Piscirickettsia-salmonis-like organisms have recently been isolated from non-salmonid fish (7-9). The relationships of most of these organisms to P. salmonis have not been fully elucidated, but the organism isolated from white sea bass (Atractoscion nobilis) (7) is genetically and serologically indistinguishable from P. salmonis.
 
The mechanisms of transmission are still under investigation. The disease has been primarily reported in marine fish farms, and has also been observed in freshwater facilities (4, 14). Horizontal transmission occurs in saltwater and freshwater (2, 10). Transmission by vectors remains a consideration, and the role of vertical transmission is obscure.
 
Although antibacterial treatment provides some benefit, it is not entirely effective as a means of controlling the disease. Currently, oxalinic acid appears to be the drug of choice. Eggs may be disinfected as part of good hatchery practice.
 
The disease is a chronic, systemic infection that generally affects salmonids reared in sea water. All ages are susceptible, from smolts to market size fish. Signs of the disease both external and internal are well documented (10, 12, 21, 23). The disease begins approximately 1 month after fish are introduced into the seawater net pens, and the organism was thought to be a marine bacterium.
 
A range of gross signs of infection may be present in salmonids infected with P. salmonis. Severely affected fish are dark, anorexic and lethargic. They often swim near the surface or edges of the cages. Fish with milder infections often show no abnormal external signs. Infections of the brain may cause erratic swimming behaviour (25). Skin lesions, appearing as small white patches that can progress to shallow ulcers, may also be present on some fish. Perhaps the most consistent external signs observed during P. salmonis infections are pale gills resulting from a significant anaemia, but this is not pathognomonic for the disease.
 
Consistent with many systemic, chronic inflammatory diseases of salmonids, the internal signs are a swollen and discoloured kidney and an enlarged spleen. Ascites in the peritoneum may be present and haemorrhages on the visceral fat, stomach, swim bladder, and body musculature can also occur (10, 24). Hallmark internal lesions of the disease are found in the liver, which may exhibit large, whitish or yellow, multifocal, coalescing, pyogranulomatous nodules. These lesions often rupture, resulting in shallow crater-like cavities in the liver. Whereas these liver lesions are somewhat unique to piscirickettsiosis, many fish with the disease do not exhibit them.
 
The most prominent histological changes are found in the liver, kidney, spleen and intestine, but pathological changes in the brain, heart, ovary and gill can also be observed (3, 7, 10, 22, 24). Multifocal necrosis of hepatocytes, accompanied by a chronic inflammatory infiltrate of mononuclear cells, is observed in the liver. Vascular and perivascular necrosis are also evident in the liver, and intravascular coagulation resulting in fibrin thrombi within major vessels is a common finding. The focal areas of necrosis underlie the pale circular lesions observed grossly in more chronically infected fish. In more acute infections, the coalescence of areas of necrosis results in a more mottled appearance to the organ rather than discrete nodules. Granulomatous inflammation also occurs in the interstitium and parenchyma of the kidney and spleen, respectively. Vascular changes similar to those in the liver may also be observed in the kidney and spleen. Meningitis, endocarditis, peritonitis, pancreatitis, and branchitis may be observed with accompanying chronic inflammatory and vascular changes similar to those in the liver and haematopoietic organs. The ovary was reported to be involved in certain infections in coho salmon (10).
 
High magnification examination of lesions reveals aggregates of the organism in the cytoplasm of degenerated hepatocytes and in macrophages. Infected macrophages are usually hypertrophied and replete with cellular debris. In tissue sections stained with haematoxylin and eosin (H& E), the organism appears as basophilic or amphophilic spheres, about 1 µm in diameter.
 

DIAGNOSTIC PROCEDURES

Gross and microscopic changes resulting from piscirickettsiosis are not are unique enough to allow for definitive diagnosis of the disease. Therefore, screening for and diagnosis of piscirickettsiosis is based on detection of the causative agent. Presumptive diagnosis can be achieved by the visualisation of the causative agent within macrophages or hepatocytes in histological sections or tissue imprints. Confirmatory diagnosis is achieved by isolation of Piscirickettsia salmonis in cell culture, but it does not grow on any known artificial bacteriological media. Confirmation of P. salmonis in culture may be made by indirect fluorescent antibody test (IFAT) or polymerase chain reaction (PCR) assay.
 
