Environmental assessment for the Canadian licensing of Huvepharma's Clostridium perfringens type A vaccine, Live Salmonella Vector

July 15, 2021

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 Canadian Centre for Veterinary Biologics if you have any questions.

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Note

The Clostridium perfringens type A vaccine, Live Salmonella Vector was submitted for licensure in two different formulations: frozen (USDA Code 1U11.R0, CCVB File # 800BA/C1.0/H17) and lyophilized (USDA Code 1U11.R1, CCVB File #800BA/C1.1/H17). This environmental assessment was done for the licensure of the frozen version. However, as both formulations are derived from the same Master Seed bacteria, the study conducted on the Master Seed Bacteria applies to both products. It also follows that all conclusions and conditions of this environmental assessment apply to both products.

Summary

Clostridium perfringens type A vaccine, Live Salmonella Vector is a recombinant attenuated live Salmonella vectored vaccine for use in chicks day-of-age and older for active immunization against the bacterium C. perfringens type A, the causative agent of necrotic enteritis. The vaccine is recommended to be administered by coarse spray to day-of-age chickens and a second vaccination may be administered in drinking water to chickens at 11 days of age. The vaccine was evaluated by the Canadian Centre for Veterinary Biologics of the Canadian Food Inspection Agency for licensing in Canada. As part of the requirements for licensing this product in Canada, an "Environmental Assessment" was conducted, and a public document containing information on the molecular and biological characteristics of the live genetically modified organism, target animal and non-target animal safety, human safety, environmental considerations, and risk mitigating measures was prepared.

1. Introduction

1.1 Proposed Action

The Canadian Centre for Veterinary Biologics (CCVB) of the 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 the Health of Animals Regulations. Any veterinary biologic manufactured, sold or represented for use in Canada must comply with the requirements specified by the CFIA regarding the safety, purity, potency, and efficacy of the product. Huvepharma, Inc., Lincoln, Nebraska has submitted an application to license the following vaccine in Canada:

Clostridium perfringens type A vaccine, Live Salmonella Vector,
USDA product code 1U11.R0, CCVB file no. 800BA/C1.0/H17

1.2 Background

Clostridium perfringens type A vaccine, Live Salmonella Vector is manufactured by Huvepharma, Inc. (US Veterinary Biologics Establishment License No. 605), and is currently licensed for sale in the United States under USDA product code 1U11. RO. This vaccine is a recombinant attenuated live Salmonella vectored vaccine expressing C. perfringens toxin X (where X represents the first toxin) and toxin Y ( where Y represents the second toxin). The vaccine is intended for use in healthy chicks one day of age and older for active immunization against the bacterium C. perfringens type A, the causative agent of necrotic enteritis (NE).

Necrotic enteritis occurs commonly in rapidly growing strains of broiler chickens between two and six weeks of age. The peak risk period for broiler chickens to get NE is 3 weeks of age, which correlates with declining titers of maternal antibodies. This disease causes considerable losses to the poultry industry, both in terms of morbidity and mortality of birds resulting in significant economic and environmental costs. Clinical signs include depression, dehydration, diarrhea, ruffled feathers and lower feed intake. The gross lesions of the small intestine range from thin and friable walls to frank and extensive necrotic lesions, with a typical roughened necrotic epithelial surface. Mortality can be as high as 40%. Additionally, the subclinical form of the disease is associated with reduced feed conversion and economic losses. NE is a complex and multi-factorial disease (Moore, 2016; Prescott et al., 2016). The factors affecting development of the disease include a diet high in protein, loss of maternal immunity, immunosuppression, and coccidial infection (Eimeria spp.) (Moore, 2016). Historically, feeds that contain antibiotics have been used for treatment or prevention of NE. However, an increasing number of C. perfringens poultry isolates have been shown to be resistant to at least one of the commonly used antibiotics. In recent years, the emphasis on reduced use of antimicrobials have led to a trend for increase in the incidence of NE. The prophylactic use of antibiotics has been banned in poultry production in Europe (Butaye et al., 2003). There is a need for safe and effective control measure for NE in poultry.

