Effect of oxolinic acid on bacterial flora and hatching success rate of rainbow trout, Oncorhynchus mykiss, eggs

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.4quaculture. 9 1 ( 1990) 205-222 Elsevier Science Publishers B.V., Amsterdam

Effect of oxolinic acid on bacterial flora and hatching success rate of rainbow trout, Oncorhynchus mykiss, eggs G.A. Barker”, S.N. Smith” and N.R. Bromageb aDepartment qfPharmaceutical

Sciences (Biology Division). ‘4ston Univewty, Birmingham, B4 7ET. Greaf Brifam blnstitute qfiiquaculture, University o_fStwirng. Stirling, FKY 4LA, Great Britarn (Accepted 30 May 1990)

ABSTRACT Barker, G.A., Smith, S.N.. and Bromage. N.R., 1990. Effect of oxolinic acid on bacterial flora and hatching success rate of rainbow trout. Oncorh.wzehus mykiss, eggs. Aquaculfure, 9 1: 205-222. During incubation in small-scale, totally enclosed, recirculatory water systems. rainbow trout. Oncorhynchus mykiss, eggs were exposed to oxolinic acid at two concentrations. The subsequent bacterial flora associated with surfaces of treated and untreated eggs was examined at regular intervals throughout incubation. Overall. eggs incubated in the presence of oxolinic acid were colonized by significantly fewer bacteria than untreated eggs. The bacterial flora of treated eggs tended to be composed of Cyfophaga sp., while in contrast, untreated eggs were colonized by high numbers of both Fy-Vfophagasp. and Pseudomonasjluorescens. Egg mortalities were recorded daily and although oxohmc acid was considered successful in lowering bacterial numbers on the surfaces of eggs, a corresponding increase in egg hatching was not observed.

INTRODUCTION

Rearing of both salmonid eggs and fry often results in varying degrees of success. Bromage and Cumaranatunga ( 1988), citing data obtained from commercial trials, estimated that the following percentage survivals might be expected at each of the five successive stages of development; fertilization 90%, eyeing 80%, hatching 75%, swim up 60% and after 4 months 35%. Even assuming some variation within these figures, it is obvious that considerable levels of mortality can be expected to occur within hatcheries. Possibly, due to such high mortality rates, the U.K. has annually imported ever-increasing numbers of eyed eggs to meet the expected demand for table fish. This imporAddress for correspondence: G.A. Barker, Ministry of Agriculture, Fisheries Diseases Laboratory, The Nothe. Weymouth, Dorset DT4 8UF, Great Britain

0044-8486/90/$03.50

0 1990 -

Elsevier Science Publishers

B.V.

and Food, Fish

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tation of eyed eggs is not only costly but also renders the U.K. industry vulnerable to the introduction with eggs of non-endemic diseases such as viral haemorrhagic septicemia (V.H.S. ) (Vestergaard-Jorgensen, 1970) and infectious haematopoietic necrosis (I.H.N. ) (Wolf, 1976). Therefore, it is of some importance that U.K. hatcheries maximize the successful rearing of eggs and fry, potentially leading to a reduction in the numbers of eyed eggs imports. LOSSthrough death of incubating eggs and fry is likely to be due to a wide range of causes. These include factors affecting water quality such as flow rate, dissolved oxygen, pH and temperature. In addition, efficacy of husbandry practices will also influence egg and fry survival. For example, excessive illumination of eggs (Leitritz and Lewis, 1976), physical disturbances of eggs before eyeing (Smirnov, 1975; Laird and Wilson, 1979) and failure to prevent fungal colonization through malachite green treatment (Cline and Post, 1972) will all result in “premature” loss. The role of microorganisms other than fungi in influencing egg and fry survival is less clear. However, Sauter et al. ( 1987) revealed the presence of a wide range of bacteria within chinook salmon, Oncorhynchus tshawytscha Walbaum, eggs and considered that amongst other factors, bacteria might also be important in determining “early life stage” death of eggs and fry. Similarly, Barker et al. ( 1989) demonstrated that a close correlation existed between numbers of bacteria present on surfaces of incubating eggs and egg mortality. In this study it was therefore proposed to determine whether a reduction in numbers of bacteria colonizing incubating egg surfaces, achieved by antimicrobial treatment, could in turn lead to an increase in egg hatching success. Potentially, a range of techniques exists to reduce bacterial numbers on egg surfaces including ultraviolet light sterilization of incubation water, treatment of eggs with iodine disinfectants (iodophors) and treatment of eggs with antibiotics/antimicrobial agents. Initially, the two latter methods would seem the most desirable, due to their low cost and established use on fish farms. In other aquaculture industries, for example the culture of marine bivalve larvae, the routine use of antibiotics within hatcheries is well established (Jeanthon et al., 1988). Thus, it was proposed to screen a range of antibiotics/ antimicrobial compounds for their suitability to inhibit the growth of bacteria such as Pseudomonas jluorescens and Cytophaga sp., both genera frequently found colonizing surfaces of incubating salmonid eggs (Bell et al., 197 1; Trust, 1972; Yoshimizu et al., 1980; Barker at al., 1989). However, one major disadvantage of the prolonged use of antibiotics/antimicrobial compounds is the development of resistant bacterial strains. Consequently, it was considered necessary to design and construct a unique, albeit small-scale egg incubation system, capable of securely containing eggs, water and particularly antimicrobial compounds, thus avoiding the release into the environment of antibiotics and any bacteria that may have been exposed to them. In addition, the design of such incubation equipment was also gov-

