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Environmental assessment for licensing bacterial kidney disease vaccine, arthrobacter species nov., live culture in Canada

For Public Release

October 18, 2000

The information in this environmental assessment was current at the time of its preparation. It is possible that the situation may have changed since that time. Please consult the VBS if you have any questions.


Table of Contents

  • Summary
  • 1. Introduction
    • 1.1 Proposed Action
    • 1.2 Background
  • 2. Purpose and need for proposed action
    • 2.1 Significance
    • 2.2 Rationale
  • 3. Alternatives
  • 4. Molecular and biological characteristics of parental and recombinant organisms
    • 4.1 Identification, Sources, and Strains of Parental Organisms
    • 4.2 Source, Description and Function of Foreign Genetic Material
    • 4.3 Method of Accomplishing Genetic Modification
    • 4.4 Genetic and Phenotypic Stability of the Vaccine Organism
    • 4.5 Horizontal Gene Transfer and Potential for Recombination
    • 4.6 Host Range/Specificity, Tissue Tropism and Shed/Spread Capabilities
    • 4.7 Comparison of the Modified Organisms to Parental Properties
    • 4.8 Route of Administration/Transmission
  • 5. Human Safety
    • 5.1 Previous Safe Use
    • 5.2 Probability of Human Exposure
    • 5.3 Possible Outcomes of Human Exposure
    • 5.4 Pathogenicity of Parent Microorganisms in Humans
    • 5.5 Effect of Gene Manipulation on Pathogenicity in Humans
    • 5.6 Risk Associated with Widespread Use of the Vaccine
  • 6. Animal Safety
    • 6.1 Previous Safe Use
    • 6.2 Fate of the Vaccine in Target and Non-Target Species
    • 6.3 Potential of Shed and/or Spread from Vaccinate to Contact Target and Non-Target Animals
    • 6.4 Reversion to Virulence Resulting from Back Passage in Animals
    • 6.5 Effect of Overdose in Target and Potential Non-Target Species
    • 6.6 The Extent of the Host Range and the Degree of Mobility of the Vector
    • 6.7 Safety in Pregnant Animals and to Offspring Nursing Vaccinated Animals
  • 7. Affected Environment
    • 7.1 Extent of Release into the Environment
    • 7.2 Persistence of the Vector in the Environment / Cumulative Impacts
    • 7.3 Extent of Exposure to Non-Target Species
    • 7.4 Behaviour of Parent Microorganisms and Vector in Non-Target Species
  • 8. Environmental Consequences
    • 8.1 Risks and Benefits
    • 8.2 Relative Safety Compared to Other Vaccines
  • 9. Mitigative Measures
    • 9.1 Worker Safety
    • 9.2 Handling Vaccinated or Exposed Animals
  • 10. Monitoring
    • 10.1 General
    • 10.2 Human
    • 10.3 Animal
  • 11. Consultation and Contacts
  • 12. Conclusions and Actions
  • 13. References

Summary

The Bacterial Kidney Disease Vaccine, Renogen, consists of a live lyophilized culture of Arthrobacter sp. nov. that is reconstituted with sterile saline prior to use. This vaccine was evaluated by the Veterinary Biologics Section (VBS), Canadian Food Inspection Agency (CFIA) for licensing in Canada. As part of the requirements for licensing this product in Canada, an 'Environmental Assessment' was conducted and a public document which contains information on the molecular and biological characteristics of the live organism, target animal and non-target animal safety, human safety, environmental considerations and risk mitigation measures prepared.

1. Introduction

1.1 Proposed Action

Veterinary Biologics Section (VBS), Animal Health and Production Division, Canadian Food Inspection Agency (CFIA) is responsible for licensing veterinary biologics for use in Canada. The legal authority for the regulation of veterinary biologics in Canada is provided under the Health of Animals Act and Regulations. Any veterinary biologic manufactured, sold or represented for use in Canada must comply with the requirements specified by the CFIA regarding safety, purity, efficacy and potency of the product. Aqua Health Limited has submitted the following veterinary biologic for licensing in Canada:

  • Bacterial Kidney Disease Vaccine, Arthrobacter sp. nov., Live Culture,
    CFIA File 870VB/R5.0/A8

The Environmental Assessment was prepared by VBS as part of the overall assessment for licensing the above vaccine in Canada. CFIA consulted with the Department of Fisheries and Oceans for the environmental safety aspect of this vaccine.

