Mortalities in Red Claw Crayfish Cherax Quadricarinatus Associated with Systemic Vibrio Mimicus Infection

DISEASES OF AQUATIC ORGANISMS Vol. 19: 233-237.1994 Published August 25 Dis. aquat. Org. Mortalities in red claw crayfish Cherax quadricarinatus associated with systemic Vibrio mimicus infection Lorraine E. Eaves, Peter J. Ketterer Queensland Department of Primary Industries, Animal Research Institute. Yeerongpilly, Queensland 4105, Australia ABSTRACT: Two cases of lnortalities in cultured red claw Materials and methods. Since 1989, sick or dead red crayfish Cherax quadricarinatus associated with systemic claw from aquaculture operations have been sub- Vibno mimicus infections are described. In these cases, mitted to our laboratory for disease diagnosis. Tissues V rnirnicus appears to have been an opportunistic pathogen following stress caused by either overcrowding or mis- for histological examination were fixed in Davidson's management and poor water quality. Humans consuming solution (ethanol31.7 %, formalin 22.2 %, glacial acetic raw or improperly cooked infected crayfish could be at risk of acid 11.1 % in an aqueous solution). Sections (5 pm) contracting gastrointestinal disease. were cut and stained with haemotoxylin and eosin. Selected sections were stained with Brown & Brenn's KEY WORDS: Red claw . Cherax quadricarinatus Crayfish . Vibrio mlmjcus . Vibriosis modification of the Gram stain (LiLlie & Fuller 1976). Bacteriological culturing was done on Blood Agar (containing 5 U/o sheep blood) and Trypticase Soy Agar The red claw crayfish Cherax quadricarinatus is an plates (Becton Dickinson, Cockeysville, MD, USA) in- Australian tropical freshwater crayfish belonging to cubated aerobically at 25 "C. Following overnight incu- the family Parastacidae, its habitats varying from shal- bation, single colonies were streaked onto another low clear-flowing creeks to deep turbid clay-based Blood Agar plate and incubated overnight. The follow- water holes (Rouse et al. 1991).In Australia, it is found ing day, the cultures were checked for purity and an in watercourses ranging from the Daly River in the oxidase test done on them using Oxidase Reagent Darwin region of Northern Territory through the Gulf Droppers (Becton Dickinson). The cultures were subse- of Carpentaria rivers to the westerly flowing rivers of quently inoculated into oxidation/fermentation media Cape York Peninsula (Morrissy et al. 1990). Red claw (Difco, Detroit, MI, USA) and into either the API 20E or are highly suitable for intensive aquaculture in tropical the API 20NE identification system (Bio Merieux, and subtropical zones (Jones 1990). They tolerate low Marcy-llEtoile, France) for presumptive identification. oxygen levels and a wide range of other water quality Later, comprehensive identification of Vibrionaceae parameters including ammonia, nitrite, hardness, alka- was made using a modification of tests described by linity, salinity and pH. In addition, they are not territo- Cowan (1974) as well as tests described by Furniss et rial, do not dig burrows, accept food items ranging al. (1978), Lee et al. (1979), Lee & Donovan (1985) and from hay to formulated rations and are capable of West & Colwell (1981). Identification was based on a breeding several times a year (Rouse et al. 1991). They range of tests selected by Bryant et al. (1986a) for the have been cultured in Australia since 1985 and are numerical classification of Vibrionaceae and further now farmed in Queensland and northern New South developed into a computer matrix for the probabilistic Wales. To date, infectious disease has not caused sig- identification of species of Vibrionaceae (Bryant et al. nificant mortalities in Australian crayfish. Stock losses 1986b). have been primarily due to problems associated with Case histories. Case 1: In November 1989, 3 dead site selection, water quality and management (Morrissy red claw from a comn~ercialcrayfish enterprise in et al. 1990). This paper reports 2 cases of mortalities northern New South Wales containing a hatchery and in cultured red claw in Australia associated with a ponds were frozen and submitted for examination. systemic infection of Vibrio mimicus. Upon draining the ponds, a large number of the excess O Inter-Research 1994 234 Dis. aquat. Org. 19: 233-237, 1994 adults and juveniles were recovered and placed in integument of the tails exhibited epithelia1 necrosis holding tanks. Following mortalities in these con- and cuticular erosion with evidence of bacterial gested tanks, overcrowding was reduced by the use of invasion. additional tanks. Despite this, further mortalities were Discussion. Vibrio mimicus was first described by observed. The submitted crayfish were necropsied and Davis et al. (1981) as a pathogenic species of the genus the haemolymph and hepatopancreas were sampled comprising sucrose negative varian.ts of biochemically for bacteriological examination. atypical strains of V. cholerae. Phenotypic and bio- Case 2: In July 1990, 3 live red claw with carapace chemical properties of the 5 V. mimicus isolates de- lengths ranging from 6.7 to 7.5 cm were submitted from scribed here were identical, conforming with the a commercial crayfish farm in southeast Queensland. original species description except for their lipolytic The farm had a history of low-level mortalities over a ability as they hydrolysed both Tween 20 and Tween 2 yr period which increased to 20 % when stocks were 80 (Table 1). However, this difference could be due to transferred to cement tanks. Heaviest losses occurred the fact that Davis et al. (1981) used corn oil as their during hot weather and were initially observed follow- lipid substrate rather than the Tween compounds used ing the addition of hydrated lime to the ponds to raise in this study. This is supported by the positive lipase the pH. Affected cravfish were unable to walk and reactions reported for V m!