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A LOOK AT THE PRINCIPAL BACTERIAL, FUNGAL AND PARASITIC DISEASES OF FARMED SHRIMP

James A. Brock, Ph.D Aquaculture Development Program Department of Land and Natural Resources State of Hawaii

Honolulu, Hawaii 96813 USA

and

Brad Lea Master , Ph.D, D.V.M.

The Oceanic Institute Makapuu Point Honolulu, Hawaii 96795 USA

SUMMARY

Bacterial, fungal and parasitic diseases are major causes of shrimp hatchery and farm mortality and production losses, This paper considers the major diseases attributed to non-viral pathogens of marine shrimp. The emphasis of the discussion is on current diagnostic and management strategies for these diseases.

INTRODUCTION

Diseases have long been recognized as one of the constraints to the development, expansion and intensification of shrimp farming. Sano and Fukuda (1987) reported the annual disease loss in 1984 from Kuruma shrimp hatcheries and farms in Japan was 3.4% (70.2 tonnes) of the total production. In most, ifnotall, of the major shrimp farming regions of the world, shrimp hatchery and farm production losses due to diseases are apparently increasing (Lightner in press). In some instances, the disease problem has contributed to closure of'hatcheries or farms (Wyban and Sweeney 1991) and in one culture region precipitated the near collapse of a farming industry (Lin 1989). However, with the exception of Japan (Sano and Fukuda 1987) there are apparently few statistics reported on the regional industry production and economic impacts from shrimp diseases.

A variety of agents, both biological and non- biological, have been implicated in the causation of shrimp diseases (Couch 1978; Johnson 1978; Lightner 1983, 1988; Bell and Lightner 1987; Baticados 1988a; Overstreet 1990; Brock 1991). Viruses, rickettsia/chlamydia, bacteria, fungi, protozoa, metazoa, feed factors (limiting nutrients or toxics), environmental factors (soil and water - physical, chemical, bio-toxins and pesticides) and husbandry features are all contributors to causation of shrimp diseases. In many serious disease outbreaks there is an interplay of several causal factors making timely identification of the

controlling factor(s) problematical (Brock and Lightner 1990). The purpose of this review is to discuss selected aspects of the diagnosis and management for the shrimp diseases (Table I) caused by the following biological (biotic) agents: bacteria (including rickettsia/chlamydia), fungi and parasites.

Table 1. Principal bacterial, fungal and parasite diseases of farmed shrimp

Disease or Syndrome

Cause

Texas Necrotizing Hepatopancreatic Syndrome (lNHPS)

Unclassified rickettsia or bacterium

Hepatopancreatic Rickettiosis

Unclassified rickettsia

Vibriosis

Vibrio spp.

Filmentous Bacterial Fouling

Leucothris mucor and other filamentous forms

Larval Mycosis

Lagenidium sp. Sirolpidium sp.

Fusariosis

Fusarium solani

Microsporiosis

Arneson sp., Agmasoma sp & Pleistophora sp,

Haplosporiosis zoan

Unclassified proto-

Intestinal Gregarines

Nematopsis sp.

Epicommensal Protozoans

Zoothamnium sp., Epistylis sp., etc.

Wyban, J., editor. 1992 Proceedings of the Special Session on Sltrimp Fanning. World Aquaculture Society. Baton Rouge, LA USA.

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DIAGNOSTICS: METHODS, FACILITIES AND SERVICES

Methods

For the most part, field observations and laboratory procedures are needed for the diagnosis of the principal bacterial, fungal and parasitic diseases of marine shrimp. Observational findings on distincti ve po pulation attributes and indi vidual shrimp behavioral signs and structural changes provide important, though usual I y not definitive, information concerning disease causation. Microscopic examination of wet-mount preparations of the gills, oral appendages, whole animals (larvae and post1arvae), hemolymph or organ tissue smears, all provide a rapid, reliable means for demonstration of the majority of bacterial, fungal and parasitic disease causing agents of farmed shrimp (Table 2). Histopathologic evaluation is useful as a general diagnostic tool, for research applications, for the demonstration of rickettsial infections, for diagnosis of certain viruses, and for multiple pathogen ornon- infectious (abiotic) disease invol vement. Transmission electron microscopy is another diagnostic tool required for the identification of certain rickettsial infections and plays an important role in microbial disease research.

Table 2. Shrimp diseases that are readily indentified by microscopic examination of wet-mount preparations of larvae or shrimp tissues.

Larval Bacterial Sepsis and Necrosis Larval Bacterial Fouling

Larval Mycosis

Filamentous Bacterial Infestation Ciliate Infestation

Fusariosis

Microsporiosis

Intestinal Gregarines

Metazoan Parasites

In general, the primary bacterial pathogens of shrimp grow readil y on wide! y available agar medias for marine bacteria. The filamentous bacteria such as Leucothrix mucor are the exception to this generalization as these microorganisms require a specialized medium for in vitro growth. However. filamentous bacterial infestation can be identified microscopically and recovery in pure culture on artificial media is not needed for routine diagnosis of diseases caused by these agents. Standard isolation, enumeration, and identification techniques (colony morphology, presence of luminescence, individual bacterium shape and other physical characteristics, and biochemical reactions [test strip systems provide a convenient means]) are used to study bacterial pathogens of shrimp. Antibiotic sensitivity tests by medical microbiological procedures are frequently carried-out on bacteria recovered from diseased shrimp.

The principal fungal pathogens of shrimp also grow readily on routinely available laboratory medias with the addition of antibiotics to control bacterial overgrowth. Thus, as with the majority of the bacterial pathogens of shrimp, special techniques for recovery of the mycotic pathogens are not required.