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emed by a range of other considerations, including replacement of water and compounds during incubation without excessive disturbance to eggs. This factor was considered particularly necessary since it was expected that effrcacy of compounds would gradually decline with time and would thus need to be renewed at regular (pre-determined) intervals. This study outlines the effect of one potentially suitable compound (chosen from three), oxolinic acid, on microbial populations colonizing surfaces of incubating salmonid eggs and the subsequent egg hatching rates. MATERIALS AND METHODS

Selection and preliminary screening of antimicrobial compounds A limited range of antibiotics and antimicrobial compounds were screened for their potential ability to inhibit the growth of P. fluorescens and Cytophaga sp., both commonly found on incubating salmonid egg surfaces (Trust, 1972; Yoshimizu et al., 1980; Barker at al., 1989). This first selection of compounds was based on their reported effectiveness against pathogens both from the field of human medicine and specific fish pathogens found on commercial fish farms (Fig. IA). The protocol for the testing of compounds (Fig. 1B ) was based on that of Austin et al. ( 198 1). Compounds were dissolved in distilled water or in 10% v/v dimethyl sulphoxide to aid solubility where appropriate. Compounds in a range of concentrations were incorporated into melted cooled tryptone soya agar (T.S.A., Oxoid) or low nutrient Cytophaga agar (Anacker and Ordal, 1959), poured into 90-mm diameter sterile petri dishes and dried inverted overnight at a temperature of 37 ‘C. Subsequently, four evenly spaced 4-mm diameter plugs were removed from the agar using a “cork” borer. A plug of a young actively growing culture was placed into each of these holes and plates were incubated for 20 days at 10°C. After this time the presence or absence of microbial growth around the plug was noted. Plugs were removed and placed into fresh agar plates (lacking compounds). Growth on this occasion would indicate that earlier inhibitory activity in the presence of the antimicrobial compound was bacteriostatic rather than bacteriocidal. Secondary screening of antimicrobial compounds Utilizing the results from the above trials the most promising compounds were selected and further assessed to determine their suitability for use in the experimental system (Fig. 1C). The most appropriate compounds would be those whose efficacy declined only slowly with time, reducing the number of occasions on which they had to be renewed, in turn limiting disturbance to incubating eggs. Decline in efficacy of compounds was therefore tested microbiologically in a similar manner to that outlined by Evelyn et al. ( 1986). The assay organism in these trials was a strain of Escherichia coli (Laboratory

‘08

Fig. 1. Sequential screening of selected antimicrobial

G.A. BARKER ET AL.

compounds.

isolate W 3 110). Test plates were prepared by first pouring 20 ml of assay medium (Oxoid, Antibiotic Assay Medium No. 1) into 90-mm diameter sterile petri dishes. These were then topped with 6 ml of the same medium amended with 0.1 ml of a phosphate buffered saline suspension of the assay organism ( 1.0 O.D. at 460 nm; pH 7.0). Efficacy of compounds was subsequently determined by regular sampling over 36 h of circulatory water derived from the egg incubation systems (as described below). Dry, sterile blotting paper disks (6mm diameter) (Oxoid) were briefly soaked in circulatory water, placed onto culture plates prepared in the manner outlined above and incubated right side up at 25 “C for 5 days, after which time the diameter of growth inhibition was carefully measured. From a previously constructed standard curve for each compound, based on zones of inhibition with known amounts of compound, a guide to the decline in concentration of each com-

