Viral haemorrhagic disease of rabbits

Rev. sei. tech. Off. int. Epiz.,
1991, 10 (2), 471-480
Viral haemorrhagic disease of rabbits:
vaccination and immune response
J.L. ARGÜELLO VILLARES *
Summary: Results are presented for the large-scale use of a tissue vaccine,
inactivated with beta-propiolactone and containing aluminium hydroxide
adjuvant, against viral haemorrhagic disease of rabbits.
The kinetics of haemagglutination-inhibiting
antibodies over eighteen
months, the response of vaccinated animals to challenge infection with field
virus between six and fifteen months after vaccination, the serological response
to revaccination, and the immunological mechanisms involved in primary
vaccination and revaccination were investigated.
KEYWORDS: Immune response - Immunisation - Rabbit diseases - Vaccines Viral diseases - Viral haemorrhagic disease of rabbits.
INTRODUCTION
The appearance of viral haemorrhagic disease (VHD) among A n g o r a rabbits
imported from Germany into the People's Republic of China in 1984 (14), and the
subsequent spread of the disease to E u r o p e (possibly from Eastern countries) and
then to N o r t h Africa and the Americas, where Mexican rabbits were affected,
immediately led to numerous proposals to control the disease.
Although vaccination was the first measure recommended by some authors, the
implementation of sanitary measures, under which the unaffected areas tried to remain
free from the disease while affected areas were conducting control and eradication
measures, was the most commonly adopted procedure as soon as outbreaks occurred
in a country.
These opinions are reflected in the notice issued by the O I E on 20 January 1989
(21), when reports of the new disease arrived from various E u r o p e a n countries,
including Italy (16), Spain (2), France (19) and Germany (15). Similar occurrences
were notified in countries of Eastern E u r o p e .
A m o n g disease control measures recommended in this notice were emergency
slaughter of entire populations of rabbits on affected breeding farms, with
repopulation after an interval of four weeks. General control procedures were
recommended for disease-free regions or premises.
* Laboratorios Ovejero S.A., Apartado 321, 24080 Leon, Spain.
472
When Mexico was confronted with a severe outbreak and lacked access to
vaccination, the eradication p r o g r a m m e included quarantine of infected premises,
prohibition of movement and sale of rabbits, voluntary slaughter of sick animals with
cleaning and disinfection of affected premises, and an interval of at least two months
before repopulation ( 1 , 17). These measures resolved the problem and were followed
by a significant decline in the rabbit population.
Considering the Mexican experience, the United States Foreign Animal
Disease
Report (10) recommended the concept of "sentinel r a b b i t s " , introduced in small
numbers before complete repopulation of affected premises and after an unpopulated
period of two m o n t h s .
Abundant information exists on precautions to be adopted, particularly with regard
to the early stages of investigation of the new disease; however, developments in
research have somewhat modified the previous approach. For instance, Rosell (25)
reported the appearance of the disease in well-fed rabbits which were free from
intercurrent disease; general disease precautions h a d been strictly implemented in the
colonies where these rabbits lived. Cancellotti (7) reviewed the situation and concluded
that severe outbreaks of viral haemorrhagic disease, with 9 0 % mortality, could occur
even in intensive establishments with good technical assistance and where disease
control precautions were in force. Despite isolation, disinfection and safe disposal
of carcasses, cases of the disease continued in relentless progression.
In the opinion of Rosell (26), hygiene control is indispensable before and after
outbreaks of V H D , although the adoption of such measures cannot guarantee
protection.
All these findings agree with the experience gained in the People's Republic of
China (29) which showed that the disease could be easily controlled by the slaughter
of affected animals, strict quarantine and, most importantly, vaccination.
Vaccination was contemplated in the beginning, in the hope of controlling the
disease (2). Not until research had been conducted (3, 4, 5, 6, 22, 23) and the results
of more than two years of field trials of general vaccination h a d been completed,
however, was it possible to conclude that vaccination was valuable in controlling the
new disease. The reduction in the number of new outbreaks and in the spread of
the disease since vaccination has been adopted confirms the efficacy of vaccination
as a control m e t h o d .