2. Purpose and need for proposed action

2.1 Significance

The labelling for C. perfringens type A vaccine, Live Salmonella Vector vaccine indicates that the product is recommended for vaccination of chickens one (1) day of age or older for active immunization against C. perfringens type A, the causative agent of NE.

2.2 Rationale

The CCVB evaluates veterinary biologic product submissions for licensure under the Health of Animals Act and the Health of Animals Regulations. The general criteria for licensing are as follows: a) the product must be pure, safe, potent and efficacious; b) vaccine components must be relevant to Canadian disease conditions; c) foreign products must be licensed in the country of origin; and d) the product must be produced and tested in accordance with generally accepted "good manufacturing practices." This US origin vaccine meets these general criteria and thus was evaluated for licensing by the CCVB.

3. Alternatives

The two alternative options being considered are:

  1. to issue a Permit to Import Veterinary Biologics to Huvepharma Canada Corporation, Inc. (Ottawa, ON) allowing the importation of Clostridium perfringens type A vaccine, Live Salmonella Vector vaccine, if all licensing requirements are satisfactory; or
  2. not to issue a Permit to Import Veterinary Biologics 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 vaccine strain is a recombinant Salmonella enterica serovar Typhimurium bacterial host vector carrying a plasmid, which includes a construct encoding the C. perfringens type A toxin X and toxin Y. The vaccine strain is designated as Recombinant Attenuated Salmonella Vaccine (RASV-Cp/01). The RASV-Cp/01 contains a bacterial vector, which is derived from a chicken-passaged isolate of a highly virulent S. enterica serovar Typhimurium isolated from an infected horse. Vaccines derived from the parent strain have been previously licensed for use in poultry in USA and Canada.

4.2 Source, description and function of foreign genetic material

The DNA sequences corresponding to the C. perfringens toxins were synthesized in vitro. The toxin X is considered an important virulence factor for the development of NE in chickens and is also known to stimulate protective immunity (Prescott JF et al., 2016; Jiang Y et al., 2015). The toxin Y is no longer considered an essential virulence factor of NE in chickens (Timbermont et al., 2011), however its precise role or mechanism in stimulating protective immunity is not fully known (Cooper KK et al. 2009; Wilde S et al. 2019; Jiang Y et al., 2015).

4.3 Method of accomplishing genetic modification

The DNA sequences corresponding to the C. perfringens toxin X and toxin Y were inserted into an expression plasmid. The bacterial vector is engineered with various deletion and deletion-insertion mutations from parent S. enterica serovar Typhimurium strain, which have contributed to the following properties: 1) delayed regulated lysis; 2) delayed regulated full length LPS synthesis; and 3) delayed regulated antigen expression. The plasmid was inserted into the bacterial vector to produce RASV-Cp/01.

RASV-Cp/01 was fully characterized at each step in its construction. Standard methods for DNA isolation, restriction enzyme digestion, DNA cloning and use of PCR for construction and verification of vector were used. The plasmid construct was evaluated by DNA sequencing and its ability for synthesis of the heterologous proteins was verified by gel electrophoresis and western blot analyses. Details of methods of genetic modification and construction of plasmid and RASV are on file with the CCVB.

4.4 Genetic and phenotypic stability of the vaccine organism

The manufacturer verified genetic and phenotypic stability of the Master Seed bacteria (MSB) up to 10 passage (MSB + 10) in vitro. The following parameters were used for determining genetic stability: plasmid retention by the bacterial vector, restriction digest comparison of plasmid extracts, sequencing of plasmid extract and confirmation of proteins (toxin X and Y) expression (by western blot) in pre-MSB, MSB, and MSB+10. The phenotype for MSB and MSB + 10 passages was verified by Gram staining, slide agglutination test, API 20E strip test and dependency on a specific carbon source. These studies showed stability of the plasmid carrying C. perfringens type A toxins X and Y up to 10 passages in vitro. The data support the genetic and phenotypic stability of the recombinant bacteria.

