Fish 19

689
CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
19
Applying Epidemiology to Infectious
Diseases of Fish
M.A. Thorburn
Department of Population Medicine, Ontario Veterinary College, University of
Guelph, Guelph, Ontario N1G 2W1, Canada
INTRODUCTION
Epidemiology is the study of the frequency, distribution and determinants of
health and disease in populations (Martin et al., 1987). Epidemiologists
endeavour to describe patterns of disease occurrence and, in food-producing
species, production losses in populations, and to identify factors that influence
these patterns. This chapter will focus on the use (or abuse) of epidemiological
principles to assess the contribution of infectious agents and their control on the
occurrence and severity of fish disease.
To evaluate the impact of an infectious agent on populations, researchers
and clinicians must be able to determine the proportion of animals (or groups of
animals) which are currently (prevalence) or newly (incidence) infected with the
agent. A large portion of this chapter will be devoted to the challenges of
estimating these proportions in fish populations. Relevant areas include: (i)
formulae used to describe the frequency of a disease and their interpretation; (ii)
methods for evaluating diagnostic tests and assessing the effect of diagnostic
errors; and (iii) aspects of sampling theory related to detection of disease and
estimation of disease frequency.
Various study designs used in epidemiological research will be presented. In
addition, methods for measuring associations between pathogens and disease
and causal inference will be briefly discussed.
There are few published examples where epidemiological principles have
been rigorously applied to fish populations (Klontz, 1993). Therefore, most of
the numerical examples presented in this chapter will be based on theoretical data.
MEASUREMENT OF DISEASE FREQUENCY
The occurrence of disease may be measured at different levels, including that in
an individual fish, a holding unit, a farm-site, a body of water or a watershed.
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The level to which one wishes to infer the results of a study, i.e. the unit of
concern, is generally the lowest level that is individually treated or exposed and
individually measured. The unit of concern may range from an individual to an
aggregate of individuals. For instance, if brood stock are individually injected
with erythromycin and individually tested for bacterial kidney disease (BKD),
the fish is the unit of concern. However, if a sea cage of salmon is medicated as
a group with oxytetracycline and the BKD-specific mortality rate of the cage is
measured, the cage is the unit of concern. In the current discussion, formulae are
presented at the fish level.
For epidemiological purposes, occurrences of diseases (or other related
events) in populations should be expressed as fractions of the number of units
which are biologically capable of experiencing the event of interest. The
numerator of the fraction, a, is the number of units that experience the event and
the denominator, a + b, also includes the b units, which are at risk for, but do not
experience, the event.
Incidence rates
It is important to distinguish between incidence rates and prevalence
proportions. Incidence rates describe the dynamic occurrence of new cases of
disease or infection. To calculate incidence rates, each fish must have been
measured at least twice, most commonly to observe a change in status from
negative (e.g. free of infection) to positive (e.g. infected). Two general types of
incidence rates are used in epidemiology, true rates (or incidence density rates)
and risk rates (or cumulative incidence rates). The true rate describes the speed at
which the event (e.g. infection or death) occurs per fish time at risk. It is useful
for describing extremely dynamic populations (with additions and/or
withdrawals), such as feral populations or hatchery populations of very young
fish. For an approximate true rate, one must determine the average number of
fish that are at risk (i.e. are susceptible) during the study period. This average
number at risk (NAR) is usually obtained by averaging the NAR at the beginning
and at the end of the study period. If the population is extremely dynamic, a more
accurate count is obtained by averaging the NAR over several shorter time
periods. During the study period, those fish that experience the event (e.g.
infection or death) are diagnosed and counted. The formula for calculating the
approximate true rate (Martin et al., 1987, p. 53) is:
no. of fish that experience the event
average NAR no. of time intervals

If, for instance, the true rate is to be expressed in fish-months, the no. of time
intervals in the denominator would be the number of months in the study period.
The time interval should be short enough for the fish that experience the event
(and hence are no longer at risk) not to be able to re-experience the same event
during the same interval.
Suppose a hatchery manager wishes to determine the true rate of mortality
691 Applying Epidemiology to Infectious Diseases
from bacterial gill disease (BGD) in rainbow trout, Oncorhynchus mykiss, from
swim-up to 10 cm and the study period is from September through December.
The data are as in Table 19.1. The average NAR for the study period equals
108,000 fish [(91,000 + 111,500 + 120,500 + 109,000)/4]. The true rate is then
calculated as:
(0 + 298 + 782 + 1021) fish
= 0.005 per fish-month
108,000 fish 4 months
True rates can range from zero to infinity. They are useful for describing
events in populations, but are not amenable to statistical analysis.
The risk rate provides an estimate of the probability of a fish experiencing
the event during a given interval of time. This requires that each fish at risk be
observed from its entry into the study until it experiences the event or is
otherwise withdrawn from the study, or until the study period ends. The formula
for calculating the risk rate (Martin et al., 1987, p. 51) is:
no. of fish that experience the event
initial NAR 1/2 withdrawals

where withdrawals are fish that are lost from the population during the study
period for reasons other than the event of interest.
Suppose an oral bivalent vaccine against Vibrio anguillarum is being tested.
Prior to transfer to a cage at a salt-water site, 4000 Atlantic salmon, Salmo salar,
smolt have been vaccinated. The investigator wishes to determine the risk rate
for V. anguillarum-specific mortality in these fish during the first 4 months in
salt water. During the 4 months, 520 fish die or are culled. Of these, 360 are
diagnosed with V. anguillarum, whereas the remaining 160 died from other
causes. The risk rate is:
360
= 0.092
4000 (0.5 160)
Hence, these fish had a probability of 0.092 (9.2%) of dying from vibriosis
during the first 4 months in salt water.
Risk rates, being probabilities, vary between zero and one. They may be
used to make predictions at the individual and the population level and are used
extensively in statistical analyses in epidemiology.
Table 19.1. Data for determining the true rate of mortality from BGD.
1 Sept. 1 Oct. 1 Nov. 1 Dec. 1 J an.
No. BGD deaths in
<10 cm fish during
previous month 0 298 782 1,021
No. fish <10 cm 85,000 97,000 126,000 115,000 103,000
Monthly average
NAR 91,000 111,500 120,500 109,000
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M.A. Thorburn
Prevalence rates
The prevalence proportion (also called the point prevalence rate) is a function of
existing cases of the disease. Hence, it is a static measure of disease frequency,
requiring only one measurement of each fish. The prevalence proportion, p, is
expressed (Martin et al, 1987, p. 55) as:
no. fish with disease (or infection) X at a point in time
no. fish at risk for X at that point in time

As an example, suppose that 50 salmon are randomly sampled from a cage site
and tested for antibodies to Renibacterium salmoninarum. Eighteen fish test
positive for antibodies and the remainder test negative. The prevalence
proportion of seropositive fish is:
18/50 = 0.36, or 36%
Note that the point in time in the prevalence proportion formula may
actually be a short interval of time due to the logistics involved in collecting
samples. However, prevalence data collected over an extended period of time
(such as, the BKD survey published by Sanders et al., 1992) will be misleading
if the incidence of the disease, composition of the population or sampling
procedure changes during that period.
Relationships between incidence and prevalence
The prevalence proportion is the most common approach to measuring the
frequency of fish disease in the field. The term incidence has been frequently
misused by authors referring to a prevalence type of measurement (see, for
instance, Ossiander and Wedemeyer, 1973; Worlund and Taylor, 1983; Griffiths
et al., 1991; Klesius et al., 1991). Incidence and prevalence, however, measure
quite different things. For a disease to exist (prevalence), the fish must first
develop the disease (incidence) and the disease must persist and the fish must
survive. Hence, prevalence is a function of both the incidence and the duration of
a disease. Generally, prevalence proportions will exceed risk rates for chronic
diseases, whereas the reverse is true for diseases with short duration due to
recovery or death.
There are several reasons why it is critical to distinguish between incidence
and prevalence. For one, the risk factors associated with getting a disease
(incidence) may not be the same as those associated with having the disease
(prevalence). Hence, programmes designed to prevent disease should be
evaluated specifically for their impact on incidence, whereas disease control
programmes should affect both incidence and prevalence. Another important
distinction is that prevalence data are not useful for determining causality. It is
only possible to ascertain whether a putative risk factor actually preceded
disease occurrence (and, hence, to infer causality) with incidence data.