Facilities and Services

Diagnostic laboratory services are available to shrimp farmers in some farming regions either through small laboratories on hatcheries and farms, government sponsored diagnostic laboratories or through private consultants. Large production shrimp hatcheries often have a microbiology section because there is a constant need for routine bacterial monitoring services. Similar laboratory capabilities are available on some larger farms or in central locations for companies that operate a number of farms.

The on-site microbiology laboratory provides a variety of services in the areas of quality control and microbial disease diagnosis. For an operation where dozens of bacteriological samples are tested daily, the in-house laboratory is a necessity. The microbiology laboratory is commonly equipped with some of the following: one to several light microscopes, incubators for growing microorganisms on artificial media; petri plates, various microbiological medias suitable for the recovery of non-fastidious marine bacteria; an autoclave; etc. In addition, instruments and equipment are often available to carry-out routine water analysis. Histopathology capability is occasionally available in these laboratories.

Personnel that carry-out the diagnostic tests in hatchery or farm laboratories characteristically have had university level training in microbiology and, often, these individuals have had some specialized instruction in shrimp disease diagnostics. Depending on the company, private shrimp disease diagnostic laboratories can offer a full range of services to the farming community and may be competitive with larger, government sponsored or out-of-country consultation service laboratories.

The contribution to shrimp hatchery and farm management gained from information obtained from a diagnostic laboratory is at several levels. In the shrimp hatchery, a very important function is to test the effectiveness of sanitation and husbandry practices. For example, counts can be made of viable bac tcria at differen t points in the system to check sanitation practices. When bacterial counts go up, this suggests a loss in the effectiveness of sanitation procedures and the need to identify the exact nature of the problem and institute corrective measures. Putative bacterial pathogens are often more easily visualized by their characteristic colony color and morphology. Thus, if daily larval culture water samples show an increase in a particular known pathogen, the hatchery manager may be able to initiate management steps to either prevent or reduce losses from an impending bacterial epidemic.

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Another function provided by in the on-site microbiology laboratory is testing bacterial sensitivity to chemotberapeutants, (ie., antibiotics), For example, if drug therapy is used in the system, data on the sensitivity ofkey bacterial isolates provides important information concerning the likely biological response to certain drugs. Antibiotic sensiti vity testing is frequently used in regions where drugs are administered to support hatchery based production of postlarvae. In some cases, where antibiotics may be applied to the feed used in shrimp growout ponds. monitoring of drug sensitivity may also be conducted.

There are, however, potential problems associated with a hatchery or farm-based microbiology laboratory. In hatchery monitoring programs, there is a tendency to generate lots of data. Analysis of this information is time-consuming and in some cases provides little or no beneficial guidance to the management of the hatchery. Moreover, the accuracy of the information can further complicate data interpretation. To our knowledge, cost benefit analyses are unavailable for hatchery or farm-based microbiology laboratories. Thus, the economic questions concerning the operation of these facilities remains largely undefmed.

Finally, in the area of routine daily tank and larval population management in shrimp hatcheries, there can be a tendency to over-rely on data provided by microbiology tests and disregard findings from direct observation of larval behavior or appearance of water or feeds. The results of microbiology testing should always be considered in conjunction with water quality test data and information gained from visual evaluation of the culture system and larval behavior.

A SYNOPSIS OF THE PRINCIPAL BACTERIAL, FUNGAL AND PARASITIC DISEASES

BACTERIA AND RELATED ORGANISMS

Chlamydia and Rickettsia

Infections of cultured shrimp by chlamydia-like and rickettsia-like organisms are documented from a number of regions (Chong and Loh 1984; Lightner et al. 1985; Brock et al. 1986; Anderson et al. 1987; Brock 1988; Krol et al. 1991). Chlamydia! infections are of minimal recognized disease significance to shrimp farming (Lightner in press). Syndromes associated with rickettsial agents, alone or in combination with other microbia! agents, are described from growout culture P. monodon in Asia (Anderson et al. 1987) andP. merguiensis (Chong and Loh 1984), and wild caught, tank reared P. marginatus in Hawaii (Brock et al. 1986). A rickettsia or intracellular bacteria is described from hepatopancreas lesions in growout shrimp affected by the Texas pond mortality syndrome, more recently referred to as Texas necrotizing hepatopancreati tis syndrome orlNHPS (Lightner in press). This syndrome is of considerable importance to shrimp

farms in the region where it has occurred since 1985 with losses reported of up to nearly 100% on some locations. The infection has a chronic course and affected shrimp appear sick (weak, black gills, soft shells). do not eat and do not grow (Lightner in press).

Penaeid rickettsia-like organisms are smaller than most bacteria and have not been isolated on artificial microbiological media (Brock and Lightner 1990). Presently, diagnosis of moderate to heavy infections can be made by histopathology procedures, but histological determination is uncertain for detection of low level carrier infections (Krol et al. 1991) which require demonstration by transmission electron microscopy.

A practical treatment is reported for the lNHPS. In recently conducted field trials, oxytetracycline at 1.5 g active drug per kilogram of feed significantly improved survivals in the medicated pond groups (Bell and Frelier 1991). Practical treatments are not reported for the other penaeid rickettsial infections. Prevention of rickettsial diseases by vaccination has not been attempted.

Besides the significant disease in South Texas, rickettsia infections are unreported from farmed shrimp populations elsewhere in the Western Hemisphere. Moreover, rickettsial infections remain undocumented from hatchery raised shrimp anywhere in the world. Lightner (in press) maintains that due to the difficulties in diagnosis of some forms of rickettsial infection, these organisms may be more widely occurring in shrimp fanning than is presently realized.

The Ubiquitous Gram-Negative RodsEspecially Vibrio spp.