A back passage study was conducted in specific pathogen free (SPF) chickens to assess reversion to virulence and genotypic and phenotypic stability of the vaccine organism. The RASV-Cp/01 MSB, at the lowest passage level (approximately 108 CFU of MSB), was orally inoculated in day-of-hatch chicks. Ceca and reproductive tissues from euthanized birds were collected and homogenized. For the second passage, day-of-hatch birds were inoculated with the tissue homogenate from the first passage birds. A few days after inoculation, some birds were euthanized for tissue collection and remaining birds were kept for observation. The procedure was repeated for a total of 5 passages. The RASV-Cp/01 was successfully recovered from tissue (ceca and reproductive tissues) homogenates of the first passage birds, confirmed by bacterial culture. It was not isolated from any subsequent passages except from one bird in the third passage. This one specific bird died two days after inoculation and RASV-Cp/01 was successfully recovered. The manufacturer conducted further studies and attributed the cause of death to early chick mortality. Other than one mortality, no clinical abnormalities were observed in any bird throughout the study. Together, the genotypic and phenotypic characterization of the isolate from the back passage study showed that in vivo passages did not results in detectable alteration in the vaccine organism.

4.5 Horizontal gene transfer and potential for recombination

It is well known that bacterial species can exchange genetic material among each other by three known mechanisms, namely transformation, transduction and conjugation. The studies submitted by the manufacturer showed that the potential for horizontal gene transfer or recombination is low as the vaccine organism will not be found in large quantities either in the host or in the environment. This is because the vaccine organism is dependent on the presence of certain carbon sources for its growth, which are not readily available in vivo or in the environment. It only persists transiently in target and non-target animal species, which makes it an unlikely donor or recipient.

If recombination were to occur, it would likely not introduce a new gene to the environment because donor genes as full length sequences have been found in the environment as a normal part of the C. perfringens genome. During the genetic engineering of RASV Cp/01, several genes were deleted and others were placed under the control of a promoter which requires a specific carbon source for gene expression. As a result, while the likelihood of recombination between the vaccine strain and a wild-type bacterium is considered to be low, if it occurs it could be detrimental to the recipient organism if it confers such dependency for proper gene expression.

The manufacturer used an in silico approach to analyse the MSB for presence of antimicrobial resistance genes. The results showed that the Salmonella vector at genomic level does not contain any acquired AMR genes suggesting that RASV-Cp/01 may not serve as a donor of genes that result in increasing AMR in birds/animals or in the environment.

4.6 Host range/specificity, tissue tropism and shed/spread capabilities

To determine tissue tropism and shedding and spreading, SPF chickens at day-of-hatch were inoculated with the MSB, RASV-Cp/01. Multiple tissues were collected from vaccinated birds at various time points post-inoculation. The vaccine organism colonized both intestinal and systemic organs. The peak tissue tropism was observed at 1 day post-inoculation in the ceca and reproductive tissue. The shedding in the feces was not examined. The vaccine was recovered from the tissues for 6 days post-inoculation from the vaccinated chickens but not thereafter. The short term persistence has been attributed to highly attenuated nature of the vaccine strain and dependency on carbon sources for its growth and virulence, which are not readily available in vivo or in the environment. The tissue persistence of the vaccine strain was shorter than that of the parent strains or other mutant strains that were shown to persist in the ceca of chicks for at least 5 weeks (the last time point evaluated) after vaccinations (Hassan and Curtis 1990; Porter et al. 1993; Hassan & Curtiss 1996).

The vaccinated birds were commingled with unvaccinated birds to study spread of vaccine organism to naïve birds. The vaccine was recovered, sporadically on days 2, 3, 5, and 6 from the pooled cloacal swab samples of the unvaccinated sentinel chickens that were housed with the vaccinated chickens. However, RASV-Cp/-01 was not cultured from organs (brain, spleen, reproductive organ and ceca) of naïve unvaccinated chickens at day 3, 7 and 14 post inoculation. The manufacturer did not make an attempt to culture vaccine organism from organs of naïve unvaccinated chickens on days 1, 2, 5 and 6 post infection. The study supports limited transmission or spread to unvaccinated chickens in close proximity of vaccinated chickens.