1.2 Background

The above vaccine containing live Arthrobacter sp. nov. is manufactured by Aqua Health Ltd., Charlottetown, PEI (Canadian Veterinary Biologics Establishment Licence No. 28). The vaccine is currently not licensed in any other country. Licensure is pending in Chile (submitted), USA (pre-submission field trials), and the Faroe Islands (pre-submission field trials). The vaccine is a purified and characterized lyophilized culture of Arthrobacter sp. that is reconstituted with sterile saline diluent prior to use. The vaccine strain can be identified with a specific genetic marker that differentiates cultures of the vaccine strain from Renibacterium salmoninarum, the causative agent of Bacterial Kidney Disease (BKD). The vaccine strain has not been genetically modified.

2. Purpose and need for proposed action

2.1 Significance

BKD is a chronic systemic infection of salmonids caused by a Gram positive organism, Renibacterium salmoninarum, characterized by a granulomatous inflammatory response and a marked affinity for the kidney tissue. Renibacterium salmoninarum is a pathogen that is able to parasitize cells by living intra-cellularly, growing and multiplying in the host fish, and is able to evade the protective mechanisms of the immune system. Disease pathology is associated with the formation of immune complexes consisting of antibody to the Renibacterium salmoninarum p57 antigen and extracellular complexes of Renibacterium salmoninarum p57 antigen and exopolysaccharide antigen.

Arthrobacter sp. nov. was first isolated as a companion culture to Renibacterium salmoninarum from chinook salmon. Research has shown that there is a high (97.5%) 16S RNA sequence homology of Arthrobacter with Renibacterium salmoninarum. There is also a high degree of relatedness in the cell wall carbohydrate and polysaccharide structures. (Gutenberger et al., 1991; Stackebrandt et al., 1988, Stackebrandt et al., 1979).

The label indication for Renogen is as an aid in the prevention of Bacterial Kidney Disease caused by Renibacterium salmoninarum, in healthy salmonids, 10 grams or larger in weight. The vaccine is administered intraperitoneally.

2.2 Rationale

The VBS evaluates veterinary biologics submissions for licensure under the Health of Animals Act and Regulations. General criteria for licensure are (a) the product must be pure, safe, potent and efficacious, (b) vaccine components must be relevant to Canadian disease conditions and (c) the product must be produced and tested in accordance with generally accepted "good manufacturing practices". The Bacterial Kidney Disease Vaccine, Arthrobacter sp. nov., Live Culture meets the general criteria and therefore was evaluated for licensure by VBS.

3. Alternatives

The two alternative options available are: (a) to issue a Canadian Veterinary Biologics Product Licence to Aqua Health Ltd. for Renogen, if all licensing requirements are satisfactory, or (b) not to issue a Canadian Veterinary Biologics Product Licence for the above product, if licensing requirements are not met.

4. Molecular and biological characteristics of parental and recombinant organisms

4.1 Identification, Sources, and Strains of Parental Organisms

The Arthrobacter sp. nov. vaccine strain is a member of the Arthrobacter genus, the members of which are ubiquitous inhabitants of soil and freshwater from various parts of the world and which are non-pathogenic to any living organism.

4.2 Source, Description and Function of Foreign Genetic Material

The organism contains no foreign genetic material.

4.3 Method of Accomplishing Genetic Modification

The organism did not undergo any genetic modification.

4.4 Genetic and Phenotypic Stability of the Vaccine Organism

The vaccine master strain was propagated through a series of back passages in vivo. The host fish (Atlantic salmon, Salmo salar) was injected intraperitoneally with the master seed which was then subsequently recoverable at a rate of at least 50% at one week post-inoculation. The recovered organism was then re-administered to naive fish for five consecutive passages with no demonstration of virulence, pathogenicity, or changes in biochemical characteristics.

4.5 Horizontal Gene Transfer and Potential for Recombination

As the vaccine strain has not been genetically modified, there is no known potential for gene transfer or recombination resulting from genetic manipulation.