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pound with the passage of time was obtained. From these calibration curves it was determined that three components - oxolinic acid, chlortectracycline and chloramphenicol - were suitable for use in the experimental system outlined below. Construction of equipment Totally enclosed, small-scale incubation systems were constructed, comprising a reservoir of 3 1 of aerated water which was pumped (Gilson Minipuls 2) at a flow rate of 22 ml/min over eggs which were placed into a modified, plastic cell culture tray (Sterilin). Water after passing over eggs subsequently drained from the rear of each tray (under gravity) back into the original reservoir below. All water, incubation systems and equipment were kept at 10°C ? 1 “C in a chilled air cabinet (Verticold). In total four such identical systems were set up in the cabinet (Fig. 2). Experimental design Chloramphenicol, chlortetracycline and oxolinic acid were assigned at random to one incubation system at minimum inhibitory concentration value (hereafter referred to as x 1 M.I.C. ), and to a second system at twice minimum inhibitory concentration value (hereafter referred to as x2 M.I.C.). The remaining two systems were both used as controls to which sterile water alone was added (hereafter referred to as control group 1 and control group 2). All systems had water and compounds (or water alone) replaced at intervals of 36-48 h as determined through the secondary screening of compounds. Collection of eggs and milt Eggs utilized in each trial were obtained from five fish held at a single commercial fish farm. Eggs of each fish were stripped into individual sterile containers. The first eggs from each fish were discarded to avoid contamination from the surrounding water or ventrolateral surface of the female. Milt, also collected in sterile containers was obtained from a mixture of sex-reversed females and normal males. Surface of eggs, coelomic fluid and milt were removed from each container for bacteriological examination. The remaining eggs were pooled and fertilized. After 5 min excess milt was washed off and eggs allowed to water harden for 45 min. Small batches of eggs ( 160 in each batch) were laid down in each of the four trays, as this was considered the most suitable number of eggs for each tray to ensure adequate rates of water flow and separation of eggs. Egg mortalities (eggs that turned opaque) were recorded daily. Fertilization rates were determined after 7 days at 10” C by removing 20 eggs from each tray (80 in total) and placing them into clearing solution (acetic acid : methanol: water, 1: 1: 1 v/v) (Springate and Bromage, 1984). Fertilized eggs could be clearly distinguished by the presence of a developing neutral tube. At eyeing eggs were “shocked” by siphoning from a

G.A. BARKER ET AL.

L

, Reservoir 3

3 hues of aemed water + compound

Fig. 2. Diagrammatic representation

Reservoir 4

3 lives of aerated water + compound

of egg incubation systems.

height of 1 m into a sterile beaker and any unfertilized eggs removed. Eyed eggs were subsequently returned to their relevant sections in incubation trays. Bacteriologicalexamination Ahquots of coelomic fluid and milt from each fish were serially diluted and inoculated in 0.02-ml volumes (drop inoculation method) onto T.S.A., peptone beef extract glycogen agar (McCoy and Pilcher, 1974) and low nutrient Cytophaga agar. Plates were incubated at 20°C for up to 10 days and enu-