4.7 Comparison of the modified organisms to parental properties

The vaccine RASV-Cp/01 contains bacterial vaccine vector carrying a plasmid, which includes a construct encoding the C. perfringens type A toxins X and Y. The bacterial vaccine vector is derived from S. enterica serovar Typhimurium strain, which is a chicken-passaged isolate of a highly virulent strain. The parent strain has undergone genetic modification resulting in a phenotype with the following properties – delayed regulated lysis, delayed regulated full length lipopolysaccharide (LPS) synthesis and delayed regulated antigen expression. In brief, asd and murA genes (encoding cell wall components) were deleted from the chromosome and inserted in a plasmid under the control of a promoter, resulting in dependency for growth. Additional genetic alterations included the deletion of phosphomannose isomerase leading to additional dependency for the synthesis of full length LPS. In the absence of these, the vaccine organism will lyse and die or is unable to synthesize full length LPS making it susceptible to complement mediated lysis. All these features make it a highly attenuated strain in vivo compared to the parental strain. The RASVs based on similar concept have been discussed elsewhere in details (Clark-Curtiss and Curtiss, 2018, Kong W et al., 2012).

Additional mutations in the vaccine organism, RASV-Cp/01, preclude it from forming biofilms. The absence of these mutations in the parental strain allows it to form biofilms, which serve as a persistent source of infections and also contribute to resistance to antibiotic treatment. The donor genes added within the plasmid DNA do not influence its virulence properties because donor gene expression is regulated in such a manner that expression only occurs in the absence of a carbon source which is conversely required for the growth of the Salmonella vector (host).

4.8 Route of administration/transmission

The C. perfringens type A vaccine, Live Salmonella Vector vaccine is to be administered by coarse spray to day of age chickens and a second vaccination may be administered in drinking water to chickens at 11 days of age.

5. Human safety

5.1 Previous safe use

The RASV-Cp/01 vaccine organism has never been directly inoculated into humans. There are no reported or known cases of diseases or adverse events in humans that have been associated with the use of the vaccine in laboratory or clinical study settings. An RASV vector producing Streptococcus pneumoniae surface protein A and sharing genetic modifications in common with RASV-Cp/01 has been experimentally used in human volunteers. The vaccine was given orally and was demonstrated to be safe (Frey et al., 2013).

Due to the specific genetic modification and low biosafety risks associated with the genetically modified recombinant vaccine, the National Institute of Health (NIH) Office of Biotechnology has permitted all studies, even immunizations, with RASV strains developed in Dr. Roy Curtis lab to be conducted in BSL-1/ABSL-1 containment conditions, in commercial agricultural settings and even in outpatient human volunteers. The RASV-Cp/01 vaccine strain was developed with similar technology in Dr. Roy Curtis's laboratory.

5.2 Probability of human exposure

Human exposure to the vaccine organism itself is likely to be limited to employees in the manufacturing facility, veterinarians, animal technicians, poultry farm operators and employees at the abattoir. The poultry farm operators who are administering the vaccine or work in close proximity of the birds are at the greatest risk. However, the actual risk to persons administering the vaccine at the farms will be minimal since the spray application will be conducted in a closed cabinet and the booster dose will be applied through drinking water, which reduces aerosolization of the vaccine. The probability of human exposure from vaccinated birds will be limited as the vaccine organism has limited replication and can persist only for a short duration of time.

5.3 Possible outcomes of human exposure

The safety of the vaccine organism has not been evaluated in humans. Based on the genetic modification of the vaccine organism, human exposure to the RASV-Cp/01 is not expected to be a significant health concern and will likely not lead to any detectable clinical signs. The manufacturer's studies showed that the vaccine organism is sensitive to various antibiotics; Gentamicin, Nalidixic Acid, Tetracycline, Oxytetracycline, and Ceftiofur, Ceftriaxone, Amoxicillin/Clavulanic acid, Ciprofloxacin, Chloramphenicol and Ampicillin. These commonly used antibiotics can be effectively used to limit adverse effects of accidental human exposure.