At present, it is rarely feasible to determine morbidity (i.e. infection or
disease) incidence rates in individual fish. One of the major obstacles is that
most fish diagnostic tests require lethal sampling. Thus, it is generally not
possible to verify that fish are free of infection at the start of the study period, or
to determine when and how many fish become infected during the study period.
Calculating morbidity risk rates is further complicated by the need to identify
individual fish. Until these hindrances are overcome, the use of incidence rates
in fish will be primarily limited to mortality true rates and to mortality risk rates
in groups with no mixing.
In some situations, populations are known to be free of an infection. Fish
that have not been exposed to salt water will be free of V. anguillarum infection;
newly hatched fry will be free of infections that are not vertically transmitted. In
such populations, risk rates can be estimated by conducting repeated prevalence
surveys (Schwabe et al., 1977, p. 82; Thurmond, 1980).
Morbidity incidence rates may be readily calculated at the holding-unit or
farm level. It may, however, be difficult to make valid comparisons of incidence
rates among farms because of large differences in fish growth rates and in
purchasing and sales patterns. Thorburn (1991) has proposed a methodology for
standardizing incidence rates for drug usage at the farm level.
Specific rates
Mortality rates may be crude or cause-specific. Crude mortality rates include all
dead fish in the numerator, regardless of the cause of death. Cause-specific
mortality rates usually specify aetiological agents. For instance, an Aeromonas
salmonicida-specific mortality rate would include in its numerator only those
fish that died and were diagnosed with A. salmonicida.
Host characteristics are usually important determinants of disease.
Therefore, consideration should be given to restricting incidence rates and
prevalence proportions to specific ages (or sizes), smolt year-classes, strains and
sexes of the species in question. Note that the restrictions must apply to both the
numerator and the denominator of the rate.
Proportional rates and proportions
Proportional rates (and proportions) are frequently presented in fish disease
studies. One common type of proportional proportion divides the number of fish
diagnosed with (or dying from) a given disease by the total number of diseased
(or dead) fish. Another type divides the number of cases of a specific disease in
a specific species, or in a specific time period, by all cases of that disease (in all
species, or over a longer time period, respectively). Proportional morbidity
(mortality) proportions are often calculated from fish-disease diagnostic
laboratory data (Speare and Ferguson, 1989; Freund et al., 1990b). Another
example of proportional proportions was presented by Meyers et al. (1990), who
divided the number of fish with high infectious haemopoietic necrosis virus
(IHNV) antigen titres by the total number of IHNV-positive fish.
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M.A. Thorburn
Proportional rates are potentially misleading. Since the denominator of
proportional rates does not include the entire population at risk, it is
inappropriate to interpret them as incidence or prevalence rates. Different factors
may independently affect the numerator and the denominator. Consider the
following example. A diagnostic laboratory had 180 cases submitted in 1996, of
which 40 were diagnosed with furunculosis. Twenty of the furunculosis cases
were from Atlantic salmon, ten from rainbow trout and the remaining ten from a
variety of species. Of 200 submissions in 1995, 35 were diagnosed with
furunculosis; of 160 submissions in 1994, 25 had furunculosis. The
furunculosis-specific proportional morbidity percentages by species in 1996
were 50% (20/40 100) for Atlantic salmon, 25% (10/40 100) for rainbow
trout and 25% for all other species. Unless an investigator can also show, for
instance, that the distribution of Atlantic salmon in the source population is
significantly less than 50%, whereas the distribution of rainbow trout is close to
25% or more, the investigator cannot conclude that Atlantic salmon are more
susceptible to furunculosis than are rainbow trout. The proportional morbidity
percentages by year for furunculosis were 22.2% (40/180 100) in 1996, 17.5%
(35/200 100) in 1995 and 15.6% (25/160 100) in 1994. Again, it would be
wrong to conclude from these data that the prevalence of furunculosis has been
increasing. Unless the investigator can demonstrate that the pattern of submitted
cases has been representative of the distribution of disease prevalence in the
source population during the 3 years, it can only be concluded that the proportion
of diagnoses for furunculosis has been increasing. Thorburn (1993) has shown
that farmers who voluntarily use diagnostic laboratory services are not
necessarily representative of all farmers in the target population.
Comparing rates
Fish-disease specialists frequently emphasize the interaction among host, agent
and environment in disease promotion (Snieszko, 1974). Most research on
infectious diseases of fish has, however, only emphasized the causal component
of the pathogenic agent. Some research has also been on the effects of host
factors, such as genetic strain, species and age, on the occurrence of diseases
(see, for instance, Gjedrem et al., 1991; Beacham and Evelyn, 1992; Fevolden
et al., 1992). Until recently, little work had been conducted to quantify the effect
of environmental or husbandry factors on the occurrence of infectious disease in
fish. These few studies had been restricted to controlled experimental situations,
which are not readily extrapolated to natural settings and which do not allow
exploration of potential interactions among several risk factors (see, for
instance, Sanders et al., 1978; Lall et al., 1985).
One of the major purposes of veterinary epidemiology is to use field
observations to identify environmental and management factors that have
possible causal relationships with specific diseases. By quantifying the impact of
such factors, it becomes possible to determine whether economically efficient
management changes can be implemented to reduce the rate of disease in a
population. The specific goal of many epidemiological studies is to determine
whether there is an association between specific risk factors and disease
occurrence. Often, this is accomplished by comparing animals or farms/
hatcheries that have been exposed to different levels of risk factors (cohort
studies) or that have been randomly allocated to two or more treatment groups
(field trials) or by comparing diseased and non-diseased units (casecontrol
studies). In a simple comparison with one dichotomous risk (or treatment) factor
and one disease, data can be arranged in a two-by-two table, as shown in Table
19.2. In this, a sample of n units from a population are cross-classified by disease
status (present/absent) and whether they have been exposed to the risk factor or
not.
An example of a two-by-two table and of some of the epidemiological
measures of association that may be derived from it is given in Table 19.3.
Strength of association
The ratio between the rate of disease in the exposed (factor-positive) sample and
the rate of disease in the non-exposed (factor-negative) sample reveals how
many more times disease occurred in the exposed units than in the non-exposed
units. This provides an indication of the strength of the association between the
factor and disease. This ratio, known as the relative risk (RR), is calculated
(Martin et al., 1987, p. 130) as:
RR = [a/(a + b)]/[c/(c + d)]
Theoretically, the relative risk ranges between zero and infinity. If there is
no association between the factor and the disease, the relative risk equals one
(excluding sampling variability). The more the relative risk differs from one, the
stronger the association between the factor and the disease. The statistical
significance of the relative risk should be tested; appropriate statistical methods
are described in several textbooks (Fleiss, 1981; Kleinbaum et al., 1982). A
relative risk greater than one suggests that the factor may be causal, while a
relative risk less than one suggests that the factor may be sparing.
The odds ratio (OR) is calculated as the ratio: (i) between the odds of disease
in the exposed sample and the odds of disease in the non-exposed sample; or (ii)
between the odds of exposure in the diseased sample and the odds of exposure in
the non-diseased sample (Greenberg et al., 1996, pp. 126127).
(i) OR = (a/b)/(c/d) = ad/bc
(ii) OR = (a/c)/(b/d) = ad/bc
The odds ratio is interpreted in the same way as the relative risk. It may be used
to measure the strength of association in any study design. It is particularly
useful in casecontrol studies, in which the relative risk cannot be calculated.
Table 19.2. The layout of a two-by-two table.
Diseased (D+) Not diseased (D)
Exposed (F+) a b a +b
Not exposed (F) c d c +d
a +c b +d n =a +b +c +d
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M.A. Thorburn
The odds ratio is the basic statistic obtained using powerful statistical methods
such as log-linear and logistic modelling (Hosmer and Lemeshow, 1989). If the
rate of disease in the population is low (< 5%) the odds ratio and the relative risk
are approximately equal.