4.6 Host Range/Specificity, Tissue Tropism and Shed/Spread Capabilities

(i) The vaccine strain Arthrobacter sp. nov. is not known to be pathogenic to any known species in the freshwater or marine aquatic environment. (ii) Arthrobacter sp. nov. has limited survivability in the kidney tissue of Atlantic and Chinook salmon when artificially introduced by intraperitoneal injection. There are no other known sites of tissue persistence. (iii) The vaccine strain can be sporadically detected in hatchery tank water at a concentration of 100 cells per mL of tank water up to 12 weeks post injection. The bacterial cells are likely released during tissue turnover.

4.7 Comparison of the Modified Organisms to Parental Properties

Not applicable as the organism is not genetically modified.

4.8 Route of Administration/Transmission

Arthrobacter sp. are an important fraction of the normal indigenous flora of soils from various parts of the world and form the most numerous colonies of total aerobic plate counts from soils. Arthrobacter sp. are important as soil decomposers and in soil structure building. They are also common, innocuous inhabitants of sea water. One species, Arthrobacter rhombi sp. nov., has been recovered from organs of Greenland halibut (Reinhardtius hippoglossoides). The route of administration or transmission is likely by contact or by immersion/respiration.

5. Human Safety

5.1 Previous Safe Use

The vaccine organism is a soil bacterium and has not been previously used as a vaccine in humans. The vaccine strain is not pathogenic in humans. The company has performed a study in New Zealand white rabbits and shown that neither lethal nor sub-lethal effects had resulted from immunization with the vaccine strain after two years. This laboratory animal model supported the safety of the vaccine.

5.2 Probability of Human Exposure

Human exposure to the vaccine is likely to be limited to veterinarians, animal technicians, manufacturing staff and testing laboratory staff. In large-scale vaccinations of fish, studies have documented that self-injection occurs once in every 100,000 fish per operator (vaccinator).

5.3 Possible Outcomes of Human Exposure

Arthrobacter sp. nov. is a soil bacterium and is not expected to affect humans. The label on the vaccine advises the operators to seek immediate medical attention in case of self injection. The resulting effects of self-injection would be a localized inflammatory response without septicaemia. The company has performed a study in New Zealand white rabbits and shown that neither lethal nor sub-lethal effects had resulted from immunization with the strain after two years. This laboratory animal model supports the safety of the vaccine.

5.4 Pathogenicity of Parent Microorganisms in Humans

The vaccine strain is not pathogenic to humans according to current knowledge.

5.5 Effect of Gene Manipulation on Pathogenicity in Humans

Not applicable as the organism was not genetically modified.

5.6 Risk Associated with Widespread Use of the Vaccine

The widespread use of the vaccine is not expected to have any public health significance.

6. Animal Safety

6.1 Previous Safe Use

The master seed of the vaccine strain did not cause any pathogenicity in back passage studies, safety studies and/or efficacy studies conducted in Oncorhynchus (Coho and Chinook salmon) and Salmo genera (Atlantic salmon) in laboratory and/or field trials.

6.2 Fate of the Vaccine in Target and Non-Target Species

Arthrobacter sp. nov. is culturable from salmon kidney at a 50% recovery rate one week after inoculation (p.i.), at a 10% recovery rate at two weeks p.i. and is not culturable at four weeks after inoculation. The vaccine strain has a limited survivability in the kidney tissue of Atlantic and Chinook salmon when artificially introduced by intraperitoneal injection. There are no other known sites of tissue persistence. The 16S RNA sequence data supports a grouping of closely related organisms including Arthrobacter and Renibacterium salmoninarum. Only Renibacterium salmoninarum is pathogenic and this is what definitively separates the BKD organism from the taxonomically related organisms at the genus level. The vaccine strain Arthrobacter sp. nov. is not known to be pathogenic to any known species in the freshwater or marine aquatic environment.