Diseases caused by Vibrio spp. and to a lesser extent several other Gram-negative genera, are of considerable importance to marine shrimp farming worldwide. Vibrio epidemics are a frequent problem in shrimp hatcheries (Aquacop 1977; Johnson 1978; S unaryanto and Mariam 1986; B aticados 1988a; Boonyaratpalin 1990; Prakobkit and Currie 1990; Lavilla-Pitogo 1990),intensively raised shrimp (Lightner 1983,1988; Takahashi 1985a; Lin 1989) and in some regions in pond cultured stocks as well (Johnson 1978; Lightner 1985; Guoxing 1986; Anderson et al, 1988; Nash 1988, 1990; Mohney et al. 1991). Sano and Fukuda (1987) reported that in 1984, vibriosis caused an annual production loss of 30.8 tons valued at 231.4 million yen to Kuruma shrimp farms in Japan.

The principal bacterial species identified from epidemics of bacterial diseases in marine shrimp are listed in Table 3. Typically, the species of bacteria recovered from diseased shrimp are reported to be also readily isolated from the intestinal contents of healthy shrimp (Vandersant et al. 1970; Yasuda and Kitao 1980; Dempsey and K i tting 1987) and from water and sediments in and around shrimp hatcheries and ponds (Lightner 1985. 1988; Baticados et al. 1990; Lavilla-Pitogo et at 1990). Recently an

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Table 3. The pricipal species of marine bacteria recovered from diseased shrimp,

BAC1ERIUM

Vibrio anguiilarium V. alginolyticus

V. cholerae (non-Or) v. damsela

V. harveyi

V. parahaemoiyticus V. splendidus V.vulnificus

Vibrio sp. Aeromonas sp, Pseudomonas sp.

SELECfED REFERENCES:

Barkate 1972; Lewis 1973; Lightner & Lewis 1975; Lightner 1983; Guoxing 1986; Pitogo 1988; Anderson et al. 1988; Nash 1990; Lavilla-Pitogo et al. 1990.

increasing frequency of Vibrio spp. isolated from moribund shrimp have luminescence (Sunaryanto and Marian 1986; Pitogo 1988; Prakobkit and Currie 1990; Boonyaratpalin 1990). This characteristic can aid in their rapid identification during disease outbreaks.

Historically. vibrio epidemics in tank-held bait shrimp (Fontaine 1985) or cultured shrimp have been associated with additional factors which apparently predisposed shrimp to infection and disease. Principal predisposing factors suggested by investigators include handling and injuries to the cuticle of shrimp (Lightner and Lewis 1975); primary viral infection; previous infection by other pathogens including viruses, baeuloviruses, rickettsia, Fusarium, and intestinal gregarines (Anderson et al. 1987, 1988; Nash 1990; Brock and Lightner 1990; Lightner in press); intestinal tissue damage from blue green algal toxins (Lightner 1978); ascorbateacid deficiency of shrimp (Lightner et al. 1977); exposure to physiologically stressful chemical and physical conditions such as low dissolved oxygen, elevated ammonia, temperature extremes and crowding (Barkate 1972; Lewis 1973; Lightner 1983,1985.1988; Sparks 1985; Takahashi et al. 1985a; Baticados 1988a).

In addition to the above, higher salinity and increase nitrogen levels in the culture environment have been linked to serious epidemics of vibriosis on shrimp farms in the Guayas River Delta region of Ecuador (Mohney et al. 1991; Lightner in press). This massive epidemic of vibriosis in the Guayas River Delta termed "syndroma gaviota" or "Seagull Syndrome" has been hypothesized to occur because of the drought in the region. Environmental changes have included increased salinity (20·30 ppt) and

elevated nutrient levels, particularly nitrogen, brought on by the reduced flow of water and by an increase of urea fertilization carried out by many of the shrimp fanners in the region (Lightner in press). Thus. in this ongoing epidemic of vibriosis, environmental conditions which are apparently not directly harmful to the shrimp are believed to have a major influence on the occurrence of the disease (Mohney et at. 1991; Lightner in press).

Diagnosis of bacterial infection oflarvat shrimp can be made by microscopic demonstration ofbacteria1 rods swarming through internal organs or associated with melanized cuticular lesions (Cook and Lofton 1973; Cipriani 1980) of the appendages (Fig. 1) or small, internal melanized nodules in the gills (Fig. 2) or

Figure 1. Photomicro$raph of a melanized lesion (arrow) suggestive of a localized bacterial infection ofthe anterior appendage of a mysis II Penaeus vannamei, Appendage defonnities were COIIUl1OR in this larval population. Wet.mount, no stain; 50x.

Figure 2. Photomicrograph of a gill lamellae from an adult Penaeus japonicus with a focal, internal melanized nodule suggestive of a systemic bacterial infection in this shrimp. Wet-mount, no stain; about 2000x. Original photograph by Mr. Vernon Sato.

other organs (Lightner 1983. 1988; Sparks 1985; Egusa et aI. 1988). In pond reared shrimp. vibriosis should be considered for any disease that is characterized with an abrupt onset and rapid course (several days to two weeks) with clinical signs of disori-

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ented shrimp swimming weakly, gathering along the edges of the pond: generalized opacity or cloudiness of the musculature and/ or red discoloration of the appendages; dorsal flexure at the third abdominal segment with slight rigidity; and the gathering and feeding on the weakened shrimp by fish-eating birds (Lightner and Lewis 1975; Sparks 1985; Lightner in press). Rapid demonstration of the offending bacteria can be made by microscopic examination of hemolymph specimens with or without Gram stain. Hemolymph from fatally moribund shrimp will fail to clot or will clot very slow Iy (Lightner and Lewis 1975). Isolation of the organisms on anyone of a number of medias (fable 4) and identification based on biochemical reactions by standard procedures or by a commercial biochemical profile system can be used to confirm the identity of the pathogen. When outbreaks first occur, and periodically thereafter, histopathological evaluation of moribund shrimp is recommended to evaluate for the presence of additional pathogens and diseases which may be underlying and contributing to the episode of bacterial disease.