5.4 Pathogenicity of parent microorganisms in humans

Human exposure to wild type Salmonella enterica serovar Typhimurium does occur in the field, through food sources or in laboratory settings. Salmonella infection in humans commonly causes gastrointestinal symptoms such as diarrhea, abdominal cramps, vomiting, nausea, fever, etc. These symptoms usually last for 4-7 days, and most individuals recover without treatment. Children aged 5 years and under, older adults, pregnant women or people with weakened immune systems are at higher risk for contracting serious illness. Humans can spread the disease for as long as they shed the bacterium in their feces. Additional information is available at the Public Health Agency of Canada.

5.5 Effect of gene manipulation on pathogenicity in humans

The pathogenicity of RASV-Cp/01 vaccine organism is not known in humans. It has never been inoculated into humans. As per the manufacturer, the personnel involved in construction and testing of the vaccine at the Washington University in St Louis MO, Arizona State University in Phoenix AZ, University of Florida in Gainesville FL, Southern Poultry Research Group in Athens, GA, Curtiss Healthcare in Alachua, FL, and Huvepharma in Lincoln, NE, have not reported any adverse events to date.

Genetic modification of the parent strain resulted in a phenotype that is dependent on certain carbon sources for growth and for the synthesis of full length LPS. In the absence of these the vaccine organism will lyse and die or is unable to synthesize full length LPS making it susceptible to complement mediated lysis. Thus the vaccine organism is highly attenuated and it is anticipated that the vaccine organism may not be pathogenic in humans.

5.6 Risk associated with widespread use of the vaccine

There are no identified risks associated with the widespread use of the vaccine. Due to the genetic modification of the vaccine strain, the risk of accidental exposure and subsequent widespread establishment of the vaccine strain is low.

6. Animal safety

6.1 Previous safe use

The field safety study for both the one-dose and two-dose regimens was conducted in 244,268 commercial broiler chickens by the manufacturer. The chickens were vaccinated as per manufacturer recommendations and were monitored for 21 days and 35 days post-challenge for the one-dose and two-dose regimens, respectively. The mortality rates of vaccinated flocks were similar to the control mortality rate at all sites. There were no vaccine related adverse events reported in the study.

The manufacturer also conducted a reversion to virulence study in the target species (refer to section 4.4). No adverse reactions were observed throughout the passages or for 21 days following the inoculation of last group of birds with the tissue homogenates derived from the previous group (passage 4) of birds. These studies support the safety of the vaccine in the target population.

Safety of the recombinant vaccine was also tested in the following non-target species: turkeys, quail, pigeons, mice and calves. Inoculated animals and birds were observed for 21 days post-inoculation. No clinical signs, gross lesions, or adverse reactions were observed in any of the non-target species, thus providing evidence of the vaccine's safety in case of accidental exposure of these non-target species.

The parent strain of RASV-Cp/01, is the same parent strain from which other globally distributed commercial vaccines for poultry are derived and they have history of safe use in the USA and Canada. RASV vaccines with similar genetic modifications and carrying genes from different pathogens have been previously used safely in mice and chickens (Wilde S et al. 2019; Jiang Y et al., 2015; Wang S et al., 2010; Ameiss K et al., 2010; Ashraf S et al., 2011; Juarez-Rodriques MD et al., 2012; Wang S et al., 2013)

6.2 Fate of the vaccine in target and non-target species

The manufacturer demonstrated that the vaccine strain could be detected in some tissues of vaccinated chickens for 6 days post-inoculation but it was not found to persist beyond that. This can be attributed to the fact that vaccine strain is highly attenuated and depends on carbon sources for its growth and virulence, which are not readily available in vivo.

The vaccine organism was not detected by culture in fecal/cloacal swabs and tissues of all the non-target species except quails (detected in cloacal swabs until 7 days post-inoculation only). The manufacturer's studies showed that the vaccine strain does not persist long term in target and non-target species.