VACCINE
A commercially available vaccine has been used in Spain against V H D virus
(VHDV) from the beginning. It was first used experimentally (in collaboration with
the Animal Health Sub-Directorate of the Spanish Ministry of Agriculture, Fisheries
and Food) and, subsequently, in a field trial according to protocols dictated by the
disease emergency. This vaccine has also been widely used in Germany, Belgium,
France, Greece, Italy, Portugal and Tunisia.
Like other vaccines which were either officially licensed or under trial, this vaccine
was prepared from tissues which had been obtained by inoculating susceptible animals,
then collected from the target organs shortly before death.
473
The animals used for inoculation were always selected from colonies which had
previously shown high susceptibility to the disease. The animals had been reared under
satisfactory health conditions and kept in strict quarantine upon arrival. During the
quarantine, bacterial infections were eliminated by broad-spectrum chemotherapy.
The quarantine period and the known origin of the animals guaranteed that no
animal which had experienced hyperthermia before inoculation was inoculated. Also
excluded were animals yielding a positive blood culture to standard bacteriological
testing during inoculation, as this implied an intercurrent septicaemic process.
Inoculation was performed by intramuscular injection (although other routes can
also be used) of 0.5 ml of an inoculum which h a d previously been titrated to contain
at least 100 LD50; this led to death within an average of 24-36 h. In all cases, the
inoculum was prepared from strains capable of haemagglutination (HA) at r o o m
temperature with slow elution. As reported by Badiola (6), cases occurring in the
field are associated with H A only at 4 ° C , or with rapid elution. In the experience
of the author, however, such cases are rare.
Following inoculation, the m o m e n t for collecting samples is determined by the
clinical course in the inoculated animals. Those animals which had previously
experienced pronounced hyperthermia and are in pre-agonal depression, with a
temperature below the normal physiological range, are selected for slaughter.
The target organs showing the characteristic lesions of V H D (2, 16) and which
are free from signs of intercurrent diseases, including presumptive signs, are removed
after death. The organs are triturated; phosphate-buffered saline (PBS) at p H 7.2
is added in the proportion 1:2 (w/v) and the mixture is placed in a blender, operating
at 18,000 r p m in a refrigerated environment for 5 min. It is then triturated, also at
4 ° C , for 5 min at 24,000 r p m .
The fluid resulting from clarification of the suspension is assayed for virus content
by its haemagglutinin titre against h u m a n group O erythrocytes. This is performed
by means of a microplate technique with 0.025 ml of each reagent at 4 ° C and a p H
of 6.4.
Once the number of haemagglutinating units (HAU) from the initial titration is
known, more PBS at p H 7.2 is added in sufficient volume to provide, after inactivation
of the suspension and adsorption on adjuvant, a concentration of 128-256 H A U in
the commercial p r o d u c t .
In view of these explanations, the vaccine produced by the author does not, in
principle, differ from those described by other authors in other countries (2, 3, 4,
5, 8, 11, 12, 13, 18, 22, 23, 27, 28, 29).
However, the vaccine used by the author differs from most of the products used
in the field, including those tried experimentally, in two respects.
Firstly, the inactivating agent is beta-propiolactone, originally chosen for two
important reasons:
- The product has given good results as an inactivating agent for canine
parvovirus. There are various opinions on the classification of the V H D V , but the
prevailing opinion when the author made his decision was that it belonged to the
Parvoviridae family. In any case, beta-propiolactone has since proved to be a
satisfactory inactivating agent.
474
- The product enables easy verification that it has not undergone degradation
due to partial hydrolysis before use and that it therefore remains fully active. Once
inactivation is complete, the substance is readily hydrolysed at 37°C into betapropionic acid and hydroacrylic acid, both of which are innocuous.