Table 4. Examples of artificail media sui table for the culture of invasive bactenal and fungal pathogens of farmed shrimp.

rvlEDIA FOR BACfERIA

PYG (peptone-yeast extractglucose) Agar + 2.5% NaCI and penicillin-streptomycin

MEDIA FOR FUNGI

Marine Agar - MA (Difco)

Saboraud Dextrose Agar

+ 2.5% naCI and penicillinstreptom ycin

Thiosulfate Citrate Bile Salts Agar - TCSS (Difeo)

Trypticase Soy Agar - TSA (Merck) + 1.5-3% NaCI

Nutrient Agar - NA (Merck) + 1.5-3% NaCI

Complex Seawater Medium -CSW

SELECTED REFERENCES: Lightner & Lewis 1975; Lightner 1983; Lavilla-Pitogo et al1990; Boonyaratpalin 1990. _

A variety of strategies have been applied to manage vibriosis in marine shrimp farming. For shrimp hatcheries in most regions. the prevention and control of bac terial disease is a major management consideration. Hatchery culture practices can differ substantially, depending on the training and experience of the hatchery manager as well as physical. chemical and biological attributes inherent to a particular hatchery site and design. Some of the management practices that appl y to control of vibriosis in shrimp hatcheries include batch culture of larvae and system disinfection between stockings (Aquacop 1979); disinfection (mechanical or chemical) of intake water for the hatchery system (Baticados etal. 1990); rigorous sanitation (clean water rinses) of

L

live feeds and equipment disinfection before introduction into production tanks; lower larval stocking densities; control of water temperature to avoid fluctuations; reduction of fecal contamination from broodstock in spawning tanks (K itti wattanawong et al. 1990); exclusive use of acti ve, strong swimming, phototactic, non-deformed nauplii to stock culture tanks; clean water rinsing of nauplii prior to stocking into hatchery tanks; use of periodic water exchange to reduce nutrients and bacterial population density in culture water; proper storage and handling of larval feeds to minimize bacterial proliferation prior to addition into culture tanks; use of sucrose (10 mg/l) in the culture tank water (Heres, A. personal communication); addition of'antibiotics or other chemicals to water or feeds (Aquacop 1977; Tareen 1982; Baticados 1988b; Mohney and Lightner 1990); attentive management to reduce cross transfer of water or other vectors between larval rearing tanks; application of vacci ne preparations (experimental) to the larval culture water (Newman 1992); and use of low stocking density of shrimp larvae in a biologically diverse culture medium with zero to no minimal inputs of supplemental feeds as in the Japanese or modified-Japanese method (Bell and Lightner 1987).

In the shrimp production hatchery, an outbreak of bacterial disease should be responded to as a medical emergency. Immediate management action is called for to increase the chances for effective disease control. Furthermore, continuous 24 hour monitoring of larval behavior and condition and further feed, water or treatment manipulations until the larval populations have recovered may be essential to minimize disease losses.

It should be noted that the practice of antibiotic use in shrimp hatchery disease management is controversial in part because of the perceived induction of antibiotic resistance and concerns over hatchery worker health and near-shore environmental impacts (Brown 1989). The shrimp hatchery community must work closely and cooperatively with public health and environmental safety personnel to resolve these issues. Perhaps, alternate shrimp hatchery husbandry procedures and culture designs need to be developed to minimize antibiotic use in shrimp hatchery production (Brown 1989).

Management strategies that have been applied for vibriosis control in nursery and growout culture of marine shrimp include use of medicated feeds (Corliss et al. 1979; Lightner 1983, 1988, in press; Takahashi et al. 1985b); vaccination (Lewis and Lawrence 1983; Itami et al. 1989); fertilization of the pond with 20 kg/ha sucrose (Lightner in press), partial harvest to reduce pond biomass, increased water exchange and disinfection of the pond bottom between growout cycles by drying, removal of excessive organic sediments and application of quicklime at a dosage of 0.5 kg/rn? (Anderson et aI. 1988). In extreme cases, immediate farmwide harvesting of affected ponds and partial or complete cessation of restocking for a period of time may be the best short term course of acuon to minimize economic losses.

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Filamentous Biofouling Bacteria

Microbial colonization (biofouling) of cuticle surfaces of penaeid shrimp and other crustaceans is a common occurrence in iarvae, juveniles , subadult and adult shrimp (Lightner et al. 1975; Fontaine 1985). Under natural conditions, the density of microorganisms attached to shrimp remains low without recognizable harm to the host. However, under some culture conditions, density of biofouling organisms can increase to levels on shrimp where physiological function is impaired.

The predominant bacterial fouling organism affecting shrimp culture is a filamentous bacterium, Leucothrix mucor. Other related genera that are reported from shrimp include Thiothrix sp., Flavobacterium sp., Flexibacter sp. and Cytophaga sp. (Lightner 1983, 1988, in press). Vibrio spp. and other rod-shaped organisms can also colonize cuticle surfaces (Lavilla-Pitogo et at. 1990). Infestations are usually a heterogenous mixture of filamentous and non-filamentous bacteria, blue green and other types of filamentous algae, peritrich protozoa (ie. Zoothamnium sp.) and other protozoa. Disease losses, however, are usually attributable to heavy infestation by Leucothrix mucor (Lightner 1983, 1988, in press).

Characteristically, disease occurs when there is an abundant colonization of the gill lamellae (Fig. 3) resulting in compromised respiratory function. Disease also occurs if there is

Figure 3. Photomicrograph of gilllameUae from Penaeus vannamei with extensive filamentous bacterial (Leucothrix mucor) gill fouling. Wet-mount no stain; 225x.

colonization of mouthparts or swimming appendages (larvae and postlarvae) which affects feeding or locomotion (Lightner 1983, 1985, 1988). Growth rate can slow and in severe cases, sporadic but persistent mortality of shrimp may occur. Lowenvironmental oxygen, molting, or strenuous activity, such as capture and transfer related activities, can precipitate high mortality in shrimp populations with heavy gill fouling (Brock and Lightner 1990).