-~icusby Rryar.! et al. appeared to be stuck in the residue on the bottom of (198613) and Lupiani et al. (1993) where Tween com- the ponds. Many had a white powdery coating over pounds were used as the lipid substrates. However, their bodies. Two of the submitted crayfish had blister- the isolates differed from the descriptions of both ing on the end of the telson, and one of them also had Bryant et al. (1986b) and Lupiani et al. (1993) in their blisters on the uropod. The third crayfish exhibited alginase activity and their inability to use Mannose as erosion at the end of the telson. Subsequently, alter- a sole carbon substrate (Table 1).The API 20NE system ations were made in the management practices. Lime does not have V. mimicus included in its data base and was added to a holding dam from which water was thus it identified the isolates in Case 1 as V. cholerae. drawn to fill the ponds and the residue was cleared In addition, it does not test for carbohydrate fermen- from the bottom of drained ponds. Following these tation, negating differentiation of V. cholerae and V. changes, mortalities ceased. Haemolymph from the rnimicus by sucrose fermentation. heart of each submitted crayfish was cultured bac- Vibno mimicus occurs in seawater and shellfish, and teriologically. in humans, and has been associated with gastro- Results. Case 1: A mixed culture of Escherichia coli enteritis following ingestion of seafood and with ear and Enterobacter intermedium was isolated from the infection after exposure to sea water (Ciufecu et al. haemolymph, and Aeromonas hydrophila and Citro- 1983, Shandera et al. 1983).It has also been found both bacter freundii from the hepatopancreas of 1 crayfish. in brackish and freshwater environments (Bockemiihl The haemolymph and hepatopancreas of the other 2 et al. 1986, Chowdhury et al. 1989), and was iso- crayfish yielded pure cultures of an organism initially lated from the gills and from under the carapace of identified as Vibrio cholerae by the API 20NE. The freshwater prawns Macrobrachium malcolmsonii by isolates did not agglutinate V cho1erae:Ol polyvalent Chowdhury et al. (1986). V. mimicus would thus antiserum and were presumptively called V cholerae appear to be part of the normal bacterial flora of the non: 01. However, on comprehensive characterisation, aquatic environment in aquaculture ponds. As the these isolates were subsequently identified as V mim- crayfish in both cases described in this report were fed icus. Histologically, there was severe cellular disrup- a diet of commercial chicken pellets, the water would tion due to freezing in the ovary and hepatopancreas. appear to be the most likely source of the V, mimicus However, the heart of one of the crayfish yielding causing the disease outbreaks. a pure culture of V mimicus exhibited severe inflam- The significance of the blisters observed on the tel- mation of the pericardium, and foci of small Gram- son and uropod of crayfish in the second case reported negative rods were present in the lesion. here remains uncertain. Herbert (1987) reported that Case 2: The haemolymph of each crayfish yielded a blisters on the uropods and telson of Cherax spp. most pure culture of a Gram-negative organism identified as commonly occurred following introduction into vinyl- Vibno mimicus using API 20E test kits. The isolates lined swimming pools, concrete tanks or stainless steel were not agglutinated by V. cho1erae:Ol polyvalent troughs. They considered them to be the result of irri- antiserum and were confirmed as V. mimicus follow- tation as no deaths could be solely attributed to their ing further comprehensive characterisation. On his- presence. tology, varying degrees of inflammation, indicative of Bang (1970) considered that the haemolymph of bacterial septicaemia, in the gills and hearts, and also healthy crustaceans was sterile and that the presence in the antenna1 gland of l crayfish, were observed. The of bacteria in the circulatory system was a sign of Eaves & Ketterer: Crayfish mortalities associated with Vjbrio mimicus 235 Table 1. Vibrio rnimicus. Phenotypic characteristics of isolates (5 strains, 2 from Case 1 and 3 from Case 2) compared with other published results. +: 86 to 100% of strains positive; v: 16 to 85 % of strains positive; -: 0 to 15% of strains positive; nd: not done Characteristic This study Bryant et al.

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