Effect of the risk factor
To evaluate the medical and economic importance of a risk factor, one first
determines how much of the disease rate in the exposed group can be attributed
to that factor. Some disease will almost always occur in individuals belonging to
the factor-negative group. It is assumed that the determinants which lead to
disease in the factor-negative group operate with the same frequency and
intensity in the factor-positive group. The absolute difference between the rate of
disease in the exposed group and that in the non-exposed group thus provides an
estimate of the rate of disease in the exposed group that may be explained by, or
attributed to, the factor of interest. This difference is called the attributable rate
(AR) and is calculated (Martin et al., 1987, p. 130) as:
AR = [a/(a + b)] [c/(c + d)]
Table 19.3. Example of epidemiological association statistics. Data on the
relationship between the number of hatcheries from which smolt were
obtained (F) and occurrence of clinical IPN (D) are adapted from a cohort study
of Atlantic salmon net-pen farms in Norway (J arp et al., 1994).
IPN+ IPN
>2 hatcheries 16 13
<2 hatcheries 31 61
Epidemiological measures of association
(16/29) (16)(61)
RR = =1.64 OR = =2.42
(31/92) (13)(31)
AR =(16/29) (31/92) =0.21 PAR =(29/121)(0.21) =0.05
Interpretation
The RR and OR indicate that the farm-level rate of clinical IPN is 1.64 and 2.42
times higher, respectively, among farms obtaining smolt from more than two
hatcheries than among those using one or two hatcheries. The OR estimate
differs substantially from the more valid RR estimate because the disease,
IPN, is quite common.
According to the AR, the farm-level rate of IPN that is due to having used
more than two hatcheries, in those farms which obtained smolt from more
than two hatcheries, is 21%. If the study hatcheries are representative of the
entire population of hatcheries, the PAR suggests that the farm-level rate of
IPN in the population that may be attributed to farms having obtained smolt
from more than two hatcheries is 5%. In other words, if all farms obtained
smolt from only one or two hatcheries, the rate of IPN should decrease by 5%
(from 39% to 34%).
IPN, infectious pancreatic necrosis; RR, relative risk; OR, odds ratio; AR,
attributable rate; PAR, population attributable rate.
The importance of a causal factor in the population of interest is determined
by multiplying its effect, the attributable rate, by the prevalence of the factor in
that population. This is called the population attributable rate (PAR) (Martin
et al., 1987, p. 130).
PAR = [(a + b)/n] AR
Hence, if the factor were completely removed from the studied population, the
rate of the disease of interest should decrease by an amount equal to the
population attributable rate (excluding sampling variability). Note that a valid
estimate of the prevalence of the factor must be available in order to calculate the
PAR.
EVALUATING THE USEFULNESS OF DIAGNOSTIC
TESTS IN SCREENING
Diagnostic tests may be used to screen apparently healthy populations of fish for
the presence of subclinically infected individuals. Many of the tests used to
screen fish populations often yield incorrect results. Tests based on culture of the
pathogen often produce false-negative results, whereas serological tests may
produce both false-positive and false-negative results.
Screening is used extensively in disease control and eradication
programmes. It is also used in epidemiological studies to investigate the role of
putative disease risk factors. Hence, it is critical to understand the impact that
diagnostic errors have on conclusions about the occurrence of pathogens in
screened populations.
Sensitivity and specificity
Suppose fish are correctly classified, because of their history or by the use of a
highly accurate diagnostic test (a gold standard), as either infected or not
infected with a specific pathogen. The sensitivity and specificity of a new
screening test could be determined by randomly selecting fish from each of the
two groups and testing and classifying them with the new test. The tested fish
could then be cross-classified by true infection status and screening test result in
the two-by-two table given in Table 19.4.
Table 19.4. Cross-classification by true infection
status and screening test result.
True infection status
Test result Infected Non-infected
Positive a b
Negative c d
a +c b +d
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M.A. Thorburn
The epidemiological sensitivity of the new test is defined as the proportion
of the truly infected fish that yield positive test results, i.e. a/(a + c). In
probability terms, the sensitivity may be expressed as the probability of a true-
positive test result, or one minus the probability of a false-negative test result.
The epidemiological specificity of the test is defined as the proportion of truly
non-infected fish that test negative, i.e. d/(b + d). Thus, the specificity is the
probability of a true-negative test result, or one minus the probability of a false-
positive test result.
The terms sensitivity and specificity in the fish-disease literature are
somewhat confusing, as immunologists and microbiologists often use the terms
differently from epidemiologists. Epidemiological sensitivity and specificity are
probability values, which vary between zero and one. These quantitative
estimates are useful for determining what a test result probably means in terms
of true infection status. Microbiologists and immunologists generally use
sensitivity to define detection levels (e.g. the minimum number of cells per unit
of tissue that can be detected using a test) and specificity to define the amount of
cross-reactivity with other organisms. Such information, which is critical for
identifying and improving a diagnostic test, is frequently reported in the fish
disease literature (e.g. Sakai et al., 1989; Dominguez et al., 1990; Mourton et al.,
1990). However, information on detection levels and cross-reactivity provides
little assistance in epidemiological research, nor is it readily translated into
epidemiological terms. Low detection levels (high microbiological
sensitivity) may not be required to achieve high epidemiological sensitivity
(Dixon, 1987), nor do high levels of cross-reactivity necessarily lead to low
epidemiological specificity. Information on the sensitivity and specificity of
a test, as defined by epidemiologists (and as used in this chapter, unless
indicated otherwise), is critical to the design and interpretation of field
studies investigating the frequency, impact or determinants of infection and
disease.
Antigen-binding assays provide continuous values, such as optical density,
agglutination titre or number of fluorescing cells, which must be converted to a
dichotomous outcome. This is achieved by setting a negativepositive threshold.
All values above the threshold are considered positive (infected) and all values
below the threshold are considered negative (non-infected). In such cases, there
is an inverse relationship between sensitivity and specificity. If the threshold is
increased, the specificity of the test will increase and the sensitivity will
decrease. Establishing negativepositive thresholds can be facilitated by
determining the distribution of the continuous outcome in known populations of
non-infected and infected or exposed populations (Thorburn and Jansson, 1988).
Unfortunately, for many endemic pathogens, it is difficult to confidently identify
the infection status of fish populations and it may be misleading to use
distributions to set thresholds (e.g. Meyers et al., 1993b). If the true infection
status is known, receiveroperator characteristic curves (Sackett, et al. 1991, pp.
117119) can be used to determine negativepositive thresholds that optimize
test sensitivity and specificity, as done by Newbound et al. (1995), using an
indirect fluorescent antibody test (IFAT) for plasmacytoid leukaemia.
Techniques to establish several thresholds for diagnostic tests with continuous
outputs, in order to classify the level of infection in individual fish (Elliot et al.,
1995), have not been described.
Accurate estimation of sensitivity and specificity is generally quite difficult
and several methodological approaches have been presented (Martin, 1977; Hui
and Walter, 1980; Sackett et al., 1991). Sensitivity and specificity should not be
regarded as absolute values, since the performance of fish diagnostic tests can
depend on factors such as when samples are taken (Mulcahey and Pascho, 1986)
and fish species tested (Bruno, 1987). The stage of infection can also markedly
influence sensitivity (Olesen et al., 1991; Olivier et al., 1992).
Predictive values
The predictive value of a positive test, PV+, is the proportion of infected fish
among those that test positive and is calculated as a/(a + b). It is the probability
that a test-positive fish is truly infected. It should not be confused with the
sensitivity of a test, which is the probability that an infected fish will give a
positive test result. The predictive value of a negative test, PV, equals the
proportion of non-infected fish among those that test negative, i.e. d/(c + d). It is
the probability that a fish that tests negative is truly non-infected.
Predictive values are related to the sensitivity and specificity of the
diagnostic test and to the prevalence proportion (P) of the infection. The PV+
will be low for tests with low specificity, particularly if used in populations
where the infection is rare; the PV is particularly low when tests with low
sensitivity are used in populations with a high prevalence of the infection.
Predictive values are primarily useful for understanding how tests with different
sensitivity and specificity perform in populations with specified prevalence
proportions (Martin et al., 1992).
