vibrio harveyi: a pathogen of penaeid shrimps and fish in venezuela

© 1998

Blackwell Science Ltd 313

Journal of Fish Diseases (1998) 21, 313–316

Short communication

Vibrio harveyi: a pathogen of penaeid shrimps and fish inVenezuela

J D Alvarez 1, B Austin 2, A M Alvarez 1 and H Reyes 1

1 FONAIAP, Instituto de Investigaciones Veterinarias, Maracay, Venezuela2 Department of Biological Sciences, Heriot-Watt University, Edinburgh, Scotland

Since 1980, silver mullet, Mugil curemaValenciennes, pompanos, Trachinotus carolinus L.,permits, T. falcatus L. and penaeids, i.e. Penaeusschmitti (Burkenroad), P. (Litopenaeus) vannamei(Boone) and P. (Litopenaeus) stylirostris (Stimpson),farmed and caught in Venezuelan waters, havebeen severely affected by bacterial haemorrhagicsepticaemia, coined vibriosis (e.g. Cairoli & Conroy1982). Initially, it was considered that Vibrioanguillarum was the aetiological agent, with foodcontaining non-pasteurized Anchoa regarded as theprincipal source of the pathogen (Cairoli & Conroy1982). However, since 1993, work which formsthe basis of this report indicates that widespreadmortalities among Venezuelan fish and penaeidshave been caused by V. harveyi.

Between February 1993 and December 1996,fish and penaeids were obtained from five locations.In total, 149 juveniles, adults and broodstock ofChaetodipterus faber (Broussonet), commonlyknown as Atlantic spadefish, cultured in corrals,floating cages and tanks, were obtained from theVenezuelan government’s marine station in SucreState. Ten juvenile and adult silver mullet werecaptured from wild populations at Puerto Colombia,Aragua State. One hundred and forty-eightP. schmitti were obtained from feral populations atMedano Blanco Beach (Falcon State) and PariaPeninsula (Sucre State). White leg shrimp P.(Litopenaeus) vannamei (n 5 120) and blue shrimpP. (Litopenaeus) stylirostris (n 5 74) were obtainedfrom a farm in Anzoategui State. The life stages ofpenaeids examined were juveniles and adults.

Correspondence Professor B. Austin, Department of BiologicalSciences, Heriot-Watt University, Riccarton, EdinburghEH14 4AS, Scotland

The Atlantic spadefish were examined at thecoastal site. All the other specimens were transportedto the fish pathology laboratory in the Institute ofVeterinary Research, Maracay, Aragua State. Thelive animals were placed in plastic bags withsupplemented oxygen and then sealed with rubberbands. The bags were transported in insulatedcontainers. The temperature of the water and theair was maintained at ~25 °C using ice cubes insidethe bags and in the insulated boxes.

The specimens were weighed and measured,examined for signs of disease using standard methods(see Austin & Austin 1989) and then dissected.Diseased (in groups of 10) and healthy (in groupsof 30) juveniles, adults and broodstock were killedby decapitation, dissected and samples taken fromkidneys (fish), hepatopancreas (penaeids) andintestines (Dear 1989). If lesions were observed inthe skin, exoskeleton, eyes or in any other region,samples of the diseased tissues were also removed.Samples were homogenized in 3 ml volumes ofphysiological saline (PS) in a tissue blender (Griffithstube; Jencons, Leighton Buzzard, England). Then,10-fold dilutions were prepared to 10–5 in PS, and0.1 ml volumes were spread onto the surface oftriplicate plates of tryptone soy agar (Difco, Detroit,Michigan, USA) supplemented with 1.5% (w/v)sodium chloride (TNA) and incubated for 24–72 hat 30 °C. A representative of each colony type waspurified by streaking and re-streaking on freshmedia. Pure cultures were stored at 4 °C in agarstabs with subculturing every 6–8 weeks.Identification was achieved after Baumann, Furniss& Lee (1984) and Austin & Lee (1992). The typeculture of V. harveyi (NCIMB [National Collectionof Industrial and Marine Bacteria, Aberdeen,Scotland] 1280T) was included for comparison.