Secondly, the final component to be incorporated in the vaccine, the adjuvant,
can be selected from the two options most frequently employed: Freund's incomplete
adjuvant and aluminium hydroxide.
The selection of aluminium hydroxide, which is stable in the final product over
a long period, is supported by reports in the literature (4, 8, 9, 11, 12, 13, 18, 27,
28, 29) and by the results of experiments conducted by the author. Given the special
features of the antigen, there is no doubt that stability is better in the presence of
aluminium hydroxide than in an oil emulsion and that the use of aluminium hyroxide
avoids the risk of anaphylactic p h e n o m e n a upon revaccination.
VACCINATION PROCEDURE
Previous studies (4) have established the dose at 1 ml for adult animals and 0.5 ml
for animals vaccinated at weaning. The latter amount is based on the protection
conferred by a reduced dose, taking into consideration the short period of protection
required during the production cycle, which corresponds to the fattening period.
Revaccination was initially recommended after six months. Although data existed
to indicate that protection would last for a long period, experimental evidence to
support this was lacking. Now, however, evidence has been obtained from
revaccination in the field and from vaccination experiments which indicates that
revaccination is unnecessary. For this reason, it is not used much.
SEROLOGY
All the serological data for the present study, whether from experimental animals
or from intensive rabbit colonies, were obtained by using the haemagglutination
inhibition (HI) test described above (2). This technique underwent a number of
modifications, such as the choice of p H 6.4 and a reaction temperature of 4 ° C , in
different laboratories.
Such modifications are justified because the first observations by the author were
made so soon after the disease had first been notified. This meant that the author
had to incorporate all the data obtained up to that time in order to establish the kinetics
of the antibodies and that he was unable to use those techniques, such as E L I S A ,
which appeared later (20). In any case, given the similarity between the results of
the H I test and the ELISA technique (20), the author believes that his data reflect
the true serological situation.
RESULTS O F V A C C I N A T I O N
Earlier publications (4, 5, 6, 11, 12, 13, 22, 23, 24, 26, 27, 28) reveal two distinct
aspects of the response to vaccination, depending on whether it was performed
experimentally or in the field. The results indicated below reflect current knowledge.
475
Innocuity
I n n o c u i t y in a b r o a d sense is d e m o n s t r a t e d a n d c o n f i r m e d by the
adequate inactivation of a virus originally lethal for rabbits, by the safety of
the inactivating agent, and by the absence of reaction to and the suitability of the
adjuvant.
At the control stage, before each batch of vaccine is offered for sale, at least ten
rabbits older than ten weeks are inoculated; when possible, rabbits of country origin
are chosen because of their high susceptibility to the virus. Each is inoculated with
5 ml of vaccine. The absence of clinical manifestations, including hyperthermia, and
a weight increment similar to that of the unvaccinated controls, demonstrates both
the adequate inactivation of the virus and the absence of undesirable side effects.
When the animals are slaughtered seven days later, the absence of definite or suspect
lesions of V H D confirms the inactivation.
Finally, the absence of local reactions at the inoculation site confirms that the
excipient suitably stimulates bodily defences.
Vaccination experiments were conducted on animals of different ages and on
females at various stages of gestation, between mating and parturition. In all cases,
the number and viability of offspring were completely normal.
These laboratory findings were fully confirmed in the field and included less
carefully controlled, though unproblematic applications, such as the vaccination of
all categories of animals encountered in commercial rabbit breeding: animals bred
for meat, breeding males and females, and does at various stages of pregnancy and
lactation.
Serology of vaccination
The serological response of vaccinated animals has been examined in previous
studies (3, 4, 5). W h a t follows are the most commonly encountered features of
serological behaviour:
—
Before vaccination, and during the first three days thereafter, the animals show
no serological response as measured by inhibition of H A by 4 H A U of V H D V .