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meration performed only from drops where total separation of colonies occurred. Eggs were initially sampled at the green stage (unfertilized) and subsequently 7 days, 14 days, 2 1 days and 28 days after fertilization. At each sampling time 10 eggs were removed at random from each tray and sampled individually. Each egg was rinsed in four changes of sterile water to remove any detritus or loosely adhered bacteria and added to 0.5 ml of sterile diluent (peptone 0.l%, saline 0.85%) and shaken vigorously for 2 min on a vortex stirrer, in a manner adapted from Evelyn et al. ( 1984). The number of viable cells transferred to each of the three different media was determined by plate count. By taking into account the dilution factor, the volume of diluent and the egg surface area, the number of viable colony forming units (cfu’s) per mm2 was estimated. Identification of bacteria Bacteria were identified by a variety of techniques, procedures and schemes outlined by Krieg and Holt ( 1984)) Cowan ( 1974), the tables of Allen et al. ( 1983 ) and Stanier et al. ( 1966 ) . Some Gram-negative bacteria, especially A. hydrophila, were more easily identified using API 20E and 20NE identilication strips (API laboratory products). Statistical analysis Number of colony forming units (cfu’s) per mm2 egg surface for treated and non-treated eggs were compared using a two-way split-plot analysis of variance (Ridgman, 1975; Snedecor and Cochran, 1980). Differences between means were compared by calculating the standard errors and 95% confidence limits appropriate to the split-plot design (Snedecor and Cochran, 1980). Numbers of hatched and non-hatched eggs were compared using a chi square (x2) test. The effects of bacterial presence on egg surfaces were analysed by multiple regression, chosen to compare numbers of surface bacteria with egg deaths by relating mortality to time (age) and bacterial numbers. RESULTS

Egg development Of the three antimicrobials assessed oxolinic acid appeared most efficacious, therefore, only results from trials of this compound are outlined in detail below. Eyeing and hatching rates obtained during the study are summarized in Table 1. It can be seen that estimated fertilization rates of 85% were obtained, in turn leading to hatching rates ranging from 49% to 58%. The number of fertilized eggs lost during the trial represents the number of eggs that potentially should have hatched but, instead suffered “premature” mortality. From Table 1 estimated fertilized egg losses can be calculated by subtracting numbers of “estimated unfertilized eggs” from the total number of

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TABLE I Eyeing and hatching rates for control eggs and eggs exposed to oxolinic acid Egg survival

x I M.I.C.

x 2 M.I.C.

100 85 56 54

100 85 61 58

100 85 59 49

29 13 3 45

36 8 2 46

24 15 3 42

21 20 10 51

15 30

15 31

15 27

15 36

Control (group 1)

Control

100 85 58 55

Egg loss Deaths to eyeing Deaths at shocking Deaths from eyeing to hatching Eggs that failed to hatch Unexplained losses Estimated unfertilized eggs Estimated fertilized egg losses

Total number of eggs Estimated fertilized eggs Actual eyed eggs Actual hatched eggs

(grow 2 1

“eggs that failed to hatch” and ranged from 27% to 36%. Eggs treated with oxolinic acid at x2 M.I.C. suffered the highest number of eggs lost in this category, mainly due to the high number ( 10) of eggs lost between eyeing and hatching. In contrast, eggs exposed to oxolinic acid at x 1 M.I.C. suffered the least numbers of eggs lost compared to all other groups. However, analysis by x2 revealed no overall significant difference in hatching success between all four groups (x2 = 1.69, P> 0.05 ) . Daily mortalities of eggs are presented in Fig. 3, from which it can be seen that the two control groups show slightly higher rates of mortality during the mid-point of incubation (7-2 1 days) than treated egg groups. Control eggs (group 2 ) suffered significantly greater mortalities than treated eggs ( x 1 M.I.C.) between 7 and 14 days (x2=4. 14, PcO.05) and the same control group (group 2 ) also showed significantly greater mortalities than treated eggs (x2 M.I.C.) between 14 and 21 days (x2=4.84, PcO.05). Bacteriological examination

Bacteria were isolated from the coelomic fluid of all fish examined. Genera recovered included Pseudomonas, Aeromonas, Staphylococcus, Corynebacterium and yellow pigmented Gram-negative rods of the Flavobacterium-Flexibacter-Cytophaga group. Bacteria of similar genera to the above were also isolated from all samples of milt but were generally present in greater numbers than in coelomic fluid. It should be noted that, as milt in this trial was obtained from sex-reversed females, the slightly higher numbers of some bacteria may in part be due to external contamination involving the protracted

EFFECT OF OXOLINIC

ACID ON BACTERIAL

Tlmr

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(days)

Fig. 3. Accumulative total of dead eggs recorded daily, for control eggs and eggs exposed to oxolinic acid. A control ( 1), + control (2), 0 oxolinic acid ( x 1 M.I.C. ), A oxolinic acid ( X 2 M.I.C.)

