Water quality parameters associated with Aeromonas spp-affected hatcheries

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Water quality parameters associated with Aeromonas

spp-affected hatcheries

C Ortega, Jl Muzquiz, Arnaud Fernandez, I Ruiz, I de Blas, Mc Simon, Jl

Alonso

To cite this version:

Original

article

Water

_quality

_parameters

associated

with Aeromonas

_spp-affected

hatcheries

C

_Ortega

JL

_Muzquiz,

A Fernandez

I Ruiz,

I

De Blas, MC Simon JL Alonso

Enfermedades Infecciosas y

Ep!demíolo!ía,

Dep

Patología

Animal, Facultad de Veterinaria,

Miguel

Servet 177,

50013 Zaragoza, Spaí!

(Received

6

April

1995;

accepted

25

April 1996)

Summary ―

This

_{study investigates}

the role of water

_quality

_parameters as

_possible

risk or

_protection

factors in Aeromonas

spp-affected

rainbow trout hatcheries in northeastern

Spain.

The results revealed

an _{association between oxygen} concentration, ammonia concentration and total dissolved solids

(TDS)

with the

prevalence

of Aeromonas spp cases on the affected fish farms. The oxygen and

ammo-nia concentrations acted as risk factors when their values were lower than 7.06

_mg/L

or

_higher

than 0.05

_{mg/L, respectively, generating}

odds ratio

(OR)

values of 2.0 and 2.3. TDS levels, however, acted

as a

_protective

factor

_(OR

=

_0.39),

_from an

_{epidemiological point}

of view but not

statistically,

when the values were outside the normal range, 199-261

mg/L.

On a multivariate level, fish farms

_exposed

to all the associated factors at the same _time, had a risk of

_being

affected

_by

Aeromonas spp that

was 1.74 times

_higher

than farms with normal

_quality

parameters; the risk increased when TDS had a

normal value and it decreased when oxygen and/or ammonia concentrations had normal values.

epidemiology

/ odds ratio /

_Oncorhynchus

_mykiss

/ water

_quality

/ Aeromonas

Résumé ―

Étude

du rôle des

_paramètres

de

_qualité

de l’eau dans les

_{piscicultures}

de truite

(Oncorhynchus mykiss)

affectées par Aeromonas spp dans le nord-est de

l’Espagne.

Nous

avons effectué une étude sur le rôle des

paramètres

de

qualité

de l’eau, comme facteurs

_potentiels

de

risque

ou de

_protection,

en

_{piscicultures}

_{affectées par Aeromonas spp.} Les résultats ont démontré l’exis-tence d’une relation entre la concentration

_{d’oxygène, d’ammoniaque}

et les solides totaux dissous

(STD)

dans l’eau et la

présence

dAeromonas spp. Les concentrations

d’oxygène

et

_{d’ammoniaque}

ont

agi,

comme des facteurs de

risque quand

les valeurs étaient inférieures à 7,06

mg/L

et

_supérieures

a

0,05

mglL,

respectivement.

Les valeurs

correspondantes

du Odds Ratio étaient de 1,97 et 2,6. En revanche les STD

_agissaient

comme un facteur protecteur

quand

les valeurs se trouvaient hors de l’inter-valle considéré comme normal

_(dans

le milieu d’étude : 199-261

_mglL

avec un Odds Ratio de 0,39).

Dans

_l’analyse

multivariée nous avons observé que les _{piscicultures}

_exposées

aux trois facteurs en

*

même temps ont un

_risque

1,74 fois

supérieur

d’être infectées

par Aeromonas

spp que celles non

exposées

à ces facteurs simultanément. Le

risque augmentait quand

les valeurs de TDS étaient

nor-males et diminuait

quand

les valeurs de la concentration

_d’oxygène

oulet

d’ammoniaque

étaient

nor-males.

épidémiologielOdds Ratio/Oncorhynchus mykisss

/ qualité

de 1 &dquo;eau / Aeromonas

INTRODUCTION

Disease outbreaks due to _{Aeromonas spp,}

especially

in the case of

Furunculosis,

where Aeromonas salmonicida is the

_aetiological

agent,

cause the most serious economic losses in

_Spanish

salmonid hatcheries

(Ortega

et

al,

1993a,b;

Sanz

et al,

1993).