Epicommensal bacteria are non-invasive (Solangi et al. 1979) and the cuticle surfaces of shrimp serve as an attachment substrate for these organisms (Couch 1978). In a given culture situation, the biomass or abundance of bacterial and algal biofouling on shrimp likely reflects the availability of essential soluble nutrients and in some cases the underlying health of the shrimp host as well. Shrimp that are ill from a variety of systemic diseases fail to preen normally. Consequently, epicommensal organisms proliferate on the cuticle of these impaired hosts (Lightner 1983,1988). Thus, underlying systemic disease may be a complicating factor in severe epicommensal fouling and appropriate diagnostic tests should be carried-out if these diseases are suspected.

Diagnosis of bacterial biofouling on shrimp is usually accomplished by microscopic examination of whole shrimp (larvae orpostlarvae) or gilVmouth pan appendages in wet-mount preparations. Infestation severity can be numerically graded (Lightner 1983) as an aid to assist decisions on the need to implement control procedures.

The need for control of biofouling disease of shrimp typically depends on the husbandry and intensification of the culture system. Clinical epibiotic fouling is rarely a problem in well managed semi-intensive or extensive growout culture of shrimp. For intensive to super-intensive systems, management of biofouling becomes a necessary husbandry procedure (Wyban and Sweeney 1991). Routine monitoring of giH and mouthpart fouling levels, adjustments in feeding or rate of water exchange, and periodic chemical treatment with soluble copper compounds have been applied in the management of biofouling disease (Lightner et al. 1975; Lightner 1983, 1988, in press; Bell and Lightner 1987). Antibiotic bath treatment has been administered to control bacterial fouling of tank-reared larval and post1arval shrimp (Aquacop 1977; Lightner 1983. 1988, in press; Bell and Lightner 1987).

The Fungi

Fungi are important pathogens in shrimp culture with two distinct diseases recognized that adversely affect shrimp production. One of these diseases, larval mycosis, is a ubiquitous problem that if left uncontrolled can result in catastrophic losses. The other mycosis, Fusarium disease is a problem limited primarily to intensive growout culture of certain penaeid species (Egusaand Ueda 1972; Hoseetal. 1984; Bell and Lightner 1987), but occasionally is seen in maturation systems broodstock shrimp.

Larval Mycosis

Larval mycosis was one of the first diseases described from penaeid hatcheries (Lightner and Fontaine 1973). In the earlier years, fungal infections spread rapidly within hatchery tanks and catastrophic losses were commonplace. Effective preventative treatments were discovered (Bland et al. 1976) and now for

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experienced shrimp hatchery managers, the threat of serious epidemics from larval mycosis is minimal. Several genera of phycomycete fungi are reported to cause the disease. The more common genera are Lagenidium and Sirolpidium; however, Hali phthoros and Atkinsiella are also reported. These fungi share a similar life cycle and are distinguished by differences in sporangia structure and vegetative hyphae diameter.

The phycomycete fungi that cause larval mycosis may be saprophytic and possibly occur naturally in the hatchery water supply or on broodstock shrimp. Infections are transmitted by motile zoospores released by the hyphae of the fungi. Lagenidium infections more often occur in nauplii and protozoeal stages. while the onset of Sirolpidium disease is usually in the late protozoeal to mysis stages (Brock and Lightner 1990). Juvenile or older shrimp are not susceptible to fatal infection by the phycomycete fungi, presumably because the thicker exoskeleton is an effective barrier to penetration by the zoospore germ tube.

Diagnosis oflarval mycosis is based on microscopic demonstration of the characteristic fungal mycelia that fills the body cavity of dying or dead infected larvae (Fig. 4), These fungi will

Figure 4. Photomicrograph of the caudal abdomen and uropods of a protozoea II Penaeus I'annamei infected by Lagenidiurn sp. Note the branched fungal hyphae (arrows) throughout these tissues. Wetmount, no stain: 450x.

grow on standard mycological media (Table 4) [Lightner and Fontaine 1973; Bland et al. 1976; Baticados et al, 19771. Culture of the fungus is not required to confirm a diagnosis of larval mycosis as a cause of larval mortality. Identification to specific genera is not a prerequisite in order to implement control of diseases caused by these fungi.

Once a larval shrimp becomes infected by a phycomycete fungi, chemical treatment has no effect on the survival of this individual animal. However, chemical treatment of culture water with trifuralin, an herbicide, effectively inhibits the motility of

the phycomycete zoospores and widespread larval population infection is prevented (Lio-Poet al.1982; Lio-Po and Sanvictorcs 1986; Bell and Lightner 1987). Trifuralin is unstable in seawater (Williams et al. 1986) and continuous or frequent re-dosage is the usual approach to maintain trifuralin at therapeutic levels (Williams and Lightner 1988). Likewise, malachite green oxalate has also been reported (Bland et al. 1976; Lio-Po ct al. 1982) to be an effective treatment that inhibits the spread of phycomycete infection in larval culture tanks.

Hatchery management routine is also helpful in the control of larval mycosis (Lightner 19H 1; Bell and Lightner 1987). Careful but thorough rinsing of spawned eggs with sterile or near sterile seawater; collection and use of only the phototactic nauplii; thorough cleaning and sanitation of equipment, pipes and tank surfaces between uses also helps to reduce the presence of the fungal reservoir in the hatchery environment. However, these fungi are rclati vel y resistant to chlorine and high levels (500 ppm for 24 hrs.) were found to be needed to completely destroy Lagenidiurn sp. (Lightner 1988).

