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African Journal of Biomedical Research
Ibadan Biomedical Communications Group
ISSN: 1119-5096
Vol. 5, Num. 1-2, 2002, pp. 87-89
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African Journal of Biomedical Research, Vol. 5, No. 1-2, Jan & May,
2002, pp. 87-89
THERMOSTABILITY OF RECONSTITUTED NEWCASTLE DISEASE VIRUS STRAINS AT 360C
TEMPERATURE
NSSIEN, M.A.S1 AND
D.F. ADENE2
Departments of Veterinary
Anatomy1* and Veterinary Medicine2, University
of Ibadan, Nigeria.
*Author for Correspondence
Received: January 2001
Accepted
in final form: September 2001
Code Number: md02018
Haemagglutination (HA)
test was employed to determine the stability of HA titers of reconstituted
form of Hitchner B1
(B1), LaSota (L) and Komarov (K) strains of Newcastle Disease Vaccine
(NDV) at 360c. The temperature treatment method was through incubation
(in water bath) of the reconstituted vaccines at selected temperature and sequential
sampling of each
vaccine vial for the determination of pre and post temperature exposure HA
titers. Thus, on the basis of a two-step (2log2) decline in titer
as evidence of loss of stability of HA titers (LST), the LST therefore, occurred
at 50th, 24th and 95th hour for BI, L and K
strains, respectively, post temperature exposure. The data, therefore, showed
that the NDV K strain was the most stable at the test
temperature. It is believed that the findings will enhance the understanding
of the potential of this strain in the developed and application of a thermostable
MDV target on village poultry. In rural settings.
Keywords: Haemagglutination,
Thermostability, Newcastle Disease Virus, Reconstituted and
Strain.
** Due to technical difficulties,
some figures and images associated with this article may not be available.
**
INTRODUCTION
Newcastle disease
(ND) is a ribonucleic acid (RNA) virus infection of birds, which can cause
up to 100 percent mortality in susceptible chickens. It is world wide in
distribution (Lancaster, 1966; Lancaster and Alexander, 1975). While many
avian species may become infected, dramatic losses are seen most often in the
domestic fowl and to a lesser extent in turkeys and pheasants (Rosenberger,
et al., 1975; Hanson, 1978; Lancarter, 1981; Vickers and Hanson, 1982; Gordon
and
Jordan, 1982). ND is caused by a paramyxovirus, 100 200nm in diameter and
is classified as avian paramyxovirus type 1 (Alexander, 1980; Nagai et al.,
1976b). The known surface antigens are the haemagglutinin and the enzyme neuramidase
(Nagai., et al., 1976a). The haemagglutinin causes dimer formation with avian
and other red blood cells and the resulting haemagglutination is the basis of
the most commonly used laboratory tests, the identification of viral haemagglutinin
and the demonstration of its specificity using the haemagglutination inhibition
(HI) test (Allan and Gough, 1974).
The stability of the
ND virus is crucial to its
value as a vaccine strain (Allan., et al, 1973). Recent developments on ND
vaccine studies have been focussed on the thermostability of vaccine strains
and its suitability for village operational settings. Available reports on
the only widely known thermostable ND vaccine, the Australian V4 strain, indicate
the need for further work on aspects like the vaccine strain, immunogenecity
and compatibility with feed or vaccine-delivery medium (Copland, 1992; Spradbrow,
1992). This study was designed to provide information on the thermostability
of three reconstituted strains of ND virus at a temperature (360)
which equates that of the tropical environment especially Nigeria with the aim
of establishing and
adopting a vaccination programme suitable for rural poultry.
MATERIALS AND METHODS
Vaccine Strains and Temperature
(360) Treatment
Representative vials
of three common strains of ND vaccine i.e. Hitchner B1 and LaSota (both lentogenic
strains) and Komaro (mesogenic strain) but with varying degrees of immunogenecity
were procured from proprietary outlets
of the manufacturer Nigeria Veterinary Research Institute (NVRI), Vom, Jos, Nigeria.
A 200 dose vial of each strain
of the lyophilized vaccine was reconstituted in 8ml of fresh normal saline
as diluent, recapped and then gently shaken to homogenize before placement
on a rack inside a water
bath at 360C. A pre treatment sampling had been taken so as to
determine the initial (stock) haemagglutination (HA) titer for each vaccine
strain.