P sensitivity
PV+ =
(P sensitivity) + [(1 P) (1 specificity)]
(1 P) specificity
PV =
[(1 P) specificity] + [P (1 sensitivity)]
As an example, suppose a Gram stain is used to screen spawning female
brood-stock for R. salmoninarum, in order to cull eggs from positive fish. If the
sensitivity is 0.75 (25% of all R. salmoninarum-infected females test negative)
and the specificity is 0.70 (30% of all R. salmoninarum-free females test
positive), the predictive values in a population with a 45% prevalence would be:
0.45 0.75
PV+ = = 0.67
(0.45 0.75) + [(1 0.45) (1 0.7)]
(1 0.45) 0.7
PV = = 0.77
[(1 0.45) 0.7] + [0.45 (1 0.75)]
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M.A. Thorburn
Hence, there is a 33% chance that a test-positive fish is actually free of infection
and a 23% chance that a test-negative fish is actually infected. If the prevalence
in the screened population was only 2%, the corresponding error rates would be
95% and 1%, respectively.
When investigators apply a diagnostic test to each fish in a sample that has
been randomly collected from a population, they obtain an estimate of the
apparent prevalence proportion (AP) of infection, which is the proportion of test-
positive fish in a population. The AP is a function of the truly infected fish that
correctly test positive and of the non-infected fish that falsely test positive
(Martin et al., 1992).
AP = (P sensitivity) + [(1 P) (1 specificity)]
If estimates for the sensitivity and the specificity are available, the true
prevalence proportion in the sample may be estimated from the observed
apparent prevalence, using the equation (Schwabe et al., 1977, p. 78):
ap (1 specificity)
p =
1 [(1 specificity) + (1 sensitivity)]
(Small letters are used to designate prevalence, or other, values estimated from
random samples, whereas capital letters are used to designate population
parameters.)
As an example, suppose that chinook salmon, Oncorhynchus tshawytscha,
brood stock are being screened for IHNV and that together the cell culture and
virus identification technique have a sensitivity of 80% and a specificity of
99.9%. The observed prevalence percentage in the sample is 30%. The estimated
true prevalence proportion (p) is:
0.30 (1 0.999)
p = = 0.374
1 [(1 0.999) + (1 0.8)]
Hence, approximately 37% off the sampled brood stock are infected with IHNV.
In general, surveys conducted using diagnostic tests with low sensitivity
will tend to underestimate the prevalence proportion, whereas those with low
specificity will tend to overestimate P, particularly if P is quite small.
Measuring agreement
Most current fish diagnostic tests cannot be considered gold standards;
consequently, it is often difficult to establish the true state of infection.
Hence, quantitative estimates of sensitivity and specificity are not
available for most fish diagnostic tests. As an alternative, new tests are
frequently compared with current, standard, but imperfect, test. The results
of a comparison may be presented in the two-by-two table given in
Table 19.5.
The numbers represented by r and u are of fish which gave the same results
for the two tests (positive and negative, respectively), whereas s and t indicate
the number of fish which gave conflicting test results. If the two tests show high
agreement, they are probably measuring what they purport to measure. If they
disagree, one or the other, or both, may be fairly useless.
To evaluate agreement, it is important to note that a certain number of test
results will agree by chance alone. Consider an example in which r = 80, s = 110,
t = 90 and u = 720. The apparent prevalence in this sample of 1000 fish as
estimated by the standard test equals 0.17 (80+90)/1000 and that for the new
test equals 0.19 (80 + 110)/1000. At this level, the tests appear to agree quite
well. Provided that the two tests are independent of one another, the probability
of both tests being positive, by chance alone, is given by the product of the two
apparent prevalences: 0.17 0.19 = 0.032. The probability of both tests being
negative equals the product of one minus the apparent prevalence yielded by
each test: (1 0.17) (1 0.19) = 0.672. The proportion of total agreement
expected by chance is thus 0.704 (0.032 + 0.672), while the observed proportion
of agreement is: (80 + 720)/1000 = 0.80. The observed proportion of agreement
is 0.096 higher than that expected by chance alone. The maximum possible
agreement beyond chance is given by one minus the proportion of total
agreement expected by chance; in this example, it is 0.296 (1 0.704). The
observed agreement beyond chance relative to the maximum possible agreement
beyond chance can be calculated by dividing 0.096 by 0.296. This quotient,
called kappa, equals 0.32. No agreement beyond chance gives a kappa of zero
and perfect agreement gives a kappa of one. Thus, in the present example, it
appears that the agreement between tests in not as good as indicated by the
simple comparisons of apparent prevalences.
There are very few published examples of kappa being used to compare two
fish diagnostic tests. Saksida (1995) estimated very low kappas between
histology and IFAT for diagnosing plasmacytoid leukaemia. Kappa has also been
used to compare fish diagnosticians using the same tests. Armstrong et al. (1989)
calculated kappas to compare the agreement between the results of IFAT tests for
R. salmoninarum conducted by two different laboratories. As in the example
presented above, the two apparent prevalences obtained were very similar, and
yet the kappa statistics obtained indicated that agreement between the two
laboratories was very bad. Kappa values ranged from 0.03 to 0.23, depending on
the number of cells used for the negativepositive threshold. Stephen et al.
(1995) used kappa to show poor agreement among histopathologists diagnosing
plasmacytoid leukaemia, although Saksida (1995) later suggested that this result
was due to the use of vague histological criteria. Stephen and Ribble (1996)
Table 19.5. Comparison between a new test and
a standard test.
Standard test
New test +
+ r s
t u
702
M.A. Thorburn
developed a different set of histological criteria and found good intra-observer
agreement (kappa = 0.84), leading them to suggest that the new criteria were
valid and repeatable.
Unfortunately, most published comparisons of diagnostic tests applied to
fish only present the apparent prevalences obtained with each method. While
this approach allows some discussion of relative sensitivities (e.g. Cipriano
et al., 1985; Pascho et al., 1987; Hsu et al., 1991; Sakai et al., 1991; Meyers et
al., 1993a), it does not, as indicated, provide a statistically valid estimate of
agreement. This approach ignores the problem of false-positive test results,
which serves to make a test with imperfect specificity appear more sensitive than
it actually is.
See Fleiss (1981) and Kraemer (1992) for further details on kappa and other
measures of agreement and disagreement.
Test interpretation at the group level
In aquaculture, diagnostic tests are most frequently used on individual fish (or
pooled individuals) to reach conclusions about populations of fish (Thorburn,
1996). Martin et al. (1992) expanded the principles of sensitivity, specificity and
predictive values to encompass group- (usually farm-) level testing. Hence, it is
possible to assess the impact that diagnostic test errors have on decisions made
about groups of fish. Farm-level sensitivity (specificity) is defined as the
probability that an infected (non-infected) farm is declared positive (negative),
given a specific sampling and diagnostic protocol. Farm-level positive
(negative) predictive value is the probability that a farm that is declared positive
(negative) is infected (free of infection). Estimates of farm-level sensitivity,
specificity and predictive values are critical to the design and interpretation of
results from regional disease control and eradication programmes.
The farm-level sensitivity (FSENS), i.e. the probability of observing at least
one or more reactors (test-positive fish) in a randomly selected sample of n fish
from an infected farm, is calculated as follows:
FSENS = 1 (1 ap)
n
.
For instance, if a diagnostic test with 80% sensitivity and 99.9% specificity is
used to test 60 fish randomly selected from a farm where the prevalence of
infection is 2%:
ap = (0.02 0.8) + [(1 0.02) (1 0.999)] = 0.016 + 0.001 = 0.017
FSENS = 1 (1 0.017)
60
= 1 0.357 = 0.643
Hence, there is a 36% chance that an infected farm will not, under these
conditions, be declared positive (i.e. that no test-positive fish will be included in
the sample).
Farm-level sensitivity increases with increasing diagnostic-test sensitivity,
prevalence and numbers of fish sampled. It decreases, however, with increasing
diagnostic-test specificity, since there will be a lower probability of non-infected
fish falsely testing positive. On an infected farm, a false-positive test result will
result in that farm being correctly classified as infected; note, however, that
lower test specificity will result in lower farm-level positive predictive value.
The farm-level specificity (FSPEC), i.e. the probability of detecting no
reactors in n fish sampled from a non-infected farm, is calculated as follows:
FSPEC = specificity
n
For example, if the above diagnostic test with a specificity of 99.9% was used on
60 fish randomly selected from a non-infected farm:
FSPEC = 0.999
60
= 0.942
Hence, there is a 6% chance that a non-infected farm will, under these
conditions, be falsely declared infected. Farm-level specificity increases with
increasing test specificity and decreasing sample size.