process of testis removal and its subsequent handling by farm staff (Woodroffe and Shaw, 1974). Few bacteria were obtained from the surfaces of unfertilized eggs, Stuphylococcus epidermis and Pseudomonas sp. were occasionally isolated. In contrast, during incubation large numbers of bacteria accumulated around egg surfaces of all groups. However, generally greater numbers of bacteria were isolated from surfaces of untreated eggs compared to those exposed to oxolinic acid. Estimates of total colony forming units (cfu’s ) isolated during the trial are summarized in Fig. 4. It can be seen from this figure that treated eggs ( x 1 M.I.C.) were colonized by the least number of bacteria when sampled at both 14 and 2 1 days incubation. Treated eggs ( x 2 M.I.C. ) were colonized by the least number of bacteria when sampled at 7 and 28 days but, during

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A8

600

500

400

.* 5 ‘j

300

h 5 z

200

100

0

I

0

10 Ttme

I

I

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ldaysl

Fig. 4. Estimated total colony forming units/mm* egg surface for control eggs and eggs treated with oxolinic acid. 0 control ( 1), n control (2), 1 oxolinic acid ( x 1 M.I.C.), A oxolinic acid (X 2 M.I.C.). A=95% confidence limits for differences within a treatment and B=95% confidence limits between two treatments at the same or different times.

the mid-point of incubation ( 14-2 ldays ) they supported bacteria in similar numbers to untreated eggs (group 1). Untreated eggs (group 2) mostly supported the highest numbers of bacteria throughout incubation. Analysis of numbers of cfu’s recovered for all egg groups (by two-way splitplot analysis of variance) revealed overall a highly significant difference in numbers of egg surface bacteria between groups (PC 0.00 1). Bacterial numbers on egg surfaces were highly significantly affected by time (P-C 0.001) but no significant interaction between time and bacterial numbers was found. It should also be added that although bacterial numbers were significantly different between groups, they were all subject to great fluctuations over the experimental period, as demonstrated in Fig. 4. An examination of species of bacteria found on egg surfaces during incubation revealed that both untreated groups (Fig. 5a, b) exhibited a very similar flora with greater numbers of Pseudomonas fluorescens recovered, followed closely by Cytophuga sp. Numbers of both organisms reached their maximum after 21 days’ incubation and declined after 28 days’ incubation.

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AB

0 10

0

30

20 Time

(daysI

AB 400 -I

300

-

200

-

lb1

1 3 z ;

00 0

10 Ttme

20

30

(days1

Fig. 5. Estimated total colony forming units/mm’

egg surface for each bacterial genus. 0 Pseu-

domonas sp., n Cytophugu sp., A “others”. A= 95% confidence limits for differences within a treatment and B = 95% confidence limits between two treatments at the same or different times. (a) Control group 1. (b) Control group 2. (c) During treatment with oxolinic acid at x 1 M.I.C. (d) During treatment with oxolinic acid at x 2 M.I.C.

All remaining bacterial genera that were isolated were accumulated and referred to as “others”. Within this group Aeromonas hydrophila was the most abundant organism. Within egg groups exposed to oxolinic acid (Fig. 5c, d) numbers of P. fluorescent and Cytophagu sp. were again seen to reach their maximum after 21 days’ incubation. In contrast to untreated groups Cytophuga sp. were seen to predominate on surfaces of eggs exposed to x 1 M.I.C.

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AB

(cl

O:b Time

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lriaysl

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20 Ttme

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Fig. 5. (continued)

However, Cytophagu sp. were only isolated from eggs exposed to x 2 M.I.C. in greater numbers than P. fruorescens after 2 1 days’ incubation. Overall, analysis by multiple regression of egg death demonstrates a marked correlation between egg mortality and the numbers of bacteria on incubating egg surfaces (r~0.8 1). However, time (6.36) rather than numbers of bacteria ( 1.87) showed the greatest correlation with egg mortality. DISCUSSION

In common with Barker et al. ( 1989) this study has shown that after fertilization salmonid eggs are not heavily “loaded” with bacteria. They are, how-