Sampling
and Titration
A pre treatment
HA titer for each vaccine strain was determined according to the procedure
described by (Allan and Gough, 1974a; Brugh and Beard, 1980) and slightly modified
through the use of micro-titration tools as
stated below.
Haemagglutination: In the
HA test, the microtitration format was employed,
using U bottom polystyrene microtiter plates, 50µl calibre steel-alloy microdiluters
and 50µl plastic micropipettes. Thus, a two-fold serial dilution was carried
out from 1:2 dilution, in wells A1 and B1 down to 1:1,024 dilution in wells A12
and B12. Finally using the 50µl plastic micropipette, a drop of 0.5 percent
chicken red blood cell (rbc) suspension (as indicator) was
added to each well. The control consisted of a pair of wells with same volumes
of normal saline and rbc but no antigen. The microtiter plate was shaken for
about five seconds on a microshaker, then incubated at room temperature and read
after 30 45 minutes or as soon as the RBCc in the
control wells have settled. The end-point was taken as the dilution in the
last pair of wells that showed complete (100 percent end point)
haemagglutination. The HA titer was the reciprocal of that value as recommended
by (Allan, et al., 1973).
Subsequently, sampling and
titration was done at
selected interval every hour (minimum of 3 hours daily) post temperature exposure,
until the haemagglutinative activity of the investigated antigen(s) titer had
declined by two-step (2log2) titers or more.
RESULTS
The persistence of haemagglutination
(HA) titer was
adopted as a measure of antigen stability. The starting titer and the trend
in decline in titer when the three strains of ND vaccines were maintained at
360c
are shown in figure1.
Hitchner B1 strain with
an initial (stock) titer of
5log2, within a few hours of post temperature exposure (PTE) experienced
onestep decline in titer to 4log2. The said titer was stable uptil
the 50th hour when a two-step decline in titer to 3log2 was
observed and subsequently, complete loss of titer was recorded on the 71st hour
PTE.
Similarly, the L strain with
a pre-temperature
exposure titer of 6log2 soon lose its titer after the first three
hours to 5log2. The antigen was fairly stable at this titer uptil
the 24th hour when a further decline to 3log2 and below
was experienced.
The K strain with a stock
titer of 5log2 was indeed stable at this titer right from the onset
of exposure at 360C
uptil the 28th hour PTE. A 2nd phase of stability at
4log2 was maintained by K strain, which lasted uptil the 95th hour
when a two-step decline in titer was recorded. Complete lose of HA titers were
observed subsequently thereafter.
Using the criterion of decline
by at least two-step
(2log2)in titer as a signal for the loss of stability (LOS), the figure
shows that the LOS started at the 50th, 24th and
95th hour PTE for B1, L and K strains, respectively. This result
thus portrays K strain as the most stable of the three strains of ND vaccine
at
360 C PTE.
DISCUSSION
Considering the trend in
HA titers of the three ND strain from the initial (i.e. pre-temperature exposure)
phase up to the 49th hour, showed that although the L strain had
an initial advantaged titer of 6log2, the rapid decline in titer
portrarys it as a less stable strain than the B1. Meanwhile, the B1 strain
was maintained at a reasonably stable titer of 4log2 from the 5th to
49th hour post-temperature exposure
before experiencing a decline. The sum of results from this study which suggest
the K strain as the most stable is perhaps indicative of reversal of the comparative
stability of the K and B1 strains at lower temperatures (Nssien
and Adene, 2000).
There is a death of current
information on investigation on the thermostability of HA titers of ND virus
strains in
reconstituted form at 360C. Nevertheless, previous report by Frerichs
and Herberts (1974) on International Reference Preparation of lyophilized ND
live vaccine stated that titers were reduced by 26% after one
month at 370C. Panina and Nardelli (1965). Likewise, working with
F strain, reported that storage at 370C for one week produced a
reduction in titer. In this study, the vaccines were investigated in their reconstituted
form at 360C and the results were presented
graphically. Furthermore, K strain portrayed a stability period, which lasted
between 49 95 hours PTE in reconstituted form at a temperature (360C)
equating that which obtains in a country like Nigeria. This, therefore, has
lend allegiance to guaranteed availability of the antigen to rural poultry
prior to loss of stability and
immunogenecity.
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© 2002 - Ibadan Biomedical Communications Group
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