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Journal of Fish Diseases 1998, 21, 313–316 J D Alvarez et al. Fish and shellfish pathogenic Vibrio harveyi

A random group of bacterial isolates from diseasedanimals were grown on TNA slopes for 18–24 h at30 °C, suspended in PS, washed twice (1000 g for15 min at 4 °C), and suspended in fresh PS to 108

bacteria ml–1, as determined by comparison withthe density of the MacFarland standard no. 1(Branson 1974). Dilutions were prepared, and0.1 ml volumes injected into groups of six fish orfour penaeids to achieve doses of 104–107 cellsanimal–1. Fish were injected intraperitoneally,whereas the penaeids received the bacteriaintramuscularly between the third and fourthabdominal segments on the ventral side (Song,Cheng & Wang 1993). Additional groups wereinoculated with PS, as controls. The animals weremaintained in aerated sea water (salinity 5 36‰)at ambient temperature, i.e. 28–30 °C, withexamination daily for 14 days. Dead and moribundspecimens, and the survivors after 14 days, wereexamined bacteriologically, as before. Mortalitieswere considered to be attributed to the isolate onlyif it was recovered in virtually pure culture growthfrom dead animals. The LD50 dose was calculatedby the Probit method, after Wardlaw (1985).

Throughout the sampling period, most (73%)farmed Atlantic spadefish appeared to be healthy,although some juveniles showed darkening of thebody pigment (5 melanosis), eroded fins andhaemorrhaging in the eyes. There was no evidenceof other disease signs. However, during April 1994,10 of the Atlantic spadefish broodstock, which weremaintained separately in floating cages and in tanks,demonstrated bilateral exophthalmia, haemorrhagesin and around the eyes (when this condition wassevere, corneal opacity appeared), and occasionallyhaemorrhages in the fins. From the eyes of thediseased specimens, a single bacterial colony-typewas recovered in dense, virtually pure culturegrowth. Similarly in July 1995, 10 immature adultAtlantic spadefish showed clinical disease, with signsof bilateral exophthalmia and haemorrhaging in eyesand at the base of fins. Again, virtually pure bacterialculture growth was recovered from all the fish. Inaddition to the bacteria, occasionally some of theseAtlantic spadefish were observed to contain theparasitic copepod Aniloca laticauda in the mouthcavity, a monogenetic trematode resemblingDactylogyrus sp. in the gills, and (sporadically) heavyinfections of a monogenetic trematode, morpho-logically resembling Neobenedenia sp., on the skin.

During December 1996, a feral population ofsilver mullet displayed signs of haemorrhagic

septicaemia. From the 10 diseased animals, a purebacterial culture growth was obtained.

In July and August 1995, three batches of juvenilemarine shrimp (P. vannamei and P. stylirostris) fromthe commercial shrimp farm in Anzoategui Stateappeared to be healthy upon transfer to the aquariumin Maracay. However, within 3 days in the aquarium,many black spots developed on the exoskeleton.From these lesions, dense, virtually pure culturegrowth of bacteria was again recovered. Subsequentlyin December 1996, the farmed shrimp (P. vannameiand P. stylirostris) began to die in large numbers.These mortalities were correlated with overflightsby large numbers of migratory birds. Many juvenileshrimp sent for examination were dead on arrivalat the laboratory in Maracay. Other specimens wereweak. From the specimens, virtually pure culturebacterial growth was recovered on TNA.

In total, 49 pure bacterial cultures recovered fromdiseased fish and penaeids produced cream-coloured,occasionally translucent, raised, shiny colonies,which were subsequently equated with a singlespecies, i.e. V. harveyi. These cultures containedluminescent motile Gram-negative fermentativerods, which produced catalase, indole, lysinedecarboxylase, nitrate reductase and oxidase, but notarginine dihydrolase, swarmed on TNA, requiredsodium chloride for growth, and were sensitive tothe vibriostatic agent 0/129. The Voges Proskauerreaction was negative. Most isolates (ù 80% of thetotal) degraded chitin, gelatin and starch, but notelastin. Growth occurred at 40 °C. Cellobiose, D-gluconate, L-serine, sodium acetate, sodium citrateand sodium propionate were utilized. Thus, thecultures possessed the key characteristics ofV. harveyi, and were identical to the type strainNCIMB 1280T (Baumann et al. 1984; Austin &Lee 1992).

Six isolates, albeit at dosages equivalent to ù105

cells animal–1, were pathogenic for fish and shrimp(Table 1). There was no evidence of disease nor wasany isolate recovered following infection with 104

bacteria animal–1. The highest levels of mortalitywere recorded for V. harveyi isolate 3 (100%mortalities in fish and shrimp) and isolate 24 (83 and75% mortalities in fish and shrimp, respectively). Itis noteworthy that V. harveyi isolate 3, which wasisolated from a skin ulcer on an Atlantic spadefish,produced total mortalities within 24 h of injectionwith 106 cells shrimp–1. In fish (Atlantic spadefishand silver mullet), this bacterial isolate produced100% mortality within 24 h of challenge with 106

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Table 1 Pathogenicity of the V. harveyi isolates from Atlantic spadefish and Penaeus spp.