EFFECT OF OXOLINIC ACID ON BACTERIAL FLORA .AND HATCHING OF RAINBOW TROUT EGGS

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ever, exposed to them from a very early stage of development as bacteria were present in both coelomic fluid and milt samples. During incubation egg surfaces were colonized by considerable numbers of bacteria even though equipment and water were either thoroughly disinfected or sterilized by autoclaving to reduce contamination. Such bacterial proliferation is probably the consequence of initial introduction of bacteria to incubation systems either adhering to egg surfaces or with residual traces of coelomic fluid and milt. Subsequent increase in microbial biomass is in turn supported by the accumulation of nutrients close to egg surfaces, which as noted by Zobell and Anderson ( 1936) commonly occurs around exposed surfaces of aquatic environments. The actual numbers of bacteria on surfaces of eggs from all four groups, as revealed by plate count, were shown to be subject to great variation throughout incubation. Similar fluctuations in numbers of aquatic bacteria have been found to occur seasonally (Jones, 1973; Allen et al., 1983; Austin and AllenAustin, 1985; Iriberri et al., 1987). In addition, Bell et al. ( 197 1) reported fluctuations in microbial numbers on surfaces of stream-incubated salmon eggs, and Trust ( 1972) demonstrated a similar pattern of bacterial colonization on eggs maintained in a vertical upwelling incubator. In this study it is also likely that changes in bacterial numbers on egg surfaces were further accentuated by the unique experimental design. In particular, the regular refilling of systems may perhaps have led to the partial removal of unattached “free living” bacteria in turn reducing the regular exposure of eggs to potential colonizers. Oxolinic acid is frequently used to treat infections caused by Gram-negative bacteria and indeed was specitically developed for fisheries use in Japan (Endo et al., 1973a). Its efficacy against a wide range of specific fish pathogens, including Aeromonas salmonicida, A. liquefaciens, Vibrio anguillarum, Chrondococcus columnaris and Yersinia ruckeri has been well documented (Endo et al., 1973a,b; Austin et al., 1983; Rodgers and Austin, 1983). However, Austin and Al-Zahrani ( 1988) used oxolinic acid against dense and mixed populations of bacteria in the gastrointestinal tract of rainbow trout and found that during the course of treatment bacterial numbers increased. This was possibly a consequence of inhibiting sensitive organisms, in turn allowing the proliferation of more resistant bacteria. In contrast, during this study treatment of incubating egg surfaces with oxolinic acid resulted overall in a decrease of bacterial numbers. This may in part have been due to the limited range of bacterial genera found on egg surfaces (i.e., few Gram-negative bacteria and no Gram-positive bacteria) compared to the adult intestine. Oxolinic acid was successful in reducing the bacterial egg surface populations, mainly through lowering numbers of P. fluorescens. Oxolinic acid also proved successful in reducing numbers of “other” bacteria, a category mainly comprising Aeromonas and Pseudomonas sp. Endo et al. ( 1973a) and Jo

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( 1978) have demonstrated the effectiveness of oxolinic acid against Aerornona~ and Pseudomonas sp., respectively. Therefore, reductions in numbers of “other” bacteria are probably at the expense of these two species. In contrast Cytophaga sp. were largely unaffected by oxolinic acid treatment and numbers remained similar to those found on control eggs. Inhibition of P. fluorescent and “other” bacteria could have allowed numbers of Cytophaga sp. to proliferate due to reduced competition. However, as numbers of Cytophaga sp. did not significantly increase, it is possible that the presence of oxolinic acid reduced environmental quality in such a manner as to restrict proliferation of Cytophagasp. Furthermore, oxolinic acid is known to be effective against a wide range of Gram-negative rods (Alderman, 1988) and has been reported to be effective against Flexibacter sp., a group closely related to Cvtophaga sp. (Austin and Austin, 1987). The estimated fertilization rates of 85% obtained during this study were slightly lower than the 90% that might be expected from other predictions derived from commercial data (Bromage and Cumaranatunga, 1988 ) Subsequently, actual hatching rates for egg groups ranged from 49% to 58%, well below the “expected” hatching rate of 75% obtained by the above authors. During the present trial great emphasis was placed on preventing antimicrobial compounds (and any resistant bacteria) from entering the natural environment. The slightly poorer hatching rates obtained might therefore suggest that although adequate conditions for egg development were provided, a combination of slow water flow rate, low levels of oxygen and increased levels of egg disturbance, a consequence of the unique experimental design, may not have provided “optimum” conditions for salmonid egg survival. Potentially, eggs incubated under such environmental conditions would in turn profit from any subsequent form of prophylactic treatment which reduces bacterial pressure. However, despite the potential for oxolinic acid to lower bacterial numbers on egg surfaces, hatching success rates of treated eggs did not significantly increase, implying that the presence of bacteria on incubating egg surfaces is not detrimental to hatching success. In contrast, Barker at al. ( 1989) suggested that there might be a correlation between egg survival and number of bacteria on egg surfaces. It would appear therefore that the reduction in bacteria mediated by oxolinic acid is neither maintained for a sufficient period, nor great enough to sustain an improvement in hatching success. Other compounds or combinations of compounds rather than oxolinic acid may prove more effective at lowering the overall number of bacteria on egg surfaces. However, chlortetracycline and chloramphenicol evaluated under the conditions outlined in this study also lacked promise as neither reduced bacterial numbers significantly or increased egg hatching success. Antibiotic treatment may, therefore, not be the most appropriate method of reducing the established bacterial colonies of salmonid egg surfaces due to the growth habit of such bacteria. Geesey et al. ( 1977 ) demonstrated that in aquatic en-