If the diagnostic test is less than 100% specific, the farm-level positive
predictive value decreases and the farm-level negative predictive value
increases, with increasing n and decreasing farm-level prevalence (i.e.
proportion of farms that are infected). Refer to Martin et al. (1992) for formulae
to calculate farm-level predictive values. Other methodological issues
surrounding group testing have been elaborated by Donald et al. (1994) and
Carpenter and Gardner (1996). Thorburn (1996) investigated the impact of
apparent prevalence assumptions on FSENS and FSPEC in routine surveillance
of asymptomatic salmonid populations.
SAMPLING
To study the epidemiology of infectious fish diseases, investigators require
knowledge about certain population parameters, such as the prevalence
proportion of infected fish. Generally, data are collected from a subset (sample)
of a population, in order to make inferences about the entire population.
Prior to determining a sampling strategy, investigators should clearly define
the target population. The target population is the aggregate of units (fish,
tanks, farms, etc.) to which the investigators wish to infer results. It is often
restricted to specific species, life stages, life histories and geographical
locations.
Survey sampling
This section will focus on a few sampling strategies that may be used in surveys.
Non-probability sampling
Samples collected by methods that do not rely on formal random techniques are
termed non-probability samples. Such samples usually produce biased results.
Furthermore, it is not possible to estimate the precision (as measured by the
standard error) of the estimates obtained. Unfortunately, when fish are the
sampling unit, it is practically very difficult to conduct random sampling.
704
M.A. Thorburn
Crowding fish, or attracting fish with food and catching individuals by dip-
net are the most frequently used non-probability method of sampling fish. The
bias observed with dip-net sampling done to estimate average weight or length,
although significant, may be acceptably small if the population being
sampled is somewhat crowded (Hammell, 1992). The bias associated with
weight estimates may occasionally be further reduced by weighting
observations by the lengths of the sampled fish (Seeger et al., 1977). The
bias associated with disease prevalence estimates obtained from dip-net
samples may, however, be unacceptably large (Hammell, 1992; Stephen and
Ribble, 1995).
Probability sampling
Three methods for probability sampling will be presented. In the first two,
simple random sampling and systematic sampling, the sampling units and the
units of concern are the same. In the third, multistage sampling, at least two
levels of units are sampled; the lowest level is the unit of concern. Other
common methods of probability sampling, including stratified sampling and
cluster sampling, will not be covered here. Refer to Cochran (1977) and Levy
and Lemeshow (1980) for more detailed coverage.
SIMPLE RANDOM SAMPLING
When simple random sampling is used, every sampling unit has the same
probability of being selected. Also, every sample of size n has an equal
probability of selection. In practice, simple random sampling requires that every
sampling unit in the population be listed and that a fixed percentage of the listed
units be selected by a random process. It is feasible to use simple random
sampling if farms or holding units (troughs, tanks, raceways, cages, etc.), but not
if live fish, are the sampling units. Thorburn (1992), however, demonstrated that,
if all fish in a tank are sufficiently crowded, dip-net samples will provide
reasonable approximations of simple random samples. Simple random samples
provide unbiased estimates of all parameters and standard errors can be
estimated using the formula: se = (s
2
/n)
1/2
(Snedecor and Cochran, 1989, p. 436).
SYSTEMATIC SAMPLING
Systematic sampling is useful when the sampling units are randomly arranged
in a physical line-up, such as when fish are being transferred through a chute or
processed in an abattoir line (Hammell, 1992), or when the identities of the
population are available on a randomly ordered list.
The n sampling units are selected from the population at regular intervals of
size k. The starting point of the first unit to be sampled must be k and is
determined by a random process. Systematic sampling should only be used if
investigators are confident that there is no particular ordering or periodicity in
the line-up or list. If the characteristic of interest is either related to the interval k
or to the order with which sampling units enter the sampling process, the
estimates obtained from the sample will not represent the target population.
If the size of the target population, N, divided by the sampling interval, k, is
not an integer, some bias will result in the sample estimate. The bias is minimal,
however, if N is large and k is small relative to N. One may then use the formula
for simple random sampling to estimate the standard error.
MULTISTAGE SAMPLING
In multistage sampling, random samples are taken at several levels. For instance,
first-stage sampling units may be farms, second-stage sampling units may be
holding units within the selected farms and third-stage units may be fish within
the selected holding units. The final stage is the unit of concern, that is, the unit
which will comprise a single observation in the descriptive summary or
statistical analysis. For example, a two-stage sample might be taken from a
target population of commercially farmed female rainbow trout brood stock in a
certain region to estimate the prevalence of fish with IHNV in their ovarian
fluids. A random sample of m farms would be selected and a fixed percentage of
the female brood stock on each of the selected farms would be randomly (e.g.
systematically) sampled during the spawning season. If the number of brood
stock on each farm is known, a better procedure is to sample farms such that any
given farms probability of selection is proportional to its number of female
brood stock and then to select a fixed number of fish from each selected farm
(for a more detailed discussion, see Martin et al., 1987).
Practically, multistage sampling is the only feasible approach to sampling
populations of live production fish when the target population comprises several
holding units or several farms. Snedecor and Cochran (1989) provide formulae
for estimating the standard error of estimates derived from two-stage samples.
Sample size considerations
The following formulae should provide suitable sample size estimates for simple
and systematic random samples (Snedecor and Cochran, 1989, p. 439).
DICHOTOMOUS OUTCOMES
The true prevalence proportion of infected fish (or farms) or seroreactors in a
population is denoted as P. In order to determine an appropriate sample size to
estimate P, an investigator must provide an educated guess of the probable
prevalence level, p, as well as stating the desired level of confidence, (1 ) and
the allowable error, L. The appropriate sample size formula is:
n = Z
1 /2
2
pq/L
2
where q = (1 p)
Suppose, for instance, the manager of a government hatchery wishes to
estimate the prevalence of R. salmoninarum in returning chinook salmon. A
sample taken the previous year indicated that 25% of the brood stock were IFAT-
positive. The manager wishes the survey estimate to be within 5 percentage
points (L) of the true prevalence 95% (1 - ) 100 of the time. The required
sample size is:
n = 1.96
2
0.25 0.75/0.05
2
= 288 brood stock
If the estimated sample size n is greater than 10% of the population size N,
the required number to sample may be modified by the formula (1/n + 1/N)
1
(Cannon and Roe, 1982). For instance, if only 400 brood stock are expected to
706
M.A. Thorburn
return to the hatchery, the new estimate for n would be (1/288 + 1/400)
1
= 168
brood stock.
CONTINUOUS OUTCOMES
The sample size required to estimate the mean of a continuous variable is related
to the variance of that variable in the target population, S
2
. Suppose a farmer has
been feeding a cage of fish a specially supplemented diet to reduce the impact of
BKD and now wishes to estimate the mean weight of the fish in the cage with a
90% certainty of being within 50 g of the true mean weight. From previous
experience, the farmer estimates the standard deviation, s, of the weight to be
200 g. The required sample size is:
n = Z
1 /2
2
s
2
/L
2
= 1.65
2
200
2
/50
2
= 44 fish
TWO-STAGE SAMPLING
To obtain the same precision in a two-stage sample as in a simple or systematic
random sample, the sample size is increased. The increase in sample size is
dependent on how much the disease clusters within the first-stage sampling
units (e.g. farm sites). If there is large variation among farms in the prevalence of
an infectious disease, but the prevalences within farms tend to be either quite
high or nearly zero, the sample size should be increased five to seven times
(Leech and Sellers, 1979). This situation typically occurs with highly contagious
diseases, particularly if some farms are able to avoid infection. Less contagious
diseases may only require sample size increases of two to three times.
Snedecor and Cochran (1989) provide formulae for estimating the
appropriate numbers of first-stage and second-stage sampling units. For
instance, when tanks are the first-stage sampling units and fish the second-stage
sampling units, the optimal numbers will depend on the relative costs of
sampling each stage and on the among-tank and the within-tank variances.
Generally, the among-tank variance, termed the pond-effect by Michel et al.
(1984), of infectious fish diseases will be large enough for the most precise
sampling scheme to necessitate sampling fish from most, or all, of the holding
units on a given farm.