- Between four and seven days there is obvious seroconversion in all animals,
attaining titres of 1:320 by the end of this period.
- A titre of 1:320 is maintained for three m o n t h s after vaccination.
- When four, five or six months have elapsed since vaccination, the response
falls to a new plateau of 1:160.
- At seven and eight months after vaccination there is another fall in serum titre
by one logarithm to the base of two, with H I titres of 1:80 still capable of providing
protection, as shown by the results of challenge infection.
- The subsequent serological picture in the ninth and tenth months after
vaccination in animals kept under experimental conditions, and in the sixth to sixteenth
months in field trials, shows that the same titres become stabilised, with all animals
possessing an H I titre of 1:80 to 4 H A U .
476
Results of challenge infection
Animals kept in the laboratory or in field trials (culled for poor productivity) were
inoculated with 100 L D of virus at various times after vaccination.
5 0
Those animals vaccinated 2-3 days previously and possessing titres between 1:20
and 1:40 died after field virus inoculation. A less acute form of illness, however, lasted
six to seven days after challenge in some animals, particularly those with the higher
titre.
All animals with initial titres of 1:80 at 4-5 days after vaccination survived, a fact
which indicates that this titre conferred protection. Seven days after challenge the
antibody titre usually reached 1:640, subsequently stabilising (at 14 days) at 1:320.
In some animals the titre reached 1:1280, while in others it increased by only two
logarithms, to 1:320.
Recent vaccination followed by challenge with a field strain of virus in animals
having initial titres of 1:160 or 1:320 resulted in an increase in some animals to 1:640,
although the most c o m m o n H I titre was 1:320. This demonstrates that, with high
initial titres, the challenge infection failed to stimulate a new serological response.
A m o n g animals vaccinated some m o n t h s previously, challenge infection under
the conditions described above met with the following responses:
- animals with titres of 1:160 or 1:80 at six, seven or eight m o n t h s after
vaccination showed no significant seroconversion after challenge, the titres increasing
only to 1:320, but without any clinical illness;
- between nine and fifteen months after vaccination, challenge infection produced
increasing seroconversion, almost always without symptoms (there were some
exceptions in the periods furthest removed from initial vaccination), with titres
reaching 1:5120 one week after challenge. In every case the titres then subsided slowly,
falling to the previous titres by 3-4 weeks after challenge.
REVACCINATION
The following is a synopsis of events after revaccination of laboratory and farmed
rabbits:
- T h e vaccine was found to be entirely safe u p o n second inoculation, as there
was no local or general reaction and no difference in response from the first
inoculation.
- With regard to the serological response, revaccination after seven to eight
months invariably resulted in an increase in titres in all animals, reaching 1:320 within
2 to 3 days. There was no further increase in titre in this group and no exceptional
incidents were reported.
- T h e starting titres of 1:80 increased to 1:320 in 6 0 % and t o 1:640 in 4 0 % of
the animals revaccinated after nine to ten m o n t h s .
- The serological response always increased to 1:640 in animals which had
received a primary vaccination 11-13 months previously. Because the first test was
not conducted until seven days after revaccination, the onset of titre increase was
477
not determined in this group. This titre persisted for three weeks, however, after which
it declined (by the fourth week) to 1:320 in the majority of animals tested.
- Animals revaccinated 14-16 months after primary vaccination responded early
and intensely, giving titres between 1:1280 and 1:2560 at 2-3 days after revaccination.
The persistence of these elevated titres lasted for approximately four weeks; in most
cases, they then declined to 1:320.
DISCUSSION
The results obtained by the author and those reported by Pagés (23) proved
beta-propiolactone to be a suitable inactivating agent. While preserving the
immunogenicity of the virus, it also provided a valid test of the integrity of those
antigenic determinants involved in the i m m u n e response.
The results obtained by the author, and supported by those of Galassi (9), showed
that the adjuvant which was used potentiated the immune response and contributed
to a completely effective vaccine.