Together

with A

salmonicida,

there are other Aeromonas spp

species

such as A

hydrophila,

A

_sobria,

A

caviae,

which are

associated with disease outbreaks in

aqua-culture,

in both

_extensively

and

_intensively

cultured fish

_(Paterson,

_{1983; Barja}

and

Est6vez, 1988).

All these

_{aetiological agents}

are

_present

in a latent state on a

_large

num-ber of fish

farms,

often

_producing

asymp-tomatic carriers

_(Jensen

and

Larsen,

1985;

Austin and

_{Austin, 1987).}

_{Aeromonas spp} have been detected in salmonid and

non-salmonid fish

_species,

in both saltwater and

freshwater,

and cause

_high

_mortality

rates

(Cornik

et

al, 1984;

Austin and

Austin,

1987;

Nougayred

et

al, 1990; Wiuklund,

1990).

Disease outbreaks affect both

_growing

fish and

adults,

and the clinical

_presentation

of

the diseases is characterized

_by

such

_signs

as the presence of lesions

_resembling

boils in the skeletal

musculature,

exophthalmus,

lethargy,

blood-shot

fins,

and/or darker skin colour

_(De

Kinkelin et

al, 1985;

Austin and

Austin, 1987;

Barja

and

_{Est6vez, 1988).}

Diseases

_produced

_by

_{Aeromonas spp}

have been detected in association with envi-ronmental factors such as water

tempera-ture, season of the year, or with

_biological

factors such as the

_{immunological}

status

and stress level of the fish

_(Michel

and

Dubois-Darnaudpeys,

1980;

Ohtsuka et

al,

1984; Thorburn,

1987; Bly

et

al,

1993;

Jarp

et

al, 1993;

Whipple

and

Rohovec,

1994;

Ortega

et

al,

1995).

Control of these factors could

_help

to reduce the effects of disease

outbreaks on fish farms and could

_provide

better information about the role of

asymp-tomatic carriers in these disease outbreaks

(Jensen

and

_Larsen,

1985;

Ortega

et

_al,

1995).

The

_objective

of this

_study

was to

evalu-ate the role of water

_quality

_parameters

as

risk factors on fish farms affected

_by

Aeromonas spp at a univariate and multi-variate level. This could then

_help

better

control these

_{damaging aetiological}

_agents,

which are the most common in

_Spanish

aquaculture

(Sanz

et

al,

1993).

MATERIALS AND METHODS

The

_study

was carried out in 12 hatcheries in northeastern

Spain,

over a 3 year

period

(1992-1994).

All the fish farms

_produced

rain-bow trout

_{fingerlings throughout}

_{the year. The} studied farms had cement and

_fiberglass

pools

depending

on the age of the fish. In all the farms,

the water was

_{supplied by}

rivers. At every farm water

_{samples (see}

_Sample

selection

_(fish))

and fish

(see

Water

quality parameters)

from three different

_pools

were taken every 3 months

(one

sampling

occasion per

season).

Sample

selection

_(fish)

The number of _sampled fish to be taken from every fish farm was determined

_using

the

com-puter programe

Episcope (Frankena

et al,

1990)

and the formulas for

sampling

selection in

Graat,

1994).

We used an _{expected prevalence} of 30%

_(the

value observed

_by

Sanz et al

₍₁₉₉₃₎

for Aeromonas spp infection in

_previous

studies in

Spain),

an _accepted error of 10% and a confi- i-dence level of 90%. This determined a

sample

size of 57 animals per

hatchery. Taking

into

con-sideration the

possibility

of losses

_resulting

from

diagnostic

and data

_analysis,

the

_sample

size

was increased

_by

5% to 60 animals for _every fish farm. This

_sample

included fish of all different ages and both sexes

living

on the farms,

using

a likelihood selection method

_(Martin

et al, 1987; Thrusfield, 1990;

Ortega

and Graat,

1994).

Aeromonas

_spp-affected

hatcheries

A

hatchery

was considered to be affected

_by

Aeromonas spp if the presence of symptoms

associated with Aeromonas spp was detected

(lesions

resembling

boils in the skeletic muscu-lature,

exophthalmus, lethargy,

blood-shot fins,

multiple haemorrhages

in musculature or darker skin colour

_(De

Kinkelin et al, 1985; Austin and

Austin, 1987;

Barja

and Est6vez,

1988))

and/or if Aeromonas spp were isolated from the organs of the fish in culture media.