GROUP TESTING
Pools of fish, as opposed to individuals, are frequently tested, particularly when
populations of fry and small fingerlings are sampled. The sample sizes required
to estimate prevalence will depend on the sizes of the groups, j, as well as on the
anticipated prevalence and desired confidence level and precision. According to
Worlund and Taylor (1983), the number of groups required may be derived by:
g = {Z
1 /2
[(1 p)/(L j)]}
2
[(1 p)
j
1]
For instance, if fish are to be tested in pools of five, the anticipated prevalence is
8% and the investigator wishes to be within 2 percentage points of the true
prevalence 95% of the time, the required number of groups is:
g = {1.96 [(1 0.08)/(0.02 5)]}
2
[(1 0.08)
5
1]
= 18.03
2
0.52 = 168
Hence, (168 5) = 840 fish should be randomly collected and tested in pools of
five fish each.
Sampling to detect disease
An integral part of most fish disease control or eradication programmes is to test
farms to ascertain the presence or absence of specific pathogens. A random
sample of n fish taken from a farm will give a certain confidence of yielding at
least one test-positive fish if the apparent prevalence of the pathogen on infected
farms is at (or above) a specified level. Ossiander and Wedemeyer (1973) and
Simon and Schill (1984) have published tables of the sample sizes needed in
populations of different sizes and different assumed minimal prevalences on
infected farms. These tables, however, assume that the diagnostic test used has
100% sensitivity and specificity. Formulae are available which incorporate
diagnostic error, by using the apparent prevalence (Martin et al., 1992).
In small populations, the appropriate sample size formula is:
n = [1 (
1/(ap N)
] {N [(ap N) 1]/2}
where ap is the lowest apparent prevalence proportion anticipated on infected
farms, N is the size of the population being sampled, is the probability of
detecting no test-positive fish in the sample when at least (ap 100)% of the fish
in the target population are test-positive.
In large populations an approximate sample size formula may be used:
n = ln ()/ln (1 ap)
As an example, suppose egg exporters from a certain region must
demonstrate absence of IHNV in their brood stock. The participating diagnostic
laboratory estimates that cell culture of reproductive fluids followed by serum
neutralization has a sensitivity of 60% (40% of randomly selected IHNV-
infected brood fish will falsely test negative) and a specificity of 100% (no false
positives). It is assumed that, if the brood stock population is infected, at least
10% of the fish will be infected. Hence the minimum apparent prevalence is:
ap = (0.10 0.60) + [(1 0.10) (1 1.0)] = 0.06
For a 99% probability ( = 0.01) of having at least one test-positive fish in a
sample taken from a population of 500 fish, the number of fish that would need
to be tested is:
n = [1 (0.01)
1/(0.06 500)
] {500 [(0.06 500) 1]/2} = 69 fish
The number of fish that would need to be sampled from a large population is:
n = ln (0.01)/ln (1 0.06) = 75 fish
Unfortunately, few appropriate data are available from which to estimate
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M.A. Thorburn
expected ap values. Thorburn (1996) analysed data from several Canadian fish
diagnostic laboratories, which showed that the ap values of some pathogens in
asymptomatic salmonid populations vary widely, depending on factors such as
species and life stage.
ANALYTICAL OBSERVATIONAL STUDIES
Epidemiologists most frequently develop or test hypotheses by conducting
observational studies in natural environments. Very few analytical observational
studies of fish diseases, using valid epidemiological methodology, have been
published. Aquaculture researchers have thus far relied heavily on
questionnaires, which have inherent problems, such as bias due to poor recall
and non-response, to collect data used in epidemiological studies (Jarp et al.,
1993). However, several regional and national systems are currently being
developed to routinely collect disease and production data from aquaculture
sites. Data generated through these systems may prove useful for analytical
observational studies. Most epidemiological studies of farmed fish disease have
been conducted at the site level (i.e. the site has been the unit of concern).
Three types of epidemiological studies cross-sectional, cohort and case
control will be discussed. For ease of presentation, it is initially assumed that
only one exposure (risk) variable and one disease variable are being investigated
and that exposure and disease status are classified as dichotomous variables. An
exposure or risk variable may be dichotomous in that it is either present or
absent, or present at one of only two possible levels. The general methodology
presented, however, is easily transposed to studies that have continuous
outcome(s) or independent variable(s). For instance, the presence or absence of a
specific pathogen is a dichotomous (independent) variable, which may be
hypothesized to influence growth rate, a continuous variable. Conversely,
stocking density, a continuous (independent) variable, may be hypothesized to
contribute to the occurrence of clinical infectious pancreatic necrosis (IPN). At
the level of a holding unit, clinical IPN could either be handled as a dichotomous
(yes/no) or as a continuous (rate or proportion) outcome.
Cross-sectional studies
In a cross-sectional study, a sample is collected from the target population,
preferably using a probability-survey sampling method. Each sampled unit is
then classified according to its exposure status and disease status. The main
objective of cross-sectional studies is to investigate the association between
putative risk factors and disease. This is achieved by calculating appropriate test
statistics and estimating epidemiological measures of association, including the
relative risk, the odds ratio, the attributable rate and the population attributable
rate. The prevalences of disease and of exposure in the target population can also
be estimated from cross-sectional data.
Cross-sectional studies are not appropriate for rare diseases, since very large
samples are required to obtain an adequate number of cases. A disadvantage of
cross-sectional studies is that they provide prevalence rather than incidence data.
Since it is rarely possible to know if the exposure factor actually preceded the
disease, it is generally not appropriate to infer causality from cross-sectional
studies. Hence, cross-sectional studies are best used to investigate the effects of
permanent or constant exposure factors.
Cross-sectional studies may be used to simultaneously study associations
among several risk factors and several disease or production outcomes.
Thorburn (1995) conducted a cross-sectional study of Ontario, Canada, trout
farmers, which revealed associations between several farm-level variables,
including farmer experience, number of employees, type of operation, water
flow and the use of prophylactic treatments and drug treatment probability and
frequency. Bebak et al. (1997) conducted a cross-sectional study to investigate
risk factors for BGD in North American rainbow trout hatcheries. They found
that commercial (as opposed to government) hatcheries, the presence of fish in
the hatch-house water-supply and higher production were associated with an
increased probability of BGD. Researchers of diseases in wild fish populations
have used repeated cross-sectional studies to identify factors associated with
disease prevalence. Margenau et al. (1995) found that location, sex, age and
length were associated with the prevalence of blue spot disease in northern pike,
Esox lucius, in Wisconsin. Mellergaard and Nielsen (1996) identified spatial and
temporal variations in the prevalence of X-cell gill disease in common dab,
Limanda limanda, in Denmark.
Cohort studies
In cohort (longitudinal) studies, a sample is randomly selected from a defined
group (the cohort). Generally, only units that are free of the disease of interest are
entered into the study. Exposure data are collected on these units at the start of
the study period and the occurrence of disease is ascertained throughout the
study period. Prior exposure information is related to subsequent disease
experience, which (as opposed to cross-sectional and casecontrol studies) can
provide evidence that a putative risk factor has a causal role. With a single
dichotomous exposure factor, the incidence rates of disease in the exposed and
non-exposed groups are compared. If the disease and the factor are not
associated, these two rates should be equal. If they are associated, the relative
risk and the attributable rate should differ significantly from one and zero,
respectively.
It is important to follow up all units throughout the study period with equal
rigour, in order to identify units that are lost from the study group, observe new
cases as they occur and note changes in exposure status. Cohort studies are not
recommended for rare diseases. If appropriate historical records are available, it
is possible to conduct retrospective cohort studies. Refer to Breslow and Day
(1987) for details on the design and analysis of cohort studies.