In addition to enhanced efficacy due to the right sort of adjuvant, the careful
selection of a strain of virus for vaccine production, based on its haemagglutinating
property and slow elution, was an important factor in avoiding ineffective batches.
This has been a problem experienced by numerous research workers in different
countries.
The accumulation of results of hundreds of serological tests and the narrow ranges
of HI titres obtained provided solid criteria for the validity of the serological procedure
used. Nevertheless, studies on the relationship of this test to others, such as E L I S A ,
have yet to be conducted.
The innocuity of the product conforms with the experience of all those who have
used vaccine to control V H D . In none of the field trials did the author encounter
any impairment of fertility when animals were mated 5-15 days after vaccination,
as stated by Kelemen (13) when using other products.
Unvaccinated animals either possessed n o antibody or h a d titres < 1:20 in
serological response to primary vaccination. As these animals were procured in the
early stage of the disease in Spain, this result was not unexpected. The results obtained
in an endemic situation would be different, as witnessed by Morisse in France (personal
communication) and other authors, where some animals, selected randomly from
colonies free from the disease, h a d clearly positive titres (20). This, however, differs
from the experience of Xu, who found a decrease in both susceptibility and mortality
in areas where the disease had occurred, the H I test giving doubtful or mildly positive
results (29).
The precocity of the serological response, confirmed by the disappearance of
clinical cases on infected premises only 4-5 days after vaccination, was a constant
finding in the trials conducted by the author and by others. In any case, the use of
inactivated vaccine provides no easy explanations.
478
Xu mentioned the possible participation of interferon (29). The author believes that
the antigenic material to which the animals respond is comprised more of the modified
surface of virus-containing cells than of the virus itself. The response to these novel
antigenic determinants, readily accessible to the immune system through their superficial
location, no doubt occurs sooner than the response to intracellular virus.
If this happens, the vaccinal protection would involve, among other mechanisms,
cytotoxic mechanisms which destroy infected cells and stop viral replication.
Challenge infection of vaccinated animals at various intervals (prolonged intervals,
in many cases), has confirmed that, given the high proportion of replacements required
to increase yield, revaccinating rabbits is unnecessary in commercial colonies. If not
clearly expressed, this idea has been assumed in numerous earlier studies.
The absolute innocuity of revaccination is to be expected from a vaccine high
in protein derived from the organs of rabbits and free from excipient, other than
aluminium hydroxide: the protein available to the immune system of the vaccinated
animals is homologous and would not elicit anaphylactic shock.
The serological response of animals revaccinated after seven to eight months was
not typical of a secondary response. The antibody titres achieved, and the fact that
they appeared promptly and persisted, is clear proof of the activation of memory
cells and of the type of g a m m a globulin on which the secondary immune response
is based.
Enhancement of the serological response at a greater interval between primary
vaccination and revaccination may owe to the adjuvant function of an immunity which
forms its own antibodies. These antibodies consist of aggregates which are not formed
earlier by an excess of circulating antibody. Better presentation of antigenic material
to macrophages and the consequent improved management of the antigen as a whole,
which results in an enhanced immune response, may explain how this process occurs.
Finally, the response elicited by challenge infection at a variable interval
demonstrates an improved serological response after challenge. This reponse is directly
related to the time which has elapsed since the primary vaccination and does not
permanently influence the initial serological titre of the animal. The absence of clinical
signs proves that the aetiological agent has not multiplied. It also signifies that
challenge infection is the equivalent of revaccination in that the immune mechanisms
mentioned above no doubt play their part.
CONCLUSION
Without giving a detailed account of the presumably complex immunological
mechanisms linked to vaccination against V H D , but considering the actual production
cycles in rabbit farming, which usually involve the maintenance of breeding stock
for no more t h a n a year, the author concludes that vaccination of future breeding
stock at ten weeks of age will confer solid protection throughout the productive life
of the rabbits.
*
* *
479
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