PCR assays can also be conducted directly on tissues (19, 20), and thus PCR assays on tissues along with the observation of suspect organisms within macrophages or hepatocytes are also suitable methods for confirmatory diagnosis. Alternatively, P. salmonis can be detected with Giemsa-stained tissue smears, followed by IFAT for positive identification. An enzyme-linked immunosorbent assay for detecting P. salmonis is commercially available, although there is no published information using this method.
 
Piscirickettsia-salmonis-infected fish tissues suitable for examination in cell culture, PCR, tissue imprints and histology are kidney, liver and blood, collected from diseased fish during either overt or covert infections (18). Due to sensitivity of P. salmonis to antibiotics in vitro, none should be used in media during collection of tissue or the culture of cells.
 
Sampling procedures: See Chapter I.1 Section B.
 

1.	Standard Monitoring Methods for Piscirickettsiosis
	1.1.	Isolation of Piscirickettsia salmonis in cell culture (18)
		Cell line to be used: CHSE-214 or EPC (without antibiotics added)
		a)	Preparation of tissue
			i)	The kidney must be aseptically removed and transferred to a sterile container. Antibiotics must not be used at any step in the isolation procedure. Tissues must be kept at 4°C or on ice until processed, and must not be frozen.
			ii)	Kidney tissue should be homogenised homogenised at 1/20 in antibiotic-free balanced salt solution (BSS), and then, without centrifugation, further diluted 1/5 and 1/50 in antibiotic-free BSS for inoculation on to cell cultures. Final dilutions for use are 10-2 and 10-3.
		b)	Inoculation of cell monolayers
			i)	A 10-2 and 10-3 dilution of the organ homogenates should be inoculated on to cultured cell monolayers and maintained in antibiotic-free medium.
			ii)	The diluted homogenate can be inoculated directly (0.1 ml/culture) into the antibiotic-free culture medium overlaying the cells.
			iii)	The cell cultures must be incubated at 15-18°C for 28 days and observed for the appearance of cytopathic effect (CPE). The P. salmonis CPE consists of plaque-like clusters or rounded cells. With time, the CPE progresses until the entire cell sheet is destroyed.
			iv)	If CPE does not occur (except in positive controls), cultures should be incubated at 15-18°C for an additional 14 days.
	1.2.	Giemsa stain and fluorescent antibody test of cell culture supernatant
		Fluid from cell cultures showing extensive CPE can be spotted directly on to microscope slides, and stained with Giemsa or tested in the IFAT (17) as described in the following section.
2.	Diagnostic Methods for Piscirickettsiosis
	2.1.	Giemsa stain
		i)	Preparations of tissue culture supernatant, smears or impressions of the kidney, liver, and spleen should be prepared, air-dried, and fixed for 5 minutes in absolute methanol.
		ii)	Immerse slides in a working solution of Giemsa stain for 30 minutes.
			Stock solution: 0.4 (w/v) in buffered methanol solution, pH 6.9 (commercially available).
			Working solution: diluted 1/10 in phosphate buffer pH 6.0 (0.074 M NaH2PO4, 0.009 M Na2HPO4).
		iii)	Destain with tap water.
		iv)	Observe slides under oil immersion. Tissue smears from infected organs show darkly stained pleiomorphic organisms occurring in coccoid or ring forms, frequently in pairs, with a diameter of 0.5-1.5 µm.
	2.2.	Fluorescent antibody test of tissue smear (17)
		i)	The positive identity of P. salmonis isolated in cell culture or observed in Giemsa-stained smears may be determined by serological methods, for example IFAT.
		ii)	Smears or impressions of the kidney, liver, and spleen should be prepared, air-dried, and fixed for 5 minutes in absolute methanol.
		iii)	Tissues smears to be examined by IFAT must be freshly prepared or stored at -20°C.
		iv)	The sample is first incubated with anti-P.-salmonis polyclonal or monoclonal antibody, then washed and incubated with a secondary antibody conjugated with fluorescein isothiocyanate.
		v)	Following washing, apply glycerol-based mounting media and cover-slip, then examine under a fluorescence microscope.
	2.3.	Histology
		i)	Preserve visceral organs in formalin-based fixative and process for routine histology.
		ii)	Stain histological slides with H& E or Giemsa.