As with cross-sectional studies, cohort studies can investigate associations
among several risk factors and several outcomes. A retrospective cohort study
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M.A. Thorburn
found associations between each of three risk factors farm size, farm age and
date of fingerling transport to salt water and farm-level V. anguillarum-specific
mortality rates in Swedish rainbow trout (Thorburn, 1987). Another
retrospective cohort study identified associations at the farm level between farm
location, farm age and number of hatcheries from which smolt were purchased
and the occurrence of clinical IPN and between farm location and the occurrence
of furunculosis in Norwegian Atlantic salmon (Jarp et al., 1994). At the smolt-
group level, Jarp et al. (1994) found that smolt age and transportation method
were associated with mortality. Ahmed and Rab (1995) conducted a cohort study
with retrospective and prospective data to investigate determinants of epizootic
ulcerative syndrome (EUS) at the pond level in Bangladesh. The probability of
EUS was highest in ponds that had been treated using piscicides, that had not
previously contained fish or that were in poor condition. Also, silver barb
Puntius gonionotus were at higher risk for EUS. Wheatley et al. (1995)
conducted a 4-year site-level prospective cohort study of mortality rates in
Atlantic salmon postsmolt in Ireland. Cumulative mortality was higher on sites
that were not fallowed, that reared more than one generation, that slaughtered
fish on site or where farm staff moved between farm sites.
Casecontrol studies
In casecontrol studies, samples of diseased units (cases) and samples of non-
diseased units (controls) are selected. The proportion of each that has been
exposed to the factor of interest is used to estimate the corresponding population
proportion. If the difference between the sample proportions is statistically
significant (i.e. the odds ratio differs statistically from one), one concludes that
the factor and the disease are associated.
Classic casecontrol studies are conducted retrospectively; hence data are
usually collected from existing records or by interview. Ideally, both cases and
controls are representative of the corresponding diseased and non-diseased
segments of the population from which they were selected. Since casecontrol
studies are often used to study rare diseases, the case group frequently comprises
all known cases of the disease. Usually, cases are selected from one or more
sources, such as diagnostic laboratories or practitioners. Newly diagnosed cases
are preferable, as they will have better recall and be less influenced by
determinants of survival (as opposed to determinants of disease). Furthermore,
with prolonged cases of disease, it is possible to confuse the effects of a disease,
such as the occurrence of other diseases or husbandry changes, with potential
causes of that disease. Highly specific criteria should be used to select cases (i.e.
avoid false positives).
The selection of appropriate controls is one of the most difficult aspects in
casecontrol studies (Wacholder et al., 1992). Controls are often selected from
the same source(s) as the cases. With this approach, the prevalence of the
factor(s) of interest in the controls may differ from the prevalence in the non-
diseased segment of the target population and resulting estimates of the factor
disease association will not necessarily reflect the true association in the target
population. (The same issue arises if cases comprise only a sample of all
diseased units in the target population and are not selected randomly.) This
problem may be circumvented by randomly selecting controls from the entire
population. The managers of randomly selected controls (farms, holding units or
fish) however, may be less able to remember details about previous exposures
and less willing to participate in the study.
Theoretically, cases and controls should be similar in all respects, except for
the disease being investigated and risk factor(s) associated with the disease. As
such, cases and controls should be subjected to the same exclusion criteria. Also,
it is important to obtain a similar response rate from both groups. Odds ratios are
used to evaluate associations in casecontrol studies. Since units are purposively
selected in casecontrol studies on the basis of disease status, several population
parameters cannot be estimated; these include the prevalences of the disease and
of the risk factor and the probability of disease in each of the exposure groups
(and, hence, the relative risk and the attributable rate).
Casecontrol studies are more subject to bias than the other study types
presented. Also, it is often not possible to show that exposure preceded disease,
which will limit an investigators ability to infer causality. However, if good
records are available, casecontrol studies are relatively inexpensive and are
useful for investigating rare diseases and for screening the significance of
several risk factors for a given disease.
Two excellent casecontrol studies of fish disease have been published with
site as the unit of concern. Jarp et al. (1993) identified associations between the
occurrence of A. salmonicida infection on Norwegian freshwater hatcheries and
migration of anadromous fish in the freshwater supply, sharing of personnel with
other fish farms and a high concentration of A. salmonicida-infected farms in the
vicinity. Vgsholm et al. (1994) showed that the probability of infectious salmon
anaemia (ISA) infection in Norwegian sea-cage sites increased if there were
slaughterhouses or ISA-infected sites nearby, if salmon deemed unfit for
slaughter had been returned to the site, if employees worked at several sites, if
salmon previously reared in sea water had been brought to a site for further
rearing or if fish had been vaccinated by intraperitoneal injection. Refer to the
texts by Breslow and Day (1980) and Schlesselman (1982) for further details on
the design and analysis of casecontrol studies.
Sample size
Too small a sample will give an analytical study low power, i.e. a low probability
of detecting an association between a risk factor and a disease when such as
association truly exists. Searcy-Bernal (1994) reviewed a large number of
aquaculture research papers and suggested that many non-significant results
have been due to low power. The following formula provides a useful guideline
for the number of units which should be included in each group (case and
control; exposed and non-exposed; treated and control) in a casecontrol study,
cohort study or field trial (Snedecor and Cochran, 1989, p. 129):
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M.A. Thorburn
(Z
1 -
+ Z
1
)
2
(p
e
q
e
+ p
c
q
c
)
n =
(p
e
p
c
)
2
whereZ
1
= value of Z which provides /2 in each tail of the normal
curve if a two-tailed test is used or in one tail if a one-
tailed test is used; equals the probability that the study
statistically indicates a difference between the groups when
there is no true difference in the population
Z
1
= absolute value of Z which provides in one tail of the
normal curve; equals the probability that the study does
not show a statistical difference between the groups when
there is a true difference in the population
p
e
= estimate of prevalence of exposure in control group or
incidence of disease in non-exposed or control group
p
c
= estimate of prevalence of exposure in case group or
incidence of disease in exposed or treated group
q
e
= 1 p
e.
q
c
= 1 p
c.
As an example, suppose an investigator wishes to use a cohort study to
determine if the introduction of Arctic char, Salvelinus alpinus, to rainbow trout
farms will increase the likelihood that furunculosis is detected on a farm. Only
farms that do not currently have detectable A. salmonicida may be entered into
the study. During a 1-year study, 10% of farms that do not introduce char are
expected to be diagnosed with furunculosis. A farm-level incidence of 30% or
higher is expected among the farms that do introduce char. How many farms
should be followed to be 80% (1 ) certain of detecting a difference this large,
if it exists, and 95% (1 ) certain of not declaring a difference, if the true
difference is smaller? Substituting into the above formula:
(1.96 + 0.84)
2
+ (0.3 0.7 + 0.1 0.9)
n = = 61.6
(0.3 0.1)
2
The investigator should include 62 farms that do not introduce char (non-
exposed) and 62 farms that do introduce char (exposed) in the study.
If fish or holding units are the unit of concern and the study results are to be
extrapolated to more than one farm, the calculated sample size should be
increased, particularly if only a few farms are included in the study. Shoukri and
Martin (1992) provide a complex formula for estimating the number of farms to
include in field trials in which fish or holding units are randomly allocated to two
treatment groups on each participating farm.
Interpretation of study results
There are several reasons why the results of an epidemiological study might
indicate (or fail to indicate) an association between a risk factor and a disease.
Ideally, the finding of a statistical association reflects a true, causal relationship
and failure to find an association occurs because there is no true relationship.
However, an observed statistically significant (or non-significant) association
may be spurious or indirect. Spurious associations are observed due to chance
(i.e. sampling variability), bias in the study design or improper statistical
analysis. Some reasons for indirect associations include confounding and
ecological fallacy.
Causal association
In experimental research, it is fairly straightforward to restrict sources of bias,
through randomization and, except for field trials, strict control of external
factors. Hence, experiments can provide strong evidence of causality. However,
it is often difficult to extrapolate the results of experiments to real life
situations. Analytical observational studies have the advantage of being directed
toward the species of concern in its natural environment. Their main
disadvantage is that it can be difficult, if not impossible, to prove causality.
As a first step in determining causality, the design of the study should be
carefully examined. Cohort studies are better able to demonstrate causality than
are casecontrol or cross-sectional studies, because they are subject to fewer
biases and because the temporal relationship between the factor and the disease
can be established. Possible sources of bias resulting from the specific study
design should be elaborated.
The criteria used for defining exposure and disease will influence
conclusions on causality. The use of specific outcomes (e.g. A. salmonicida-
specific mortality as opposed to crude mortality) and precisely defined exposure
variables (e.g. specific water chemical variables as opposed to stocking density)
will strengthen the observed statistical association between the outcome and the
factor if the relationship is causal.
