THE MEANING OF VACCINATION TITRES
P.F.McMullin
Research Veterinarian, Veterinary Technical Services and
Research Unit,Merck Sharpe & Dohme Research Laboratories,
Campinas, Brazil
Proceedings of the 2nd Symposium of the Brazilian Chick
Producers Association (APINCO) pp 11-16 Campinas, SP, 1984
INTRODUCTION
Titration is the method or process by which the strength or
concentration of a substance in a solution is measured. The
result obtained is called the titre of the solution. The word
titre is often used in connection with vaccination of poultry. It
is, however, used to refer to two quite distinct subjects. On the
one hand we have the titre of the vaccine in use, and on the
other the titre of antibody produced in the bird by way of
vaccination.Most of the live vaccines are titrated by sucessive
dilution to find the minimal dose necessary to bring about a
given effect in a biological system (in our case usually chick
embryos our cell cultures). The titre of a given batch of vaccine
is usually a good measure of its ability to immunize animals. It
must be understood that the titre necessary is specific for each
product. The methods used in titration can have a considerable
effect on the titre obtained.The rest of this paper refers to our
main subject , that is the serological titres produced by
vaccination. In this case the titres are found by sucessive
dilution of the serum to find the minimal quantity capable of
producing a given effect in a biological system. Effects often
used in titration of antibody are inhibition of haemagglutination
and neutralization of infectivity.
THE IMMUNE RESPONSE
When a vaccine is applied to a bird, a complex biological
mechanism is set in motion which normally results in the
elevation of the birds specific defenses against the disease in
question, and it may also raise non-specifically its defenses
against other diseases.The main components of this defence system
are :
1. Antibodies circulating in the blood.
2. Antibodies secreted on the mucosae (including conjunctiva,trachea,intestine
etc.).
3. Lymphocytes and macrophages sensitized to the antigen.
4. Interferon.
The exact mechanism by whch these defenses are elicited and their
nature in poultry is a subject of active research at the moment.
We do know that the immunological mechanisms of birds are not
identical to those of mammals. For our present purposes it is
sufficient to remember that the response is complex and has more
than one facet. Not all of these facets of the birds immunity are
readily measured.
ALTERNATIVES AVAILABLE
Although some methods of studying sensitised lymphocytes and
mucosal antibodies have been developed for research purposes,
under normal circumstances these methods have not been applied to
commercial poultry. There are basically two methods which are
used to evaluate birds response to vaccination : titration of
specific antibodies circulating in the blood, and measurement of
the effect of a challenge exposure to one or more virulent
strains of organisms. Challenge has sometimes been protrayed as
the "ideal" method of evaluating the effectiveness of a
vaccine. On the surface this may correct but in fact this
approach has a number of serious defects if we are thinking about
routine flock monitoring (as opposed to quality control of
batches of vaccine in the laboratory). Facilities capable of
safely containing the challenge organisms throughout large
numbers of tests are expensive and not widely available to the
industry. Simple live/die challenge models may greatly
underestiate the effects of disease on productivity parameters in
supposedly protected birds (especially layers). It may also
underestimate the importance of virulent viral replication, in
apparently healthy birds. In view of these defects of challenge
techniques , emphasis is being placed increasingly on titration
of circulating antibody levels as a guide to response to
vaccination in comercial poultry. Among the many laboratory
methods available to measure antibody the choice should be
determined by availability of reagents, economy, and the
accumulated experience in interpreting results. When a new test
is introduced it is often necessary to start "from scratch"
when it comes to interpretation of what the results mean. It is
sometimes necessary to estimate changes in antibody titre by the
judicious use of qualitative tests. This is possible because it
is generally true that the greater the proportion of birds
positive in a qualitative test, the higher will be the mean titre
of the group. This strategy has been used with the agar gel
precipitation test for infectious bronchitis in Holland for a
number of years.
SAMPLING
In view of the fact that we often use very small samples to infer
about the condition of a large flock of birds. Appropriate
statistical techniques are available to determine the number of
samples necessary for a given purpose. It is often the economic
and practical considerations which actually determine the numer
of samples taken. Sampling should generally be stratified. That
is, the larger the degree of variability we expect in a given
class of birds, the greater the proportion which must be sampled.
Methods of sampling must be practical, easy to teach to farm
staff, and produce serum of appropriate quality for the intended
test(s).
STANDARDIZATION
There has been some discussion on the necessity of standardizing
laboratory procedures so that results from one laboratory are
comparable with those produced by others. While the objective is
obviously desirable, the means by which it may be obtained is not
so obvious. Not all laboratories have exactly the same physical
facilites or equipment, and sometimes subjective judgement can
have an effect on the results. For these reasons I personally
feel that the emphasis must be placed on standardization of
results and not necessarily the means by which they are obtained.
