Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 3.1.7.

WITHERING SYNDROME OF ABALONE
(Candidatus Xenohaliotis californiensis)

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GENERAL INFORMATION

Withering syndrome here refers only to the disease in abalone caused by Candidatus Xenohaliotis californiensis on the west coast of California, United States of America (USA) and Baja California, Mexico. However, as infected abalone have been transported to Chile, Japan, Israel and other countries, the geographical range of the aetiological agent is suspected to be broad where abalone are cultured. If detected outside the known range of Candidatus Xenohaliotis californiensis, light microscopy in combination with molecular probes, if available, must be used to identify and distinguish the detected organism from other rickettsial bacteria. The presence of these pathogens in any abalone should be regarded as potentially serious and the OIE Reference Laboratory should be consulted.
 
Candidatus Xenohaliotis californiensis occurs along the south-west coast of North America in California, USA and Baja California, Mexico. However, as infected abalone have been transported to Chile, Japan, Israel and other countries, the geographical range of the aetiological agent is suspected to be broad where California red abalone, Haliotis rufescens, are cultured.
 
Candidatus Xenohaliotis californiensis infects the gastrointestinal epithelial cells of the posterior oesophagus, digestive gland and, to a lesser extent, intestine. The dimorphic rod-to-spherical shaped bacterium measures an average of 332 x 1550 nm in the bacillus form and an average of 1405 nm in the spherical morphotype. The bacteria reproduce within intracytoplasmic vacuoles 14-56 µm in diameter (6). Severe infections result in withering syndrome, a disease that is characterised by morphological changes in the digestive gland, which vary between species and may include degeneration (atrophy of tubules, increase in connective tissues and inflammation) and/or metaplasia of the digestive tubules. The metaplasia involves the replacement of terminal secretory/absorptive acini with absorptive/transport ducts similar in appearance to the post-oesophagus. Some hyperplasia of the absorptive/transport ducts may also be involved. This morphological change is accompanied by a decrease in feeding, depletion of glycogen reserves followed by use of the foot muscle as an energy source and death. The foot of affected abalone contains fewer and less organised muscle bundles, abundant connective tissue and may contain more cerous cells than unaffected individuals. Disease (withering syndrome) occurs at elevated water temperatures (~18°C and above) (9). The incubation period of withering syndrome is prolonged and ranges between 3 and 7 months (10).
 
Cumulative mortality has been recorded at over 99% in black abalone and over 30% in red abalone (3, 5, 7). The pathogen and disease (withering syndrome) may occur year round, but losses due to the disease occur most often in the summer and autumn, after a 3-4-month period when temperatures are elevated over 15°C. Reducing densities and application of an oxytetracycline-medicated diet may reduce losses.
 
For diagnosis, the recommended guidelines for sampling are those stated in Chapter 1.1.4 and Chapter I.2. of this Aquatic Manual.
 