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vironments the majority of bacterial populations exist as enclosed microcolonies. According to Costerton et al. ( 198 1) such encapsulation is widespread in natural environments caused by a “polysaccharide component” surrounding cell walls, termed the glycocalyx. The glycocalyx may in turn act as a barrier, influencing access of molecules, ions and protons to the bacterial cell wall and cytoplasmic membrane (Cheng et al., 1970; Costerton et al., 198 1) as well as giving some measure of protection against antimicrobial compounds (Govan and Fyfe, 1978). Furthermore, the routine use of antibiotics in salmonid hatcheries cannot be encouraged due to the possible selection of antibiotic-resistant bacterial strains and their potential toxicity to developing salmonid embryos. However, during this trial no evidence of developed bacterial resistance was observed and according to Forfar et al. ( 1966) cessation of treatment gives rise to a rapid decline in resistance levels. Of greater consequence therefore is the potential toxicity of antibiotics. Excessive and prolonged use of oxolinic acid may well be detrimental to eyed eggs as a markedly greater number of eggs exposed to x2 M.I.C. failed to hatch after successful eyeing. Commercially, it could therefore prove more expedient to avoid widespread prophylactic use of antibiotics and to prevent or reduce initial bacterial colonization of egg surfaces by reducing numbers of circulating “unattached” bacteria. Water treatment methods, particularly those that avoid the long-term use of chemicals, for example, ozone disinfection (Conrad et al., 1975 ) or ultraviolet (U.V. ) sterilization (Spanier, 1978; Brown and Russo, 1979; Kimura et al., 1980) might prove suitable. Although during these trials insufficient evidence was obtained to support regular use within hatcheries of such antibiotics as oxolinic acid, chlortetracycline and chloramphenicol, there may still be occasions when limited use of antimicrobial compounds may prove to be worthwhile. Evelyn et al. ( 1986) demonstrated that exposure of eggs to erythromycin could be effective in treatment of bacterial kidney disease (B.K.D.) through limiting a possible route by which the causative agent, Renibacterium salmoninarum, is disseminated. In addition, further work investigating the exposure of near-hatching eggs to certain carefully selected (non-toxic) antibiotics/antimicrobial compounds should be considered. Results from this trial have shown that hatching fry on emergence from the egg are likely to be exposed to considerable numbers of P. fluorescens, Cytophaga sp. and A. hydrophila. These three bacterial genera are well known as opportunistic pathogens and secondary invaders of diseased and injured fish (Borg, 1960; Anderson and Conroy, 1969; Allen et al., 1983; Roberts and Horne, 1978 ) . Their suppression through antibiotic prophylactic treatment may therefore reduce premature mortality amongst fry, 40% of which can die between hatching and 4 months of age ( Bromage and Cumaranatunga, 1988 ) . Any investigations which expose eyed eggs to antimicrobial compounds

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must carefully evaluate potential short-term benefits in terms of increased fry survival against the longer term performance of such fry, in particular, their weight gain and disease resistance. However, if limiting the exposure of newly hatched fry to potential bacterial pathogens is successful in enhancing fry survival, then ultimately a reduction in the U.K. demand for eyed egg imports might be achieved. ACKNOWLEDGEMENT

The authors are indebted to Dr. R.A. Armstrong of The Department of Vision Sciences, Aston University, Birmingham, for his help and statistical advice.

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