If cooperating laboratories exchange their internal reference
sera, and, better yet, if all use the same national or
international reference serum then there will be a strong
tendency towards uniformity of results produced. Obviously there
will be considerable modification of the methods in use in some
laboratories so that they produce the "correct" result
with the reference serum. It is perfectly possible, though, to
produce comparable results from a given set of samples , using
quite different equipment, and sometimes with small variations on
the method.
REPORTING RESULTS
The way in which results of serological titrations are reported
has often been a source of confusion. The term G.M.T.(Geometric
Mean Titre) is often used. In fact this is nothing more than the
simple arithmetic mean of the logarithms of the last positive
dilution of each serum. By using a doubling dilution sequence
beginning at 2, the number of the last positive tube or well is
equal to the logarithm to the base 2 of the dilution. This makes
reading and calculation of the mean titre very simple, and is the
reason for the increasing popularity of this type of dilution
sequence. The table provided (Table 2) may be useful to those
unfamiliar with the calculation of mean titres using this system.
In view of the fact that in many cases it is not enough to have
good titres - it is also necessary that they should be reasonably
uniform within the flock - some measure of variability shoud be
included when reporting the results. We have used the standard
deviations of the logs of the titres for this purpose.
INTERPRETATION
Unfortunately there is not always a direct relationship between
the titre of circulating antibody and how birds will behave when
subject to challenge. In view of the complex nature of the immune
response it would be surprising if such a rigid relationship did
exist. There are however data in the literature which correlate
circulating antibody with results of challenge, especially when
the latter is carried out under carefully controlled conditions(Table
1). It must be remembered that birds with quite low titres of
circulating antibody may be quite well protected by localimmunity,
and, on the other hand, birds with high titres may, in the
absence of clinical signs, exrete large quantities of virulent
virus, due to a lack of local immunity.Does this mean that
measuring circulating antibody is of no use? No, just that, like
any laboratory test, care must be taken in interpreting the
results. Routine monitoring of serum antibody response to
vaccination is most appropriate for comparison between flocks
immunised with the same type and route of vaccine application.
Rather than relying on published data to interpret the results
obtained, it is recommended that each organization establish a
normal base-line curve for response to vaccination using its
laboratory and normal flocks immunised with the customary program.
This entails intensive initial testing to determine the absolute
values for antibody titre post-vaccination, and the degree of
variability obtainable within and between flocks. Such a curve
for HI(haemagglutination -inhibition) response of broilers on a
large commercial farm on which there was intensive vaccination
against Newcastle Disease, is shown in Fig.1. Subsequent testing
can and should be directed to age groups or types of birds found
to have greater variability of response and to cases of suspect
vaccine failure. The results then obtained are compared to the
base-line curve, but always remember that an apparent improvement
in vaccine response may be simply the result of exposure to field
virus.The above procedure represents an ideal situation. It is ,
however, sometimes necessary to interpret the results of small
groups of samples in relative isolation. In this case it is best
to compare the titres with those obtained in the same laboratory
with samples obtained from other flocks with a similar
vaccination program. Any attempt to use published tables of
antibody titres in order to evaluate vaccination response,
depends on the comparability of results between the laboratory
generating the data to be interpreted and that which produced the
tables.
SOLVING PROBLEMS
Any routine monitoring program will sooner or later turn up
flocks which have a very poor response in terms of circulating
antibody post-vaccination. In consideraing what to do about these
flocks it should be remembered that the response of the birds to
vaccination may be modified by three main types of factor :
1. Factors related to the vaccine, such as antigenic concentration, contaminating agents, virulence of vaccine strain, nature and quality of adjuvants.
2. Factors relating to the manner in which the vaccine is applied, such as the skill of the vaccinators, the route of application and sometimes interfering factors such as poor water quality.
3. Factors relating to the physiology and the pathology of the immunity producing systems of the bird, such as early exposure to infectious bursal disease, or Marek's disease virus, and probably stress anda number of other diseases and intoxications..
(Tables and Figure not yet available in electronic format)
Table 1.Relationship between HI titres and resistance to
challenge with viscerotropic velogenic Newcastle Disease virus.
Table 2. Derivation of the geometric mean titre for a group of
sera tested using serial doubling dilutions beginning with 1:2
Figure 1. Curve of HI titres to Newcastle Disease following a 1,18,35 (ocular,aerosol,aerosol) program in commercial M.g. positive broilers.The graph represents the mean response for all flocks (n=20) and the vertical bars represent the maximum deviations from this mean.