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 2002this 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 animalspulmonary 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.

References

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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

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