Fusarium Disease

Fusarium disease is a serious problem recognized from certain highly susceptible pcnaeid species (Egusa and Veda 1972; Lightner, 1981). For Fusarium-sensitive penaeids, and those under highly intensive culture regimes, long term cumulative mortality from fusarium disease can reach 100% (Lightner 1981, 1983). S ano and Fukuda (1987) ranked fusarium disease as the third most significant cause of production and economic loss (5.9 tonnes valued at 30.7 million yen) to Japan's Kuruma industry in 1984. Fusarium disease does not occur in larval stages of pcnacid shrimp.

Fusarium so/ani, the principal causative agent of fusarium disease in penac id shri mp has a worldwide distribution (Lightner 1981). The fungus has a saprophytic existence and grows naturally in decaying organic matter and exposed tissue of superficial wounds (Lightner 1981). Fusarium spp. produce nonmotile, stable infective spores (macroconidia) that disseminate of the fungus. Fusarium solani macroconidia have been demonstrated in water samples collected from sub-sand beach wells used for a shrimp farm (Lightner et al, 1979).

Fusarium solani infects shrimp by colonization of existing cuticle wounds (Lightner 1981; Hose et al. 1984). Apparently, macroconidia dispersed in the culture water settle into openings in the cuticle, germinate and invade into the surrounding host tissues. These infections slowly progress and are accompanied by an intense host response that result in ulcerated to raised melanized lesions (Fig. 5) which are characteristic for the disease. The cause of death in individual shrimp infected with Fusarium is thought to be due to eventual fatal bacterial sepsis by microorganisms that enter through Fusarium lesions or, possibly, from toxins produced by the fungus (Lightner 1981; Hose et al.

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1984).

Shrimp species and age susceptibility playa major role in the occurrence of fusarium disease (Lightner et al. 1979; Lightner 1981; Hose et al. 1984). Of the farmed penaeids, P. japonic us is highly susceptible, P. stylirostris and P. vannamei less so and P. mono don appears to be very resistant to attack by F. solani (Lightner in press). Older, larger shrimp are more apt to suffer from fusarium disease. Colomi (1989) has reported fusariosis from cultured P. semisulcatus.

Culture environments which favor the accumulation of organic detritus or that are heavily loaded with organic material (ie. super-intensive systems) are ideal incubators for Fusarium so/ani. The fungus rapidly colonizes decaying substrate and produces multitudes of infective macroconidia. The culture of susceptible penaeids under high stocking density which enhances the chances for cuticle wounding arc strong contributing factors for fusarium disease.

A presumptive diagnosis of a fusarium infection in shrimp can be made by noting the characteristic gross appearance of a Fusarium solani infected cuticle lesion (Fig. 5). In terms of the

6

Figure 5. Photograph of a melanized, erosive lesion (arrow) suggestive of infection by Fusarium solani of the uropod of an adult Penaeus stylirostris.

shrimp population, the significance of the infection can be assessed by counting the number of shrimp w ith fusarium lesions in a random samp Ie from the population and evaluating each dead shrimp collected for the presence of one or more of the characteristic gross lesions. A diagnosis for fusarium infection can be made by microscopic demonstration in tissue scrapings of the characteristic canoe-shaped macroconidia (Lightner 1981; Sparks 1985), histopathological preparations with special stains that reveal the fungal hyphae and macroconidia (Momoyama 1987; Colomi 1989) or isolation on mycological media and subsequent demonstration of the morphological characteristics of Fusarium spp.

Management procedures for fusariosis include use of a Fusarium resistant shrimp species in culture, low stocking density when farming sensi tive species, prevention of accumulation of organic detritus in the shrimp culture tanks or ponds and harvest of susceptible populations at a younger age before f usarium disease losses increase (Lightner 1981 ; Bell and Lightner 1987). Presently, there are not practical chemical treatments for shrimp affected by fusarium disease (Lightner in press).

THE PROTOZOANS

Invasive Protozoa

Microsporida, Haplosporida and Gregarina are three orders of Sporozoa documented from marine shrimp. Sporozoan infections of shrim p are common in nature and are known from farmed shrimp as well.

Various genera and species of Microsporida occur as natural infections of pen acids worldwide (Kruse 1959; Baxter et aL 1970; Sprague 1970; Villella ct al. 1970; Subrahmanyam 1974; Feigenbaum 1975; Johnson 1978; Couch 1978; Kelly 1979; Enriquez 1980). Certain of these parasites are reported to be of economic importance to commercial shrimp fisheries (Kruse 1959; Sprague 1970; Couch 1978, 1983; Sparks 1985). Their occurrence is sporadic in pond-raised shrimp but has been cited from cultured shrimp in North America (Villella et al. 1970; Couch 1978; Johnson 1978; Sparks 1985; Overstreet 1985; Lotz and Overstreet 1990), from P monodon in Malaysia (Anderson et al, 1989) and Thailand (Nash 1990), and may be encountered in wild-caught spawners (Lightner 1983; Enriquez ct al. 1980). Microsporida are not descri bed from larval penacids and pose no known disease threat to shrimp hatcheries.

Infection of shrimp occurs following ingestion of the infective form of the parasite. Most species of Microsporida localize in the muscle tissue but at least one species settles in the gonads and occasionally other organs as well (Kelly 1979). Once in the target tissue the parasite multiplies and eventually forms large, often visible masses of spores. Heavily infected shrimp have densely opaque muscle or white irregularly enlarged gonads and may have dark blue to black pigmentation (Lightner 1983). Diagnosis of Microsporida infection is based on gross appearanceof affected organs and, when necessary, microscopic confirmation of the presence of the characteristic spores.

Transm iss ion of the shrim p Microsporida rcq uires the spores to be ingested and be passed through the digestive tract of an appropriate species of fish (Iverson and Kelly 1976). Thus, in ponds rnicrosporida infected shrimp do not occur or remain infrequent unless a suitable [ish species is present that can serve as a final host.