Aeromonas spp isolation

The

_sampled

fish were sacrificed

_using

a

_high

dose of

_{2-phenoxyethanol,}

and

samples

were

taken from the

kidney,

liver,

spleen

and lesions in skeletal musculature.

Samples

were cultured in

tryptic

soy agar

(TSA)

and brain heart infusion agar

(BHIA),

at 22 °C for 48-72 h. Isolated bac-teria were identified

by microscopy,

biochemical and

_{staining procedures (Austin}

and Austin, 1987;

Barja

and Est6vez, 1988).

Water

_quality

_parameters

Water was

_sampled

in three different locations

(pools)

of each fish farm. Water

_samples

were

taken from the centre of the

pools (10-20

cm in

depth).

The

following

measurements were made

using

portable electronic

_{equipment, colorimetry}

reagents and a

_{portable spectrophotometer:}

water

temperature

(°C), pH,

oxygen

(mg/L),

ammonia

(mg/L),

nitrate

_(mg/L),

nitrite

_(mg/L),

hardness

(mg/L), alkalinity (mg/L),

carbon dioxide

_(C0

₂

₎

(mg/L),

chemical oxygen demand

(COD) (mg/L),

conductivity

(

N

S/cm

2

),

total disolved solids

(TDS)

(mg/L)

and oxygen reduction _potential

(REDOX)

(mV) (Stirling, 1985).

These _parameters were the continuous

independent

variables of the

study.

Epidemiological

and statistical

_analysis

The statistical

analysis

was carried out as a

cross-sectional

_{study (Martin}

et al, 1987; Thrusfield,

1990),

where the association between Aeromonas

spp-affected

fish farms and water

_quality

param-eters was

_analyzed

at three different levels.

The first level of determination was of a

pos-sible statistical association between water

quality

parameters and Aeromonas

spp-affected

fish farms. At this level,

only

factors with a P value lower or

equal

to 0.25 were selected, which meant the factors more

_likely

to be associated with Aeromonas

spp-affected

fish farms.

The second level determination used the odds ratio

(OR),

of the

quantitative

value of the

asso-ciation between Aeromonas

_spp-affected

fish farms and water

quality

parameters at an uni-variate level

_(role

of every associated factor eval-uated

_{independently).}

This second level

_required

the transformation of the continuous _parameters into dichotomic _parameters. This was made

_by

defining

cut-off

points

for every parameter

(Frankena

and Thrusfield,

_{1994) giving}

two pos-sible results for every parameter, normal values

or abnormal values. Values were considered

nor-mal if

_they

fell within the confidence interval

(CI)

defined

_by

the parameters in the

_study

area

according

to the

following

formula:

where X= = mean; SD = standard deviation; n =

population

size; Z= value of Student’s ttest at a

defined confidence level

_(Z=

1.96 at a 95%

con-fidence level for two-tailed

distributions).

This defined two cut-off

_points

for _{every parameter:} the lower and the upper CI limits.

The third level determination used the OR of the quantitative value of the association betweenAeromonas

spp-affected

fish farms and water

_quality

_parameters at a multivariate level

A cross-sectional

_study

was

adopted

rather than a cohort or case-control

study,

because every farm was

_studied

12 times. The animals and the factor values, however, were different every time, so every

sampling

visit at a

_given

farm had to be treated as a different case. The data base and statistical

analysis

of the factors

were made

_using

the _computer program EPI-INFO 5.0

(WHO)

and OR was determined

using

the

_Episcope

program

(Frankena

et al,

1990).

The statistical association between disease and factors was determined

_using

the Student’s ttest

or

_{chi-square (depending}

on the type of

_variable)

and P value. The

_quantitative

value of the

asso-ciation was determined

_by

transformation of the continuous variables into dichotomic variables and

_doing

the calculation of the OR and its

con-fidence interval

(CI) (Frankena

and Thrusfield,

1994).

P means the likelihood of a real association between the disease and the factor. An associa-tion with P values < 0.25 was

accepted

for the

preliminary

factor

screening.

OR means the

_quantitative

value of the asso-ciation, and its

interpretation

was ’animals

exposed’

to the variable

_having

an ’OR value’

indicating

a

_higher

risk for disease than

non-exposed animals. When OR = 1 association did

not exist; OR < 1 the factor acted as _{a ’protective’;}

OR > 1 the factor acted as a ’Risk’.

CI means

_accepted

limits for the OR. When the value 1 was included in the interval, a

mea-surable association at a

_quantitative

level did not

exist; an interval lower than 1 indicated a

’pro-tective factor’; an interval

higher

than 1 indicated

a ’risk factor’.