Ref. no. of Source of Animals used Dose Mortality LD50 dose Time to death Reisolation ofV. harveyi isolates isolate for challenge animal–1 (%) (days) pathogen

2 C. faber M. curema 106 0 .107 – –3 C. faber C. faber 106 100 13105 1–2 1

3 C. faber M. curema 106 100 53104 1–2 1

3 C. faber M. curema 105 100 53104 1–5 1

3 C. faber P. vannamei 106 100 13105 ,1 1

7 C. faber P. vannamei 106 25 .106 1 1

8 P. schmitti P. vannamei 106 50 13106 2 1

24 C. faber M. curema 106 83 23105 1–2 1

24 C. faber P. vannamei 106 75 43105 1–2 1

62 P. vannamei P. vannamei 106 25 .106 1 1

63 P. vannamei P. vannamei 106 50 13106 ,1 1

bacterial cells fish–1. It was noted that moribundfish, infected with all the pathogenic isolates, showedsome external signs of disease, notably melanosis,abdominal distension and slow erratic swimming.Internally, there was congestion of the viscera, withfriable organs. The lower intestine was filled with amucopurulent substance. By examination of Gram-stained preparations, short Gram-negative rods wereobserved. Bacteria were recovered as dense pureculture growth from the posterior section of kidneysand intestines of the challenged fish. In shrimp,isolate 3 caused muscular opacity around the siteof injection. Also, the animals displayed slow erraticmovement. Internally the hepatopancreas was pale.Again, bacteria were recovered as dense pure culturegrowth from the kidneys of all the diseased fish, thehepatopancreas of the shrimp and the intestines ofboth groups.

Certainly, this study has shown that V. harveyihas been recovered from diseased fish and penaeidsin Venezuelan waters since 1993. It is noteworthythat there was no evidence for the presence ofV. anguillarum, which has been reported previouslyas a fish pathogen in Venezuela (Cairoli & Conroy1982). V. harveyi has been isolated from the diseasedeyes of the common snook, Centropomusundecimalis, (Kraxberger-Beatty, McGarey, Grier &Lim 1990) and sunfish, Mola mola, (Hispano, Nebra& Blanch 1997) and from outbreaks, regardedas ‘vibriosis’, from cultured silvery black porgy,Acanthopagrus cuvieri, and brown-spotted grouper,Epinephelus tauvina (Rasheed 1989; Saaed 1995).Despite the increasing evidence of its pathogenicityto fish, more attention has focused on the role ofV. harveyi as the aetiological agent of shrimp diseases,especially in South America and Asia (Baticados,

Lavilla-Pitogo, Cruz-Lacierda, de la Pena & Sunaz1990; Karunasagar, Pai & Malathi 1994).

The significance of V. harveyi as a shrimp pathogenis reinforced by reports from other tropical countrieswhere the organism has been found to cause up to100% mortalities in shrimp hatcheries. A commontheme is that V. harveyi has been isolated fromdiseased but not healthy larvae and often fromthe water in the rearing facilities (Jiravanichpaisal,Miyasaki & Limsuwan 1994; Karunasagar et al.1994). Interestingly, Jiravanichpaisal et al. (1994)isolated V. harveyi as a minor component of themicroflora on the exoskeleton of female black tigerprawns in Thailand. Nevertheless, proof of apathogenic role for V. harveyi has rarely beenforthcoming, illustrating the comparative difficultyof carrying out infectivity experiments with shrimp.Yet in this study, isolates, which were recoveredfrom diseased animals, demonstrated some degreeof virulence to fish and penaeids. Moreover, inagreement with the results from this study, otherworkers have reported high LD50 values (4.9 3 107

and 1.56 3 109 CFU) for fish (Saeed 1995). Lowerdoses, e.g. 102–103 cells ml–1, have been determinedto be sufficient to cause high mortalities in larvalshrimp (Lavilla-Pitogo, Baticados, Cruz-Lacierda &de la Pena 1990). Interestingly, Liu, Lee & Chen(1996) used two reference strains (not originallyisolated from shrimp) and four field isolates ofV. harveyi from diseased shrimp, and noted that thelatter were more virulent in shrimp. Perhaps thesedata indicate a loss of pathogenicity with storage,which would be the situation with the referencecultures. Clearly, there is accumulating evidencepointing to the pathogenicity of V. harveyi foraquatic vertebrates and invertebrates over a widegeographical area.

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Acknowledgments

This work was supported by FONAIAP, CONICITand the British Council.

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vibrio harveyi: a pathogen of penaeid shrimps and fish in venezuela

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