Practical treatments have not been developed for microsporidan infections of farmed shrimp. Prevention of infee-

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tion is feasible. however. by excluding suitable fish species from entry into growout ponds and avoidance of feeding raw fish viscera that may contain the infective microsporidan spores. Nash (1990) recommends disinfection of microsporidan contaminated ponds to eradicate the agent Microsporida infected shrimp should not be disseminated with live shipments of shrimp into geographic regions beyond their natural range.

Haplosporeans are another class of sporozoan parasites that have been infrequently described either singly or in concert with other agents and diseases from cultured shrimp. Dykova et al. (1988) described a haplosporean infection of the hepatopancreas of quarantined juvenile P. vannamei in Cuba. Similar occurrences of haplosporean infections of the western penaeids have either not been recognized or have not yet been reported. Additionally, hepatopancreatic haplosporean infection was reported in pond reared P. monodon in the Philippines and Indonesia (Lightner in press). In the pond cultured P. monodon, poor growth, reduced survival, infection by other agents and nutritional deficiency were demonstrated that complicated the interpretation of the findings. The present information indicates that shrimp haplosporeans may be widely distributed geographically, but are an uncommon disease in farmed shrimp. Similar to the Microsporida, haplosporeans are unknown from cultured larvae of penaeids.

Currently, diagnosis ofhaplosporean infection of shrimp can be made based on demonstration of the organism in histological sections of hepatopancreas. There are no known treatments for haplosporean infection of shrimp. When encountered in cultured shrimp populations efforts to eradicate the organism from contaminated ponds or tanks should be given serious consideration. Haplosporean infected shrimp should not be transferred for restocking within or between shrimp culture regions.

As a group, the Gregarina (Protozoa, Apicomplexa) are predominantly intestinal parasites of invertebrates. Annelids, insects, molluscs and crustacea all serve as hosts for a wide variety of gregarine species (Jahn et al. 1979). Worldwide, penaeid shrimp are natural hosts for a number of gregarine species placed into either of two genera, Nematopsis or Cephalolobus (Couch 1978, 1983; Johnson 1978; Overstreet 1973, 1985; Lotz and Overstreet 1990; Lightner in press). For cultured shrimp, gregarines have been recognized both from the larval stages reared in hatcheries and farmed shrimp populations. The life cycle of shrimp gregarines is reported to involve marine snails or clams (Johnson 1978; LOLZ and Overstreet 1990) or the polychaete worm, Polydora cirrhosa, which occurs commonly in shrimp ponds in Ecuador (Lightner in press). Shrimp become infected when they ingest gregarine spores in the slime of clams or tissues of the polychaete worms.

Infection of shrimp with low numbers of gregarines is considered to have little con seq uence to the host (Johnson, 1978). However, heavy infections are recognized in pond cultured

shrimp in some regions and very heavy infection (more than 100 tropozoites per ern of midgut intestine) is reported to result in a yellow discoloration of the midgut (Lightner in press). Heavily infected shrimp populations are suggested to have sluggish growth and, possibly. reduced survival (Lightner in press).

In farming areas where gregarines are endemic, growout populations have been monitored for gregarine infection by microscopic examination in wet-mounts of the midgut intestine (Fig. 6) of shrimp sam pled from production ponds. Anticoccidial

•

.. :, '

.,;..;

11

®

, '. '.:-, .... ... ,

Figure 6. Photomicrograph ofNematopsis sp. (gregarine) trophozoite in the intestinal contents from a subadult fannraisedPenaeusvannamei. The arrow points to the anterior end of the protozoan. Wet-mount, no stain; MOx.

drugs have been gi ven in prepared feeds by some shrimp farmers in regions where gregarines are common. The benefit of this treatment for gregarines is presently unclear. However, fewer gregarines have been noted in shrimp during periods of low salinity (Currie, D. personal communication).

Additional Invasive Protozoa (ciliates, flagellates and amoebae)

Internal infection of shrimp by ciliates, flagellates or amoebae has occasionally been reported. Couch (1978) noted infection by Parauronema sp. and Leptomonas sp. during an epidemic of P. aztecus larvae and postlarvae. An unclassified amoeba was found associated with mass mortalities of larval P. stylirostris and P. vannamei (Laramore and Barkate 1979). Lightner (1988) documented an internal infection of juvenile P. stylirostris by Paranophrys sp .. Most recently, Wang and Ma (1990) mention that Paranophrys carcini is a frequent, serious pathogen in larval and over-wintered P. chinesis.

Diagnosis of these infections is based on microscopic recognition of the protozoan in wet -mounts of larvae of shrimp tissues

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or in histological sections (Lightner 1988). Effective treatment for these additional invasive protozoa are not reported.

Epicommensal Protozoa

Worldwide in distribution and ubiquitous in shrimp culture environments are a variety of epicommensal protozoa which colonize the cuticle surfaces of shrimp (Fig. 7). Although surface

C(]erlt i mttt ers

Figure 7. Photograph of a juvenile Penaeus monodon with extensive cuticular infestation by epicommensal colonial fouling organisms (arrows). The specimen was preserved in Davidson Fixative.

symbionts, epicommensal protozoa have been implicated as the cause of losses in shrimp farming (Tareen 1982; Liu 1989). Infestations are recognized in all life stages and species of fanned shrimp (Lightner 1983).

The principal protozoans described from cuticle surfaces of cultured shrimp include peritrich protozoans (Zoothamnium sp., Epistylis sp., Vorticella sp, and Lagenophrys sp.); suctorians (Acineta sp. and Ephelota sp.) and unidentified flagellates, a choanotlagellate and an apistorne ciliate (Overstreet 1973, 1985, 1990; Couch 1978, 1983; Johnson 1978; Lightner, 1983, 1985, in press). Infestations on cultured shrimp are usually a mixture of protozoans, filamentous and non - filamentous bacteria and algae.