6.3 Potential of Shed and/or Spread from Vaccinate to Contact Target and Non-Target Animals

The vaccine is administered to salmonids housed in hatchery tanks. The vaccine strain can be sporadically detected in tank water at a concentration of 100 cells per mL of tank water up to 12 weeks post injection. The manufacturer has indicated that this release of vaccine organism is due to tissue turnover. The manufacturer has demonstrated that horizontal transmission could not be demonstrated when unvaccinated and marked fish were co-habitated. In this particular experiment the vaccinated fish were given 10E6 cells per fish (greater than 10 times the dose).

6.4 Reversion to Virulence Resulting from Back Passage in Animals

The vaccine master strain was propagated through a series of back passages in vivo. The host fish (Atlantic salmon, Salmo salar) was injected intraperitoneally with the master seed which was then subsequently recoverable at a rate of at least 50% at one week post-inoculation. The recovered organism was then re-administered to naive fish for five consecutive passages with no demonstration of virulence, pathogenicity, or changes in biochemical characteristics.

6.5 Effect of Overdose in Target and Potential Non-Target Species

The vaccine organism is safe when delivered at two logs higher than the vaccine dose in the target species. No lethal or sub-lethal effects were observed in the vaccinated population over a two month period at either 12 or 18°C.

6.6 The Extent of the Host Range and the Degree of Mobility of the Vector

The vaccine is not known to be pathogenic to any known species in the freshwater or marine aquatic environment.

6.7 Safety in Pregnant Animals and to Offspring Nursing Vaccinated Animals

The vaccine is intended for use in young fish 10 grams or larger and is not intended for use in broodstock.

7. Affected Environment

7.1 Extent of Release into the Environment

Live Arthrobacter sp. nov. vaccine would only be used in an artificial tank based rearing system. Since the vaccine is specified for use at least four weeks prior to seawater transfer, and the levels that are shed are both low as well as sporadic, it is unlikely that any live microorganism would be excreted in the feces of vaccinated fish to colonize non-target species in the marine environment or to multiply in marine soil sediments. The potential for occasional environmental release through accidental spills or unintended syringe aerosols and contamination of the fish skin around the vaccination site is considered very low due to the artificial tank setting.

7.2 Persistence of the Vector in the Environment / Cumulative Impacts

The risk of dispersal in the environment is low because of the parenteral use of the vaccine and the limited survivability in the target species. The non-pathogenicity of the vaccine strain and lack of horizontal transmission in vivo indicated a low risk of spreading to other non-target species. There are no known or predicted adverse ecological effects of the vaccine strain to the aquatic environment.

7.3 Extent of Exposure to Non-Target Species

Extent of exposure to non-target species is expected to be low since the vaccine administration occurs in an artificial tank based rearing system, where the vaccinates are not in contact with non-target species. The vaccine is not known to be pathogenic to any known species in the freshwater or marine aquatic environment. Since the vaccine is specified for use at least four weeks prior to seawater transfer it is unlikely that any live microorganism is present in the feces of vaccinated fish to colonized invertebrate populations in the marine environment.

7.4 Behaviour of Parent Microorganisms and Vector in Non-Target Species

The vaccine is not known to be pathogenic to any known species in the freshwater or marine aquatic environment.

8. Environmental Consequences

8.1 Risks and Benefits

For any vaccine, risks of vaccination can be attributed to potential adverse reactions. In numerous laboratory and field studies no apparent risk has been posed by the vaccine strain, and the safety of this vaccine in salmonids has been demonstrated. The benefit is that Arthrobacter sp. nov. shares antigenic determinants with the causative agent of BKD, Renibacterium salmoninarum, and is able to confer protection against BKD without causing pathogenesis and disease. The vaccine strain can be isolated from kidney tissue of Atlantic and chinook salmon for a limited period of time post-inoculation and is known not to target other tissues.

8.2 Relative Safety Compared to Other Vaccines

Bacteria belonging to the genus Arthrobacter, are most often isolated from a terrestrial environment and, more recently, have been isolated from Greenland halibut. The vaccine strain is not known to be pathogenic to any known species in the freshwater or marine aquatic environment. There are currently no other licensed vaccines for BKD.

9. Mitigative Measures

9.1 Worker Safety

With injection vaccination using experienced and trained operators (vaccinators), studies indicate that a self injection occurs once in every 100,000 fish for each operator. The vaccine strain is not pathogenic to humans.