EXAMINATION PROCEDURES

1.	Screening Methods
	1.1.	Histology
		Histological procedure is detailed in chapter I.2 of this Aquatic Manual. Remove the shell and cut several 3-5 mm cross sections that contain posterior oesophagus (post-oesophagus), digestive gland, and foot muscle and placed in Davidson's or Carson's solutions (see Chapter I.2. of this Aquatic Manual) for 24 hours and process for routine paraffin histology. Cross sections are most easily handled when placed in cassettes prior to fixation. The ratio must be no more than one volume of tissue to ten volumes of fixative.
		Deparaffinised 3-5 µm sections should be stained with haematoxylin and eosin and viewed by light microscopy for RLP (Rickettsiales-like prokaryote) inclusions in the post-oesophagus and digestive gland, and morphological changes in the digestive gland and foot. It is recommended that sections should be examined at x200 or x400 magnification.
		Candidatus Xenohaliotis californiensis may be morphologically similar to other marine rickettsial bacteria. Definitive diagnosis of the bacterium may include molecular tools (in-situ hybridisation). Definitive diagnosis of withering syndrome by histology must include the presence of the bacterium and morphological changes to the digestive gland and may include those of the foot muscle.
		Where losses have been observed within the known geographical range of withering syndrome, visualisation of intracellular bacterial foci within digestive epithelia, by histological examination, may be considered to be a confirmatory method. However, confirmation by in-situ hybridisation is recommended to verify the identity of the rickettsial bacteria in abalone species previously not known to be susceptible to the bacterium or in a new geographical location.
2.	Presumptive Diagnostic Methods
	2.1.	Histology
		See Section 1.1. above.
	2.2.	Cytological examination: tissue imprints
		Tissue imprints may be used to detect moderate to high intensities of infection of Candidatus Xenohaliotis californiensis. However, histology is more sensitive than tissues imprints.
		Excise a section of the post-oesophagus, mince and lay on a slide, dry with a hair dryer for ~20 minutes. Stain the slides using a fluorescent stain for nucleic acid such as propidium iodide or Hoechst. Incubate in the dark for 3 minutes and view by epifluorescence at x200 magnification. Bacterial inclusions are differentiated from host nuclei by size and frequency. However, if the sample slides are to be retained for future examination, they should be thoroughly dried and stored desiccated until staining.
		Inclusions of the parasite, 14-56 µm in diameter, appear interspersed with the smaller host nuclei. An observation time of 5 minutes per slide is sufficient at x200 magnification.
	2.3.	Polymerase chain reaction
		A positive polymerase chain reaction (PCR) amplification is only a presumptive diagnosis because it detects DNA and not necessarily a viable pathogen. Other techniques, preferably histology and in-situ hybridisation, must be used to visualise the pathogen.
		The PCR primers developed for Candidatus Xenohaliotis californiensis detection specifically amplify a 160 base-pair segment of the Rickettsia-like pathogen. Primers are currently designated as: RA 5-1 (5'-GTT-GAA-CGT-GCC-TTC-AGT-TTA-C-3') and RA 3-6 (5'-ACT-TGG-ACT-CAT-TCA-AAA-GCG-GA-3'). They target small subunit ribosomal DNA and have been shown to be sensitive and specific for this pathogen (1). PCR amplification is performed in a standard 50 µl reaction volume containing 10 mM Tris, pH 8.3 (at 25°C), 50 mM KCl, 1.5 mM MgCl2, 0.001% (w/v) gelatin, 400 µM of dNTP, 5 µM tetramethyl ammonium chloride, 40 pmoles of each primers, 2 units of Taq polymerase, and template DNA. The reaction mixtures are cycled in a thermal cycler. The programme for amplification reaction is: initial denaturation at 95°C for 5 minutes, 40 cycles at 95°C for 1 minute, 50°C for 30 seconds, and 72°C for 30 seconds, and a final extension at 72°C for 10 minutes. An aliquot of each PCR reaction is checked for the 160 base pair amplification product by agarose gel electrophoresis and ethidium bromide staining.
3.	Confirmatory Identification of the Pathogen
	3.1.	In-situ hybridisation
		In-situ hybridisation is the method of choice for confirming identification because it allows visualisation of a specific probe hybridised to the target organism. DNA probes must be thoroughly tested for specificity and validated in comparative studies before they can be used for confirmatory identification.
		In-situ hybridisation has recently been developed to detect Rickettsiales-like prokaryotes in tissue sections (2). Specific labelled oligonucleotide probes hybridise with the small subunit ribosomal RNA of the bacterium. This hybridisation is detected by an antibody conjugate that recognises the labelled probes. Substrate for the antibody conjugate is added, causing a colorimetric reaction that enables visualisation of probe-parasite RNA hybridisations.
		The procedure of in-situ hybridisation is conducted as follows. Positive and negative controls must be included in the procedure.
		i)	After removing the shell, a transverse section (3-5 mm) is cut that contains posterior oesophagus (post-oesophagus), digestive gland, and foot muscle and placed in Davidson's AFA fixative (glycerin [10%], formalin [20%], 95% ethanol [30%], dH2O [30%], glacial acetic acid [10%]) for 24-48 hours, then transferred to 70% ethanol until processed by histological procedures (step ii). The ratio must be no more than 1 volume of tissue to 10 volumes of fixative.
		ii)	The samples are subsequently embedded in paraffin by conventional histological procedures. Sections are cut at 5-6 µm and placed on positively charged slides or 3-aminopropyl-triethoxylane-coated slides. Histological sections are then dried overnight in an oven at 40°C.
		iii)	The sections are deparaffinised by immersion in xylene or other less toxic clearing agent for 10 minutes. The solvent is eliminated by immersion in two successive absolute ethanol baths for 10 minutes each and rehydrated by immersion in an ethanol series. The sections are then washed twice for 5 minutes in phosphate buffered saline (PBS).
		iv)	The sections are treated with proteinase K, 50 µg/ml in PBS, at 37°C for 15 minutes. The reaction is then stopped by washing the sections in PBS with 0.2% glycine for 5 minutes. The sections are then placed in 2 x SSC (standard saline citrate) for 10 minutes.
		v)	The sections are prehybridised for 1 hour at 42°C in prehybridisation buffer (4 x SSC, 50% formamide, 5 x Denhardt's solution, 0.5 mg/ml yeast tRNA, and 0.5 mg/ml heat-denaturated herring sperm DNA).
		vi)	The prehybridisation solution is then replaced with prehybridisation buffer containing 2 ng/µl of the digoxigenin-labelled oligonucleotide probes. The sequences of the probes designated as RA 5-1, RA 3-6, RA 3-8 and RA 5-6 (2) are, respectively: 5'-GTT-GAA-CGT-GCC-TTC-AGT-TTA-C-3', 5'-ACT-TGG-ACT-CAT-TCA-AAA-GCG-GA-3', 5'-CCA-CTG-TGA-GTG-GTT-ATC-TCC-TG-3', and 5'-GAA-GCA-ATA-TTG-TGA-GAT-AAA-GCA-3'. The sections are covered with in-situ hybridisation plastic cover-slips and placed on a heating block at 90°C for 12 minutes. The slides are then cooled on ice for 1 minute before hybridisation overnight at 40°C in a humid chamber.
		vii)	The sections are washed twice for 5 minutes in 2 x SSC at room temperature, twice for 5 minutes in 1 x SSC at room temperature, and twice for 10 minutes in 0.5 x SSC at 40°C. The sections are then placed in Buffer 1 (100 mM Tris, pH 7.5, 150 mM NaCl) for 1-2 minutes.
		viii)	The sections are placed in Buffer 1 (see step vii) supplemented with 0.3% Triton X-100 and 2% sheep serum for 30 minutes. Anti-digoxigenin alkaline phosphatase antibody conjugate is diluted 1/500 (or according to the manufacturer's recommendations) in Buffer 1 supplemented with 0.3% Triton X-100 and 1% sheep serum and applied to the tissue sections. The sections are covered with in-situ hybridisation cover slips and incubated for 3 hours at room temperature in the humid chamber.
		ix)	The slides are washed twice in Buffer 1 for 5 minutes each (see step vii) and twice in Buffer 2 (100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl2) for 5 minutes each. The slides are then placed in colour development solution (337.5 µg/ml nitroblue tetrazolium, 175 µg/ml 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt, 240 µg/ml levamisole in Buffer 2) for 2 hours in the dark. The colour reaction is stopped by washing in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA [ethylene diamine tetra-acetic acid]).
		x)	The slides are then rinsed in dH2O. The sections are counterstained with Bismarck Brown Y, rinsed in dH2O, and cover-slips are applied using an aqueous mounting medium. The presence of the pathogen is demonstrated by the purple-black labelling of the parasitic cells.
	3.2.	Transmission electron microscopy examination
		Transmission electron microscopy procedures are described in Chapter I.2. of this Manual. Rod-shaped, ribosome-rich prokaryotes with trilaminar cell walls accumulated into intracellular colonies within membrane-bound vacuoles in the cytoplasm of gastrointestinal epithelial cells are observed (6).