6.3 Potential of shed and/or spread from vaccinate to contact target and non-target animals

The manufacturer conducted a study to determine shedding and spreading of RASV-Cp/01 vaccine by commingling naïve unvaccinated chicks with vaccinated chicks. The vaccine was recovered on 1, 2, 3, 4, 5- and 6-days post-inoculation from the tissues of vaccinated chickens but not thereafter. The study did not investigate feces of vaccinated birds. The vaccine was recovered, sporadically on days 2, 3, 5, and 6 from the pooled cloacal swabs of the unvaccinated sentinel chickens that were housed with the vaccinated chickens. The vaccine organism was not found in cloacal swabs of the sentinel birds beyond 7 days post-exposure. Furthermore, there was no detectable vaccine in tissue samples from brain, spleen, reproductive organs and ceca collected from unvaccinated sentinel chickens on days 3, 7, and 14 post-inoculation.

The manufacturer also quantified cultures for every positive tissue (ceca, reproductive tissue, spleen or brain) by measuring most probable number (MPN) method, which indicated that a very small number of organisms were present in each tissue. The MPN method only provides an approximation for the number of organisms present. Based on the study, the RASV-Cp/01 can spread from vaccinated birds to unvaccinated chickens and likely to non-target animals, however the shed and spread is only for a short duration of time.

6.4 Reversion to virulence resulting from back passage in animals

The manufacturer performed a back passage study to assess the reversion to virulence and genotypic and phenotypic stability of the vaccine construct. The studies demonstrated that five back passages in chickens does not alter phenotypic and genotypic characteristics of the RASV-Cp/01. The vaccine organism did not cause morbidity and mortality at any of the passage levels indicating that attenuated properties of the vaccine were retained during in vivo passages and it is not likely to revert to virulence.

6.5 Effect of overdose in target and potential non-target species

The vaccine has been administered orally to day-of-hatch chickens at doses approximately 29 times the dose in the commercial vaccine. The vaccine was found safe in the target species and did not result in any mortality or local/generalized adverse reactions when birds were observed at 5, 7 and 12 days post-inoculation. The overdose study was not conducted in non-target species, however based on other studies conducted in the non-target species and the genetic engineering of the vaccine organism, it is not anticipated that an overdose of vaccine would cause any significant hazard to the non-target animals/birds health. If inadvertently the vaccine is given in higher doses by parenteral routes, then these animals/birds can be treated with appropriate antibiotics as the vaccine strain is sensitive to antibiotics.

6.6 Extent of the host range and degree of mobility of the vector

The parent organism of the vaccine strain is capable of infecting a broad range of hosts including pigs, calves, horses, etc. There is no evidence to suggest that the host range of the vaccine strain is different from the parent organism. Studies conducted by the manufacturer shows that the vaccine strain may infect but it does not persist in the target or non-target species/host and shed and spread capabilities are limited. The vaccine organism was isolated from tissues and/or cloacal swabs of inoculated chickens and quails but not from other avian species, for example turkeys and pigeons. The exact mechanism or reason for these differences are unknown.

7. Affected environment

7.1 Extent of release into the environment

The vast majority of vaccinated chickens will be housed indoors in biosecure facilities, and thus will have little direct exposure to the environment. However, limited release of the vaccine organism may occur when poultry houses are cleaned out, or through the vented air. Moreover, even if released, the vaccine organism will likely not amplify as it requires special supplements to replicate, that are absent or available in a limited quantity in commercial poultry settings and outside.

7.2 Persistence of the vector in the environment and cumulative impacts

The manufacturer performed studies to show that vaccine organism has a low potential for survival in the environment. The survivability of the vaccine organism was evaluated in a poultry house simulated environment setting consisting of poultry litter. The titers of vaccine organism in fresh litter rapidly declined within 6 hours and were not detectable after 96 hours, suggesting that the vaccine organism may not survive or accumulate in poultry houses where the majority of this vaccine will be used. Furthermore, the vaccine organism is readily inactivated by common disinfectants including Virkon, bleach (1%) and ethanol, which are routinely used for biosecurity purposes in poultry farms/houses.