9.2 Handling Vaccinated or Exposed Animals

The risk of accidental injection of the operator (vaccinator) with the live vaccine organism Arthrobacter sp. nov. is considered to be minimal. The resulting effect of self-injection would be a localized inflammatory response without septicaemia. No additional precautions are recommended.

10. Monitoring

10.1 General

The vaccine licensing regulations in Canada require manufacturers to report all suspected adverse reactions to CFIA within 15 days of receiving notice from an owner or a veterinarian. Veterinarians may also report suspected adverse reactions directly to the CFIA. On VBS receipt of an adverse reaction complaint, the manufacturer is asked to investigate and prepare a report for the owner's veterinarian and CFIA. If the problem is resolved to the satisfaction of the veterinarian/client, no further action is usually requested by VBS. However, if the investigation is not satisfactory, VBS may initiate regulatory action depending on the case which may include further safety testing, temporary stop sale or product withdrawal from the market.

10.2 Human

No special monitoring of the human safety of the product will be carried out.

10.3 Animal

Veterinarians, vaccinators and hatchery operators should report any suspected adverse reactions to VBS as indicated above. For reporting purposes, adverse reactions are divided into Type 1, 2, and 3 reactions. Type 1 reactions are defined as any systemic adverse reaction, anaphylactic or hypersensitivity requiring veterinary treatment including: persistent fever, recumbency, persistent lethargy, decrease in activity, muscle tremors, shivering, hypersalivation, dyspnea and other respiratory problems, cyanosis, diarrhea, vomiting, colic and other gastrointestinal problems, eye problems, abortions and other reproductive problems and neurological signs. Type 2 reactions are defined as death following vaccination. Type 3 reactions are defined as local persistent reactions such as edema, abscess, granuloma, fibrosis, alopecia, hyperpigmentation and excessive pain at the injection site. Suspected adverse reactions should be reported using the form Notification of Adverse Reactions to Veterinary Biologics (CFIA/ACIA 2205).

11. Consultation and Contacts

Environmental and Human Health

Fisheries and Oceans Canada
200 Kent Street
Ottawa, ON K1A OE6

Manufacturer

Aqua Health Limited
West Royalty Industrial Park
Charlottetown, PEI C1E 2A7

12. Conclusions and Actions

Following this assessment, the Product Licence held by Aqua Health Ltd., will be amended to allow the production and distribution of the following product in Canada:

  • Bacterial Kidney Disease Vaccine, Arthrobacter sp. nov., Live Culture,
    CFIA File 870VB/R5.0/A8

13. References

Bergey's Manual of Determinative Bacteriology, Ninth Edition, 1994, Williams and Wilken, Baltimore.

Griffiths, S.G., K.J. Melville, and K. Salonius. (1998) Reduction of Renibacterium salmoninarum culture activity in Atlantic salmon following vaccination with avirulent strains. Fish & Shellfish Immunology 8:607-619.

Gutenburger, S.K., S.J. Giovannoni, K.G. Field, J.L. Fryer, and J.S. Rohovec (1991). A phylogenetic comparison of the 16S RNA sequence of the fish pathogen, Renibacterium salmoninarum, to Gram-positive bacteria. FEMS Microbiology Letters 77:151-156.

Osorio, C.R., J.L. Barja, R.A Hutson, and M.D. Collins. 1999. Arthrobacter rhombi sp. nov. isolated from Greenland halibut (Reinhardtius hippoglossoides). Intl. J. of Systemic Bacteriol. 49:1217-1220.

Stackebrandt, E., and F. Fiedler (1979) DNA-RNA homology studies among strains of Arthrobacter and Brevibacterium. Arch. Microbiol. 120:289-295.

Stackebrandt, E., U. Wehmeyer, H. Nader, and F. Fiedler (1988). Phylogenetic relationship of the fish pathogenic Renibacterium salmoninarum to Arthrobacter, Micrococcus, and related taxa. FEMS Microbiology Letters 50:117-120.


Prepared and revised by:

Veterinary Biologics Section
Animal Health and Production Division
Canadian Food Inspection Agency