The possibility that other variables could have produced the observed
association or lack of association should be considered (e.g. the study variable
may be a surrogate for, or predecessor of, more direct causes of the disease;
confounding variables may have distorted the association).
Additional guidelines that should be used to assess causality in
epidemiological studies are presented in Evans (1978) and Susser (1973, 1977).
Bias in study design
SELECTION BIAS
Selection bias occurs in casecontrol studies if the likelihood of including
specific case and/or control units is associated with the risk factor being studied.
As an example, suppose a casecontrol study is conducted in a region where
Yersinia ruckeri is rare, in order to determine whether land-based trout farmers
who experienced outbreaks of enteric redmouth (ERM) were more likely to have
purchased their young stock than were those farmers who had hatched their own
fry. Case farms and control farms are selected from a group of farmers who
submitted fish to a certain diagnostic laboratory during a specified time period.
Suppose, however, that many of the farmers with hatcheries had previously
714
M.A. Thorburn
worked in regions where ERM was relatively common and were consequently
better able to recognize and treat ERM and less likely to submit ERM cases to
the laboratory (relative to other disease cases). Farmers with hatcheries will
consequently be relatively underrepresented in the case series and a spurious
positive association between purchasing young stock and ERM may be observed
(or a true-positive association may be magnified). Freund et al. (1990b) provide
a good discussion of some of the limitations, including the potential for selection
bias, of using data obtained from fish disease diagnostic laboratories.
A type of selection bias can also occur in cohort studies or field trials if the
probabilities of being lost to follow-up (withdrawals) or of being a non-
responder are related to both exposure and disease status.
SYSTEMATIC MISCLASSIFICATION BIAS
Systematic misclassification bias occurs in cohort (casecontrol) studies when
information is obtained in a non-comparable manner between exposed and non-
exposed (case and control) units. For instance, systematic misclassification bias
may occur in cohort studies if more diagnostic effort is applied to the exposed
group. Hence, those who are making the diagnoses should be blinded to the
exposure status of the units. In casecontrol studies, systematic misclassification
bias may occur, for instance, if cases have better recall concerning their
exposure. Alternatively, controls may have kept better records.
RANDOM MISCLASSIFICATION BIAS
Random misclassification bias occurs when there is inaccurate classification of
the exposure status in cohort studies and of disease status in casecontrol studies
and the misclassification is equally probable in those which become diseased
and those which do not (cohort study) or in those which have been exposed and
those which have not (casecontrol study). Random misclassification will
always bias the relative risk and the odds ratio towards one (i.e. non-
significance).
As an example, suppose 100 cases and 100 controls are correctly identified,
and classified according to their exposure status in the two-by-two table in Table
19.6.
Now, suppose that the diagnostic test used in a casecontrol study has a
sensitivity of 0.7 and a specificity of 0.9. In this case, an average of 30% of the
cases will be misclassified as controls and 10% of the controls misclassified as
cases. The results given in Table 19.7, using the same units as in Table 19.6,
would be observed.
The OR, indicating the strength of association, has been reduced by more
than half, due to the misclassification.
Confounding bias
A variable that affects the frequency of both the exposure factor and the disease
is called a confounding variable. The uncontrolled presence of such variables in
a data set will distort the association observed between the exposure factor and
the disease. Confounding bias is a problem in almost all field data. It can be
controlled in the design of studies by randomization, exclusion or matching.
Randomization of units to the treatment and control groups is only possible in
field trials (and other experiments). When randomization is used, confounding
variables will usually be similarly distributed between the two groups. Exclusion
is achieved by limiting the study to units at only one level of a given
confounding variable (e.g. one species, one age group, one type of water source,
etc.). It is difficult, however, to extrapolate the results of such studies to a
broader target population. In matched designs, investigators first identify one or
two strong confounders. For each case (exposed) unit included, one or more
control (non-exposed) units, which have the same level of the confounder as that
case (exposed) unit, are randomly selected. As there are a number of
disadvantages to matching (Kleinbaum et al., 1982, p. 382), its use should be
limited to variables which are known in advance to be strongly related to both
exposure and disease. If matching is used in a casecontrol study, it must be
controlled for in the subsequent analysis.
It is highly recommended to control for confounding in the statistical
analysis of the data. This approach requires investigators to collect data on the
presence or level of potential confounding variables simultaneously with the
collection of other relevant data. Methods for the analytical control of
confounding variables are presented by Breslow and Day (1980, 1987), Fleiss
(1981) and Kleinbaum et al. (1982). One basic approach involves stratifying
data by the different levels of the confounding variable, as presented in the
following example. Suppose an investigator suspects that rainbow trout
fingerlings transferred in the spring from freshwater hatcheries to brackish-
water cages off the Swedish coast of the Baltic Sea are more susceptible to
vibriosis than are those transferred in the autumn. A questionnaire is sent to all
eligible cage farmers at the end of the high-risk summer period to collect
information on when specific lots of trout were transferred (spring or the
previous autumn) and on whether they had experienced V. anguillarum
outbreaks. The lot-level data given in Table 19.8, which suggest no association
between time of transfer and vibriosis, are obtained.
Table 19.6. Accurate classification of exposure status.
D+ D
F+ 40 10
F 60 90
OR =(40 90)/(10 60) =6.0
Table 19.7. Inaccurate classification of exposure status.
D+ D
F+ 29 21
F 51 99
OR =(29 99)/(21 51) =2.7
716
M.A. Thorburn
The investigator suspects, however, that: (i) lots located on farms in the
northern Baltic were less susceptible to vibriosis because of the lower salinity
there; and (ii) fingerlings were less frequently transferred to northern sites in the
fall because of harsher winter conditions. Hence, location may be a confounding
variable. When the investigator stratifies the data by location, the two two-by-
two tables given in Table 19.9 suggest that time of transfer is associated with the
occurrence of vibriosis.
Stratifying on the confounding variable, location, has removed the bias
present in the uncontrolled estimate of the relationship between season of
transfer and vibriosis.
Thorburn (1996) provides an example of potential confounding bias in a
fish-disease data set.
Ecological fallacy and clustering
When fish are exposed to a risk or treatment factor as a group (e.g. oral
antibiotics, immersion vaccination, holding-unit specific husbandry factors,
etc.), the group should be the unit of analysis and the unit of concern.
Sometimes, investigations are conducted in aquaculture in which groups of fish,
such as holding units or sites, are the unit of analysis, but the individual fish is
the unit of concern. These types of ecological studies are prone to substantial
bias, called ecological fallacy, since, lacking data on individuals, one can only
assume that what is true at the group level also holds true at the individual level.
For example, Thorburn et al. (1989) found that groups of non-vaccinated trout
with higher prevalence proportions of anti-V. anguillarum antibody-positive fish
experienced lower V. anguillarum-specific mortality rates than did those with
lower prevalence proportions. The authors emphasized, however, that then to
conclude that anti-V. anguillarum antibodies are protective for individual fish
could result in ecological fallacy. Greenland and Morgenstern (1989) provide a
good discussion of ecological fallacy.
Table 19.9. Data stratified by location.
North coast South coast
V
+
V
V
+
V
S 5 5 S 6 1
F 2 4 F 23 8
OR =2.0 OR =2.1
Table 19.8. Lot-level data.
Vibrio No vibrio
Spring 11 6
Autumn 25 12
OR =(11 12)/(6 25) =0.9
A common problem in the fish-disease literature arises when data on fish
that have been treated and/or held as a group are analysed at the fish level, as if
the individual fish were independent of one another. This problem has been
briefly discussed by Freund et al. (1990a). In the realm of fish vaccine research
alone, numerous authors have incorrectly used analytical methods that assume
independence; examples include the chi-square test (e.g. Amend and Johnson,
1984; Cardella and Eimers, 1990; Nikl et al., 1993) and survival analysis (e.g.
Lillehaug et al., 1993). When no statistical adjustment is made for the obvious
lack of independence, results will tend to be spuriously significant. McDermott
et al. (1993) have published a review of various statistical methods that can be
used to adjust for the clustering (i.e. lack of independence) of animals housed
in groups. Alternatively, the holding unit may be used as the unit of analysis
(Johnson et al., 1993; Roth et al., 1996), although this may lead to low power.
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CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

average NAR no. of time intervals

initial NAR 1/2 withdrawals

no. fish at risk for X at that point in time

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