While nearly all of these organisms are non-invasive syrnbiants (Overstreet 1990), their presence in high numbers can adversely affect shrimp health through competition when oxygen supply is limited orreduced host fitness. Protozoan epicommensal organisms arc filter feeders that prey upon microorganisms present within the water (J ahn et al. 1979; Overstreet 1983; Lotz and Overstreet 1990). In that sense, blooms of epicommensal protozoa represent a biological response to an increased supply of microorganisms in the culture water. Heavy protozoan infestations on shrimp reflect an excessive nutrient loading within the culture environment and/or a reduction of molting in the affected shrim p population (Brock 1991).

Diagnosis of epicommensal protozoan infestation is based on microscopic demonstration of the characteristic protozoan organism in wet-mount impressions of gill or appendages taken from affected shrimp (Lightner 1983). Population samples and relati ve estimates of the abundance of protozoans on individual shrimp gill and appendage surfaces as determined microscopically are useful to determ ine the severity of the infestation and the need for treatment administration (Bell et al. 1987).

Management of epicommensai protozoan gill fouling is feasible in some situations by adjustment of pond management practices including shrimp stocking rate, feeding levels and increased water exchange. In hatchery systems where epicommensal protozoan fouling is a recurring problem, procedures should be reviewed in the areas of filtration or treatment of water, handling of eggs and nauplii and sanitation of tanks and live feed cul ture areas. The peritrich protozoa should not be part of the natural fauna of intensively managed shrimp hatchery tanks. In intensive farming systems where nutrient loading, microorganism proliferation and heavy infestations of protozoans arc antici pated, routine moni toring of population samples by microscopic examination of gill and oral appendages is recommended (Lightner 1983; Wyban and Sweeney 1991). While a variety of chemical treatments have been reported to effectively control protozoan fouling organisms, controlled study findings (Bell et al. 1987) have shown that formalin is efficacious and safe as a bath treatment.

METAZOAN PARASITES

Marine shrimp in their natural environment serve as hosts for a variety of metazoan parasite groups (nematodes, cestodes, digcnean trematodes and isopods [Fig. 8]). However, in spite of the very common natural infection by these organisms, (Overstreet 1973; Couch 1978; Johnson 1978; Fontaine 1985) reports of

Figure 8. Photographof an adult, wild-caught Penaeus monodon with a conspicuous (arrow) swelling of the carapace overlying the gills. The swelling was caused by the presence of a bopyrid isopod (Epipenaeon ingens) in the branchial cavity, The shrimp was preserved in Davidson Fixative.

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significant diseases caused by helminth or isopod infections in farmed shrimp populations have not appeared. Nevertheless, metazoan parasites are known from impounded and cultured shrimp populations (Overstreet 1973; Johnson 1978; Tareen t 982). Lotz and Overstreet (1990) caution against complacency about the potential impacts from these agents. Principal concerns are loss of product value, regulation of product for public health reasons, and, possibly. reduced farm production through direct disease losses.

Helminth species that infect marine shrimp have complex life cycles that involve alternate hosts (Couch 1978; Johnson 1978; Overstreet 1990). Where shrimp are fanned in systems that effectively exclude the parasites or the host species required for development or propagation of the parasite, metazoan parasites remain self-limiting or absent altogether. Thus, effecti ve prevention of problems from these agents is usually achieved by currently employed routine management practices.

CONCLUSIONS

Disease loss from non-viral biotic agents has and continues to be of significance to shrimp fanning worldwide. Rickettsial infections are a serious problem in a few farming regions. Information on the biology of these agents is sketchy and future problems from rickettsial diseases are anticipated.

Larval mycosis, historically a very significant eause of catastrophic losses in shrimp hatcheries, is currently effectively controlled by routine sanitation and prophy lactic chern ical treatment of larval culture water. Hence. little loss to hatchery production is attributed to larval mycosis in well managed pcnaeid hatcheries. However, the phycomycete fungi that cause larval mycosis are frequently present in hatchery environments and may demolish production if operators are inexperienced or become careless.

The Microsporida and haplosporeans have occurred as significant, but sporadic problems in some shrimp farming regions. Gregarines are a group of organisms whose impact on farm production remains controversial, largely because of the lack of controlled studies to document the impact these organisms have on shrimp growth and survival. Perhaps, this information is available through the field research carried -outon various shrimp farms, but has not been disseminated to the public domain. Currently, there is little infonnation that implicates metazoan parasites as significant disease agents of farmed shrimp.

In the majority, if not all of the larger farming regions, vibriosis remains the greatest infectious disease threat to stable hatchery production, and in some areas, to growout farm production as well. In spite of this, there are only a few scientific studies that directly address the issue of control and management of vibriosis in shrimp hatcheries or farms. In the absence of more effective methods becoming available for the management and

control of vibriosis of shrimp. dependence on antibiotic medication will likely continue and may increase. However, market pressure or environmental regulations may curb this trend. The public health and environmental consequences of persistent antibiotic use in shrimp fanning remains largely unstudied and unclear.

From the standpoint of research priorities, an emphasis is suggested on studies that elucidate improved resistance (genetic, vaccination or nutritional manipulation) of shrimp to vibriosis or through increased understanding of the environmental variables that influence the abundance of specific vibrio pathogens in shrimp culture environments (hatchery tanks and growoutponds).

ACKNOWLEDGEMENTS

Mrs. Janet Yasamatsu is thanked for her assistance with obtaining reference material used in the manuscript. Support was provided for the preparation of this paper by the Aquaculture Development Program, Department of Land and Natural Resources, State of Hawaii and The Oceanic Institute, Makapuu, Hawaii.

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