In general, naturally occurring strains of Salmonella are already widely distributed in the environment and nature. It is unlikely that RASV-Cp/01 could compete with wild type Salmonella for the common environmental niches and persist.

7.3 Extent of exposure to non-target species

The host range of the vaccine organism is expected to be similar to the parent strain, which is known to infect or replicate in various species; therefore, there is a chance of spread of vaccine organism to non-target species. However, the extent of actual exposure of non-target species is expected to be curbed by the fact that vaccine administration predominantly occurs in housed domestic poultry without access to the outside. There is some risk of exposure to other avian species and wild birds if they are either closely housed or if they gain access to the poultry housing where vaccine is used. The majority of the poultry houses in Canada follow basic biosecurity measures that should reduce the risk of spread to non-target species.

7.4 Behaviour of parent microorganisms and vector in non-target species

The parental strain is known to be pathogenic in poultry, mice, horses, pigs, calves and other species (Zhang X et al., 1999; Curtiss and Hassan, 1996; Blankenship LC, 1991). In contrast, the vaccine strain is highly attenuated and is not expected to be a safety hazard in non-target species as shown by experimental studies conducted in turkeys, quail, pigeons, mice and calves.

8. Environmental consequences

8.1 Risks and benefits

NE and related subclinical diseases are known to cause significant economic losses to the Canadian broiler industry. C. perfringens type A is also associated with foodborne diseases in humans. Currently, veterinarians use preventative antibiotics to control C. perfringens induced NE, which increases the potential for anti-microbial resistance and subsequent ineffectiveness of antibiotics for treatment of human and animal ailments. There are no licensed vaccines against C. perfringens available to the Canadian poultry industry. The potential benefit of this vaccine is that it might help to improve animal health by protecting chickens against diseases caused by C. perfringens type A and subsequently may reduce zoonotic risk. The availability of vaccine will provide an alternative to the antibiotics and will improve poultry health and promote agriculture production.

The manufacturer's studies indicate that the vaccine organism has limited ability to replicate and shed in chickens and quails. There is a possibility that other avian or animal species not investigated by the manufacturer may also shed vaccine organism similar to chickens and quails. However, the risk of spread and persistence in environment is low as manufacturer studies have shown that the vaccine organism depends on multiple carbon sources for optimal fitness. The potential for horizontal gene transfer or recombination is highly unlikely due to the genetic modifications in the vaccine strain.

Overall, the benefits of the vaccine in promoting chicken health are believed to outweigh any risks associated with impact on environment by using the vaccine in chickens.

8.2 Relative safety compared to other vaccines

There are currently no other C. perfringens type A vaccines available for use in Canada. There were no vaccine related adverse events reported in the study conducted by the manufacturer. Currently, two live licensed poultry Salmonella vaccines based on the same parent strain are available in the USA and Canada. To our knowledge, there have been no significant adverse impacts on the environment as a result of their usage. We expect RASV Cp/01 to be safe for both the birds and environment.

The immune response to the bacterial host vector (Salmonella) has not been evaluated and there is a possibility that it may interfere with the Salmonella monitoring programs or other Salmonella vaccines used in poultry. Due to this reason the manufacturer has added a warning statement to the label "Interference of the vaccine with pullorum-typhoid testing has not been assessed and may occur".

9. Mitigative measures

9.1 Worker safety

The vaccine will be manufactured at a USDA licensed establishment (Est. 605) in Lincoln, NE. Individuals working with the vaccine such as employees in the production facility, as well as veterinarians, animal technicians and personnel administering the vaccine, and poultry farm operators, can be exposed to the genetically modified RASV-Cp/01 vaccine organism. The personnel administering the vaccine have the greatest exposure and risk. The poultry farm operators must follow label recommendations for preparation and use of vaccine. The direct vaccine exposure will be minimal since the spray application will be conducted in a closed cabinet and the booster dose applied through drinking water, which reduces aerosolization of the vaccine and human exposure. The poultry farms must implement and follow basic biosecurity principles to reduce direct exposure from vaccinated birds at the farm. If the vaccine is spilled, it should be inactivated by commonly available cleaning and disinfection agents. The limited shed and spread from vaccinated birds will further reduce human exposure through bird excretions. The vaccine does not contain any adjuvant and there is no risk of clinical problems due to accidental self-injection of adjuvant.