REFERENCES

1.	Andree K.B., Friedman C.S., Moore J.D. & Hedrick R.P. (2000). A polymerase chain reaction for detection of genomic DNA of a Rickettsiales-like prokaryote associated with Withering Syndrome in Black Abalone (Haliotis cracherodii). J. Shellfish Res., 19, 213-218.
2.	Antonio D.B., Andree K.B., Moore J.D., Friedman C.S. & Hedrick R.P. (2000). Detection of Rickettsiales-like prokaryotes (RLPs) by In situ hybridization in black abalone Haliotis cracherodii with Witheirng Syndrome. J. Invertebr. Pathol., 75, 180-182.
3.	Friedman C.S., Thomson M., Chun C., Haaker P.L. & Hedrick, R.P. (1997). Withering syndrome of the black abalone, Haliotis cracherodii (Leach): Water temperature, food availability, and parasites as possible causes. J. Shellfish Res., 16, 403-411.
4.	Friedman C.S., Andree K.B., Beauchamp K.A., Moore J.D., Robbins T.T., Shields J.D. & Hedrick R.P. (2000). "Candidatus Xenohaliotis californiensis" a newly described pathogen of abalone, Haliotis spp., along the west coast of North America. Int. J. Syst. Evol. Microbiol., 50, 847-855.
5.	Friedman C.S., Biggs W., Shields J.D. & Hedrick R.P. (2002). Transmission of Withering Syndrome in black abalone, Haliotis cracherodii Leach. J. Shellfish Res., In press.
6.	Gardner G.R., Harshbarger J.C., Lake J., Sawyer T.K., Price K.L., Stephenson M.D., Haaker P.L. & Togstad H.A. (1995). Association of prokaryotes with symptomatic appearance of withering syndrome in black abalone Haliotis craherodii. J. Invertebr. Pathol., 66, 111-120.
7.	Haaker P.L., Parker D.O., Togstad H., Richards D.V., Davis G.E. & Friedman C.S. (1992). Mass mortality and withering syndrome in black abalone Haliotis cracherodii, in California. In: Abalone of the World, Shepard S.A., Tegner M.J. & Guzman del Proo S.A., eds, Blackwell Scientific, Oxford, UK, 214-224.
8.	Moore J.D., Robbins T.T. & Friedman C.S. (2000). Withering syndrome in farmed red abalone Haliotis rufescens: Thermal induction and association with a gastrointestinal rickettsia-like prokaryote. J. Aquat. Anim. Health, 12, 26-34.
9.	Steinbeck J.R., Groff J.M., Friedman C.S., McDowell T. & Hedrick R.P. (1992). Investigations into mortality among populations of the California black abalone, Haliotis cracherodii, on the central coast of California, USA. In: Abalone of the World, Shepard S.A., Tegner M.J. & Guzman del Proo S.A., eds. Blackwell Scientific, Oxford, UK, 203-213.

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