		iii)	Examine macrophages within the kidney interstitium, spleen or blood, or hepatocytes within liver lesions, for the presence of multiple, spherical, basophilic or amphophilic bodies (by H& E) or dark blue (by Giemsa), approximately 1 µm in diameter in the cytoplasm.
	2.4.	Immunohistochemistry of tissue section (1)
		i)	Sections (5 µm) of formalin-fixed, paraffin-embedded tissues are deparaffinised, and treated to eliminate endogenous peroxidase activity.
		ii)	The tissue is first incubated with anti-P.-salmonis polyclonal or monoclonal antibody, then washed and incubated with a secondary antibody conjugated with horseradish peroxidase.
		iii)	Following washing, the tissue is exposed to a chromogen, counterstained with haematoxylin, dehydrated and prepared for examination under a light microscope.
	2.5.	Polymerase chain reaction amplification (20)
		A nested PCR has been developed to detect genomic DNA of P. salmonis using general bacterial 16S rDNA primers in the first amplification and P.-salmonis- specific primers in a second reaction. The two general eubacterial primer sequences are EubA (1518R) 5'-AAG-GAG-GTG-ATC-CAN-CCR-CA-3' and EubB (27F) 5'-AGA-GTT-TGA-TCM-TGG-CTC-AG-3'. The two specific P. salmonis primers PS2S (223F) and PS2AS (690R) have the following sequences: 5'-CTA-GGA-GAT-GAG-CCC-GCG-TTG-3' and 5'-GCT-ACA-CCT-GAA-ATT-CCA-CTT-3', respectively. These primers yield a specific 467 bp product. The PS2AS primer sequence has been updated to correspond to corrections in the target DNA sequence (GenBank accession number U36941).
		a)	Preparation of infected cell culture supernatant or tissue
			Use of a commercially available spin column to purify DNA from cell culture supernatant or tissue is recommended for PCR sample preparation. In addition to following the manufacturer's instructions on the use of the columns, initial digestion of the sample in lysis buffer (20 mM Tris/HC, pH 8.0, 2 mM EDTA (ethylene diamine tetra-acetic acid), 1.2% Triton X-100, 4 mg/ml lysozyme) at 37°C for 30 minutes is suggested.
		b)	Nested polymerase chain reaction
			i)	Add 5 µl of the DNA preparation to the 45 µl of reaction mixture consisting of PCR buffer (10 mM Tris/HCl, pH 9, 1.5 mM MgCl2, 50 mM KCl, and 0.1% Trition X-100), 200 µM each of dATP, dCTP, dTTP and dGTP, 1 µM EubA primer, 1 µM EubB primer, and 2.5 units Taq DNA polymerase. The mixture is covered with 50 µl of mineral oil. Denature the mixture at 94°C for 2 minutes and then amplify by 35 cycles at 94°C for 1 minute, 50°C for 2 minutes, and 72°C for 3 minutes.
			ii)	The second amplification is performed by adding 2 µl of the first PCR products to 48 µl of reaction mixture containing 2 µM each of the PS2S and PS2AS primers instead of EubA and EubB under the following reaction conditions: denature the mixture at 94°C for 2 minutes and then amplify by 35 cycles at 94°C for 1 minute, 65°C for 2 minutes and 72°C for 3 minutes.
				Amplified DNA (476 bp) (10 µl of the PCR reaction mixture) is analysed by electrophoresis in 2% agarose TAE gel (40 mM Tris acetate/1 mM EDTA) containing 1 mg per 50 ml ethidium bromide. To confirm the identification, 10 µl aliquots of the PCR amplification mixture can be digested with EcoR1 and Pst1 according to the manufacturer's instructions (expected products: EcoR1 994, 546; Pst1 541, 519, 480). If a product is obtained with the P.-salmonis-specific primers but does not cut as expected, further confirmation of the isolate should be obtained by sequencing.
		c)	Direct polymerase chain reaction
			Add 5 µl of the DNA preparation to the 45 µl of reaction mixture consisting of PCR buffer (10 mM Tris/HCl, pH 9, 1.5 mM MgCl2, 50 mM KCl, and 0.1% Trition X-100), 200 µM each of dATP, dCTP, dTTP and dGTP, 2 µM each of PS2S primer and PS2AS primer, and 2.5 units Taq DNA polymerase. The mixture is covered with 50 µl of mineral oil if the thermocyler does not have a hot bonnet. The direct PCR is performed under the same reaction conditions as for the second step of the nested PCR.
			Other PCR assays have been developed to detect P. salmonis (19). These primer sequences and reaction conditions would also be suitable for confirmation of the presence of P. salmonis.

REFERENCES

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