9.2 Handling vaccinated or exposed animals

The vaccine is labelled to be used in day-of-age chicks and with an optional revaccination at 11 days of age. As per the manufacturers studies the vaccine is shed only for a few days post-vaccination, therefore it is not expected to be shed beyond 18 days of age. The vaccinated chicks will be reared in a biosecure facility and at a younger age they are not often handled directly by humans. Moreover, poultry workers typically are expected to employ precautionary biosafety measures, therefore, there is limited exposure during handling of vaccinated chicks. Timely removal of fecal material from vaccinated birds is important to reduce spread and exposure of vaccine organism to poultry handlers and non-target species. If needed common disinfecting agents can also be used. No additional special precautions are needed to handle vaccinated or exposed animals/birds.

10. Monitoring

10.1 General

The vaccine licensing regulations in Canada require manufacturers to report all significant suspected adverse reactions to the CFIA within 15 days of receiving notice from an owner or a veterinarian. Veterinarians may also report suspected adverse reactions directly to the CFIA. If an adverse reaction complaint is received by the CCVB, the manufacturer is asked to investigate and prepare a report for the owner's veterinarian and the CFIA. If the problem is resolved to the satisfaction of the veterinarian or client, usually, no further action is requested by the CCVB. However, if the outcome of the investigation is unsatisfactory, the CCVB may initiate regulatory action, depending on the case, which may include further safety testing, temporary stoppage of product sales, 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 producers should report any suspected adverse reactions to the CCVB as indicated above. Suspected adverse reactions should be reported using Form CFIA/ACIA 2205 – Notification of Suspected Adverse Events to Veterinary Biologics.

11. Consultations and contacts

Manufacturer: Huvepharma Inc., 520 West Industrial Lake Drive, Lincoln, Nebraska USA 68528

Importer: Huvepharma Canada Corporation, Inc., 275 Slater Street, Suite 900, Ottawa ON K1P5H9

12. Conclusions and actions

Based on our assessment of the available information, the CCVB has concluded that the importation and use of Clostridium perfringens type A vaccine, Live Salmonella Vector, in Canada would not be expected to have any significant adverse effect on the environment, when manufactured and tested as described in the approved Outline of Production, and used according to label directions.

Following this assessment and the completion of the Canadian veterinary biologics licensing process, the Permit to Import Veterinary Biologics held by Huvepharama Canada Corporation, Inc may be amended to allow the importation and distribution of the following products in Canada:

Clostridium perfringens type A vaccine, Live Salmonella Vector (Frozen version - USDA Code 1U11.R0, CCVB File #800BA/C1.0/H17 and Lyophilized version - USDA Code 1U11.R1, CCVB File #800BA/C1.1/H17)

All serials of this product must be released by the USDA prior to importation into Canada. All conditions described in the Permit to Import Veterinary Biologics must be followed with respect to the importation and sale of this product.

13. References

Moore R (2016). Necrotic enteritis predisposing factors in broiler chickens Avian Pathology, 45, 275–281.

Prescott JF, Parreira VR, Gohari IM, Lepp D, Gong J (2016). The pathogenesis of necrotic enteritis in chickens: what we know and what we need to know: a review, Avian Pathology, 45:3, 288-294, DOI: 10.1080/03079457.2016.1139688.

Butaye P, Devriese LA, Haesebrouck F (2003). Antimicrobial growth promoters used in animal feed: effects of less well known antibiotics on Gram-positive bacteria. Clin. Microbiol. Rev. 16:175–188.

Jiang Y, Mo H, Willingham C, Wang S, Park JY, Kong W, Roland KL, R. Curtiss R III (2015). Protection against necrotic enteritis in broiler chickens by regulated delayed lysis Salmonella vaccines. Avian Dis. 59: 475–485

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