RESULTS

The

_study

determined that the

_prevalence

of Aeromonas

_spp-affected

fish farms

_during

the overall

_study

_period

was 19.5% in

north-eastern

_Spain.

It was observed that the

prevalence

of Aeromonas

_spp-affected

fish

farms decreased

_during

the 3 _years of the

study:

24.0% in

1992,

18.7% in 1993 and

16.7% in 1994. The results of the associa-tion between Aeromonas

_spp-affected

fish farms and water

_{quality parameters}

at the three studied levels are discussed below.

Determination of associated

_parameters

In the statistical

_analysis

of the association of the

parameters

with the Aeromonas spp

status of the fish farms at a 75% of confi-dence level

_(P

_{< 0.25),}

a

_possible

statisti-cal association was

_only

detected for

_pH,

oxygen,

TDS,

conductivity, ammonia,

nitrate,

hardness and

_alkalinity

_(see

table

_I).

These

results of the first level of the

_study

were a

preliminary

screening

of the factors that

were most

_likely

to be associated with Aeromonas

_spp-affected

fish farms. The real role of these associated

_parameters

was

_{quantified using}

OR in the second level of the data

_analysis.

Quantitative value of the association

between water

_{quality parameters}

and Aeromonas-affected fish farms at univariate level

At the second level of the

_study,

cut-off

points

for normal values of the water

_quality

parameters

were determined

_{(table II).}

Val-ues

_falling

outside these limits were

con-sidered abnormal. In the cases _{of oxygen,}

ammonia and

nitrate,

only

one CI limit was

considered as the cut-off

_point,

because it is known that low _oxygen and

_high

ammonia and nitrate concentrations are harmful to

salmonid

_species.

The

_quantitative

values of the associa-tion were determined

_using

the OR and its CI as shown in table II. The oxygen and ammonia concentrations were risk factors associated with Aeromonas

_spp-affected

fish farms because the Cls were

_greater

than one. While TDS was not

_{statistically}

associated as a

_protection

factor,

because

one was included in the

CI,

it could be

con-sidered as a

_protection

factor from an

epi-demiological point

of view

_(the

_upper

limit,

1.08,

was so close to one and the Cl was

identified as

_being

associated with Aeromonas

_spp-affected

fish

farms,

the Cls

were too wide to define the

_quantitative

value of the association. All of them included

one _{well within their ranges.}

Quantitative value of the association between water

_quality

_parameters

and Aeromonas-affected fish farms

at multivariate level

The calculated OR

_represented

the role of each associated factor at a univariate level. The

_possibility

that the factors interacted at a multivariate level was examined

_using

stratification,

but in all cases the result was

negative.

The role of these factors at a multi-variate level is

_simply

the

_product

of the indi-vidual OR values when interaction factors do not exist.

The multivariate

_analysis

results revealed that the risk of fish farms where the oxygen

and ammonia concentrations have abnor-mal values

_{(risk factors) being}

an

Aeromonas

_spp-affected

fish farm was 4.45

higher

than in a farm with normal

values,

while if the three associated factors

_together

were included in the

_{analysis (including}

the TDS abnormal values as a

protective

fac-tor),

the total risk was 1.74 times

_higher

than

in fish farms where these factors were not

present

together.

DISCUSSION

The observed

_prevalence

of Aeromonas

spp-affected

fish farms in

_{Spain (19.5%)}

was lower than that observed 3 years ago

(31 %)

(Sanz

et

al,

1993),

and it was also lower than the detected

_prevalence

of 24% in the same studied area in 1992

_(Ortega

et

al,

1993a).

We also observed that preva-lence of Aeromonas

_spp-affected

fish farms decreased over the

_{study period (24%}

in

The observed results

_implied

a

signifi-cant reduction of

_prevalence

which was

probably

due to the fact that at the

begin-ning

of this

_study

we decided to set _up new

control

_systems

in

_{hatchery management}

when

_high

_prevalence

of the disease was

detected. The disease reduction would not

be

_{possible only}

as a ’time effect’ without these new

_{management systems (these}

new

_{management systems}

were

_applied

before to the

_begining

of the

_study

and the effect of these

_management

_systems

was

observed

_during

our

_study).

