2002: Vaccine causes
antibiotic-resistant disease.
Disease now spreads almost instantly across the world and we have reason
to be concerned with our own vaccines. Reading may be slightly
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Dr.Waisees Yeung
State Key Laboratory for Biocontrol Biopharmaceutical Center
Zhongshan University, Guangzhou 510275)
020-84113793 020-84110976
Report on
the detection of antibiotic resistant Proteus mirabilis contaminant in
Intervet canine vaccine product
Waisees Yeung(Yang Dewei)1,2
Zhu Qishun1 He Jianguo1 Liu Fuan2
1. (State
Key Laboratory for Biocontrol and Biopharmaceutical Center, Zhongshan
University, Guangzhou 510275)
2. (South
China Agricultural University Veterinary College Agriculture Ministry
Poultry Husbandry and Disease Control Key Laboratory, Guangzhou 510640)
Abstract From
January to 31 May 2002 this laboratory received and tested a total of
101 samples of the same Lot Number of Intervet canine vaccines which
endusers complained as inducing death in dogs after immunization. The
result revealed that six of the samples contained Proteus mirabilis that
could cause death in canines. Molecular virology, antigenicity,
antibiotic sensitivity of contaminating bacteria, and vaccine vial
vacuum tightness testing were also done in this study.
Keywords
Intervet canine vaccine; Proteus mirabilis; canine virus
Category number
S858.292.01
1 Material and Methods
1. Material
Intervet canine vaccine
(within the expiry date) from various units such as zoos, animal clinics
and dog kennels in different provinces, cities and autonomous regions.
PCR primers for amplification of various canine viral gene fragments
kept in this laboratory. Conventional reagents for molecule biology work
and bacteria antibiotic sensitivity testing provided by this laboratory.
Experimental animals bought from the PLA Military Medical University.
1.2 Methods
1.2.1
The stopper of each vaccine vial was wiped with iodine tincture then
with 75% alcohol to remove the iodine and allowed to dry in a laminar
flow sterile hood. The vial stopper was pierced with a hypodermic needle
attached to a graduated syringe containing double-distilled water, which
is sucked into the 3 ml vial.
1.2.2
DNA template was prepared with 0.5ml of vaccine buffer solution and used
for nested PCR amplification of gene fragments (for procedures see
reference 2, 5, 6 and 8).
1.2.3
A bacterial inoculation loop was used to take up some vaccine buffer
solution and streaked on solid LB culture medium. Bacterial colonies
that appeared were subjected to cloning, identification and antibiotic
sensitivity testing. Bacteria isolated from six Intervet vaccine samples
were cloned and inoculated intranasally and orally into six groups (A1
to A6) of experimental animals, each group consisting of 5 mice and 2
virus-free SPF puppies. The control group also had 5 mice and 2
virus-free SPF puppies._ Then the following steps are carried out.
1.2.4 From the
vaccine diluted with buffer solution, 0.2 ml was aspirated diluted 10
times sterile-filtered through 0.22 mu millipore membrane then
inoculated onto WCK and MDCK cell lines. Also 0.1ml vaccine in buffer
solution was diluted 10 times and, after passing through 0.22 mu
millipore membrane, was subjected to plaque purification see reference
2,and 5.
1.2.5 Purified virus
was tested by nested PCR to identify the virus species, after which the
stock virus was stored and inoculated into a total of six groups (B1 to
B6) of experimental animalseach group consisting of 5 mice and_ two
virus-free SPF puppies. The control group also had 5 mice and 2
virus-free SPF puppies.
1.2.6 Bacteria
isolated from six Intervet vaccine samples were cloned and inoculated
intranasally and orally into six groups (C1 to C6) of 1.2.6al animals,
each group consisting of 5 mice and 2 virus-free SPF puppies. The
control group also had 5 mice and 2 virus-free SPF puppies.