With

_regards

to the

_study

of water

qual-ity

parameters,

oxygen concentrations below 7.06

_mg/L

and ammonia concentrations above 0.05

_mg/L

are risk factors associated with Aeromonas

_spp-affected

fish farms in northeastern

_Spain

at a 95% confidence level. Fish farms with water

_quality

which exceeded these limits were about twice as

likely

to _{suffer from Aeromonas spp. The}

studies of other authors also determined a

role of oxygen and ammonia concentration

as factors _{associated with disease}

preva-lence in Atlantic salmon

_(Salmo

salar

_L),

but the role of these factors was modified

over

time,

depending

on other factors

inter-acting

with oxygen and ammonia such as

environmental factors

_(season,

water

tem-perature

and water

_pH)

or

_management

fac-tors

_{(intensification}

in the

_production

and

feed) (Bergheim

et

_al,

1991;

Fivelstad,

1991

These

interactions were not detected in our

_study

in the case of environmental

factors

_using

stratification

_analysis

and in the case of

_management

we did not

_study

these factors because there were no

sig-nificant differences in

_management

between the studied farms. In contrast to our

results,

other authors did not _{find any association}

between oxygen and ammonia

concentra-tion and disease occurrence, but

_they

found

an association of some fish

_diseases,

includ-ing

diseases caused

_by

_{Aeromonas spp} with water

_temperature

and

_pH

in salmonid

(Whipple

and

_{Rohovec, 1994)}

and

non-salmonid fish

_species

_(Bly

et

al,

1993).

Another

_interesting

result was that TDS

concentrations outside the CI considered normal in our

_study

area

_{(199-261 mg/L)}

appeared

to be a

_protective

factor from an

epidemiological

point

of view but a statistical association was ’not’ detected because 1 was included in the Cl of the OR. Fish farms

with such values were

_only

39% as

_likely

to

suffer from Aeromonas spp infections as

fish farms with normal values.

TDS and

_conductivity

which are often

_closely

related. We also did not detect _any interac-tion between these two factors in the

strat-ified statistical

_analysis.

We think this

inter-action exists because the Cl of the OR for the

_conductivity

had a broad range

(a

broad

range in the CI means interaction

_(Martin

et

al, 1993;

Thrusfield,

1990)

or

confound-ing).

Perhaps

it was not detected because

an insufficient number of water

_samples

were tested

_during

the

_study

_(economical

reasons made it

_impossible

to test

_more).

It is

_possible

that normal values of TDS

act as risk factors because

_they

could

_play

some role in Aeromonas spp

activity

or

mul-tiplication.

These normal values could be

optimal

for the normal

_activity

of Aeromonas spp, while the abnormal values observed

in our

_study

area, both above and below

CI,

could deter bacterial

_activity

but not fish life. It has then to be considered that the

con-ductivity

could

_play

some similar role to that of

TDS,

because a close relation has to exist between both water

_{quality parameters.}

This

was

_suspected

but not determined in the

study.

We did not _{detect any association}

between

_temperature

_{and Aeromonas}

spp-affected fish farms as has been observed

by

other authors

_(De

Kinkelin et

al, 1985;

Austin and

Austin, 1987;

Barja

and

Est6vez,

1988).

This

_might

be because we did not

consider as affected fish farms

_only

those

farms with Furunculosis

_symptoms

and

mor-tality

but we also included the farms on

which we isolated _{Aeromonas spp. That} means we detected both fish farms with the disease

_{(Furunculosis,}

which is

_closely

related to

_temperature)

and fish farms with latent Aeromonas spp infection

(which

is

not

_closely

related to

_{temperature).}

The multivariate effect of

_combining

the

significant

risk factors

_together

indicated that farms with

_abnormally

low disolved

oxy-gen levels or

_high

ammonia

_levels,

but with normal

TDS,

were about 4.45 times as

_likely

to suffer from Aeromonas spp infections.

The presence of abnormal TDS values

decreased the risk to about

1.74,

which is lower than when oxygen or ammonia

con-centrations act as risk factors alone.

It seems

_possible

that the risk level could increase with

_{management factors,}

as was

observed in recent studies of risk factors

associated with A salmonicida infection where it was determined that environmental

parameters acting together played

a less

important

role than

_management

in

Furun-culosis outbreaks

_(Jarp

et

al,

1993)

ACKNOWLEDGMENTS

We would like to thank JL Gimenez and M Lamuela. The _govemment of

_Arag6n

and Navarra

supported

the

project economically.

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