2. Results
2.1 The stoppers of
all the 101 vials of Intervet canine vaccine were pierced with a
hypodermic needle attached to a graduated syringeafter which 1.38 to
1.61 ml of double-distilled water was sucked in.
2.2 Intervet canine
vaccines were subjected to nested PCR amplification of gene fragments,
and except for those live virus (CAV, CDV, CPV, CPIV) listed on the
packing box showing positive DNA electrophoretic bands other viruses all
showed negative result.
2.3 Inoculum from 6
of the Internet canine vaccine produced bacterial colonies. Cloned
bacterial culture were inoculated into 6 groups (A1 to A6) of
experimental animals and the principals developed pulmonary symptoms
and enteritis, all dying within 5 to 11 days. In contrast,
none of the control animals showed similar symptoms, remaining lively
and active. The isolated organism was identified as Proteus mirabilis
and found to be resistant to a variety of antibiotics.
2.4 Purified viruses
isolated from the vaccines were inoculated into 6 groups (B1 to B6) of
experimental animals; none showed clinical symptoms but from day 4
postimmunization antibodies to the corresponding viruses could be
detected in their bodies. Control animals showed no abnormal signs nor
could antibodies to CAV, CDV, CPV, CPIV be detected.
2.5 Intranasal and
oral inoculation of 6 groups (C1 to C6) of experimental animals
pulmonary symptoms and enteritis occurred and within 6 to 11 days all
died. No such symptoms were found in the control animals, all remaining
healthy, lively and active.
3.
Discussion
Intervet vaccine vials
could automatically suck in double-distilled water, attesting that the
vaccine vials maintained an up to standard vacuum state.
Bacteria could be cultured
from 6 Internet vaccine samples, and were identified as Proteus
mirabilis. Six groups (A1 to A6) of experimental animals inoculated with
the isolated bacteria developed pulmonary symptoms and enteritis and
gradually progressed to death. This situation was similar to that of
various units where disease developed and death occurred after use of
Intervet canine vaccine.
Amplification of various
canine virus gene fragment with nested PCR revealed CAV, CDV, CPV, CPIV
positive electrophoretic DNA bandsand four days after immunization with
purified virus isolated from the vaccine, homologous antibodies could be
detected in the vacinatees. This indicated that the vaccine definitely
contained the live viruses listed on the package, and could exert their
expected action.
Proteus mirabilis can
spread by horizontal and contact transmission to other mammals and it
exhibits resistance to various antibiotics. This is the reason why many
units after using Intervet canine vaccine encountered failure in
antibiotic treatment of dogs developing disease subsequently. Proteus
mirabilis can wrought severe ecological pollution, so it is of great
importance to pretest vaccines to insure its absence before vaccination.
Inasmuch as samples in this
study came from endusers of Intervet canine vaccine, and Intervet Hong
Kong Co.Ltd. refused to supply the identical Lot Number 425-1018 of
canine vaccine for testing this study could only be a partial reflection
and might not be able to give an assessment of the overall situation.
http://www.thedogplace.org/Vaccines/Deadly-Bacteria-in-Vaccine-0202_China.asp
References
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in the development of tumour genetically-engineered vaccines. Medicine
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(2) Zhu Xinchan, Zhang
Yong, Liao Xiangru. 1998. PCR technological strategy J Biotechnology
Bulletin 3 29-33
(3) Wu Hongzhuan, He
Dongsheng, Yang Dewei, Chen Feng, Qin Zhifeng, Wang Xing, Zhu Daozhong,
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Dongsheng, Yang Dewei, Chen Feng, Qin Zhifeng, Wang Xing, Zhu Daozhong,
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(5) Waisees Yeung, Yang
Dewei, Song Yanhua, Liu Fuan, 2000a Application of nested PCR to reveal
canine parvovirus in MDCK cell lines. Journal of South China
Agricultural University 3 21 81-83
(6) Waisees Yeung (Yang
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(7) Waisees Yeung (Yang
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