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Environmental Assessment for the Use of Fowl Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector

For Public Release

August 9, 2010

Prepared by:

Canadian Centre for Veterinary Biologics
Terrestrial Animal Health Division
Canadian Food Inspection Agency

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. If you have any questions, please consult the Canadian Centre for Veterinary Biologics.


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 Micro-Organisms 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 Acquisition to Virulence Resulting from Back Passages 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
  • 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 Micro-Organisms 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. Consultations and Contacts
  • 12. Conclusions and Actions
  • 13. References

Summary

Fowl Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector consists of a live turkey herpesvirus modified by the introduction of two gene sequences from a fowl laryngotracheitis virus. This vaccine is indicated for subcutaneous administration to one-day-old chickens.

The vaccine was evaluated by the Canadian Centre for Veterinary Biologics (CCVB) of the Canadian Food Inspection Agency (CFIA). As part of the requirements for licensing the product in Canada, an environmental assessment was conducted. This environmental assessment is a public document which contains information on the molecular and biological characteristics of the live recombinant organism, target animal and non-target animal safety, human safety, environmental considerations and risk mitigation measures.

This environmental assessment is based on information provided by the manufacturer (Intervet, Inc., Millsboro, Delaware, U.S. Veterinary Biologics Establishment License No. 165A), as well as information independently obtained by the CCVB reviewer.

1. Introduction

1.1 Proposed Action

The Canadian Centre for Veterinary Biologics (CCVB), Terrestrial Animal Health Division, Canadian Food Inspection Agency (CFIA) is responsible for regulating the use of veterinary biologics in Canada under the legal authority 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 the safety, purity, potency, and efficacy of the product. Intervet Inc. (Millsboro, Delaware, U.S.) through Intervet Canada Corp. (Kirkland, Que.) has submitted the following vaccine for licensing in Canada:

  • Fowl Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector (INNOVAX-ILT), USDA Product Code 16J1.R1, CFIA File 800VV/F10.0/I6.2

This environmental assessment was prepared by CCVB as part of the overall assessment for licensing the above vaccine in Canada. It is an update to a previous environmental assessment which was prepared to allow restricted use of the product in Canada prior to full licensing.

1.2 Background

Fowl Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector is manufactured by Intervet, Inc., Millsboro, Delaware (U.S. Veterinary Biologics Establishment License No. 165A), and is currently licensed for sale in the U.S. This recombinant vaccine consists of a live herpesvirus of turkeys (HVT) modified by the insertion of genetic material from an infectious laryngotracheitis virus (ILTV). The vaccine is a stand-alone product. CCVB has previously licensed a different vaccine based on a recombinant turkey herpesvirus construct containing portions of another virus for use in chickens.

Fowl laryngotracheitis is a contagious respiratory disease primarily affecting chickens, which causes decreased production and performance in commercial layer and broiler flocks (Tripathy, 2006). It has been reported in many countries worldwide, and is currently listed in Canada as an immediately notifiable disease. It is indigenous to Canada but is tracked for export certification purposes. Control of the disease is achieved by biosecurity and vaccination. Traditionally, modified live ILTV vaccine products of chicken-embryo origin and tissue-culture origin have been used. It has been shown that these chicken-embryo-culture vaccines can revert to virulence when transmitted horizontally between chickens (Guy et al., 1991). More recently, it has been suspected that strains of the virus present in conventional vaccines have been responsible for disease outbreaks due to this acquisition of virulence and the tendency of the virus to cause latent / persistent infections in the host (Oldonie et al., 2007).

Marek's disease (MD) is a viral oncogenic (neoplastic) disease of poultry found worldwide. It is considered to be ubiquitous and is presumed present in all flocks except those maintained under stringent pathogen-free conditions (Powell, 1986). The virus is extremely difficult to eradicate from flocks for several reasons. The virus spreads very quickly between individuals (whether vaccinated or not), who become infected for long periods of time, shedding the organism into the environment where it can persist for months (Fadly, 2006). Vaccination prior to exposure does not prevent infection, but rather reduces shedding of the virus (Fadly, 2006) and prevents the onset of lymphoma (Karaca et al., 2003). The use of attenuated or non-pathogenic MD viral strains as vaccines in chicken flocks worldwide has reduced mortality from 30-60 percent to less than five percent (Powell, 1986).

2. Purpose and Need for Proposed Action

2.1 Significance

The label indication for INNOVAX-ILT Fowl Laryngotracheitis-Marek's Disease Vaccine is for the vaccination of one-day old chickens as an aid in the prevention of MD and fowl laryngotracheitis.

2.2 Rationale

CCVB evaluates veterinary biologic product submissions for licensure under the Health of Animals Act and Regulations. The general criteria for licensing are as follows: a) the product must be pure, safe, potent and efficacious; b) the product must be licensed in the country of origin; c) vaccine components must be relevant to Canadian disease conditions; and d) the product must be produced and tested in accordance with generally accepted "good manufacturing practices." This U.S. origin vaccine meets these general criteria and presented no unacceptable importation risk, and therefore was evaluated for licensing by CCVB.

Since this product is a biotechnology-derived vaccine of Class II category, CCVB is required to perform an environmental assessment before its potential release into the environment. This environmental assessment has been conducted to determine whether the release of this product presents safety risks to animals, public health or the environment.

3. Alternatives

The two alternative options being considered are as follows: a) to issue a Permit to Import Veterinary Biologics to Intervet Canada Corp. for the importation of INNOVAX-ILT Fowl Laryngotracheitis-Marek's Disease Vaccine if all licensing requirements are satisfactory; or b) 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 proposed vaccine qualifies as a Class II Veterinary Biologic (live expression vectors that carry one or more foreign genes that code for immunizing antigens and/or immune stimulants). The recombinant vaccine construct consists of protein-expressing gene sequences from a fowl laryngotracheitis virus inserted into an HVT viral vector.

4.2 Source, Description and Function of Foreign Genetic Material

Gene sequences from ILTV were selected from those likely to stimulate protective immunity in chickens. Details of the actual sequences are on file at CCVB.

4.3 Method of Accomplishing Genetic Modification

Details of the methods used in the construction of the recombinant organism are on file at CCVB. The master seed virus was tested for extraneous agents, purity and safety according to tests described in the U.S. Code of Federal Regulations (9 CFR). These data have been reviewed and are on file at CCVB.

4.4 Genetic and Phenotypic Stability of the Vaccine Organism

In vitro and in vivo testing using Southern blot and RT-PCR analysis revealed genetic and phenotypic stability of the recombinant organism through five passages in a cell line and five back passages through the target species, the chicken.

4.5 Horizontal Gene Transfer and Potential for Recombination

There exists a theoretical low risk of gene transfer and recombination between the recombinant virus and other avian herpesviruses that may be present in the host, as reports can be found in the literature of experimental recombination between herpesviruses within co-infected cells in vitro as well as in vivo.

Virulence is one characteristic of a virus that may change (increase or decrease) if recombination occurs. The particular strain of herpesvirus which is the backbone of the recombinant organism is considered avirulent, but there exists a theoretical low risk of a novel recombinant organism being produced whose virulence is increased, as seen in studies with other herpesviruses. It must be noted that an MDV-2 strain and an HVT strain have been combined in existing multivalent vaccines which have been used for years with no indication of acquiring virulence. The risk of acquisition of virulence of either organism via recombination is considered to be very low for this product.

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

In vivo studies conducted by the manufacturer determined host range, tissue tropism, and shed / spread capabilities of the recombinant organism. Replication of the recombinant organism was detected in chickens and turkeys only, and the virus did not replicate in other avian species such as pigeons, pheasants, and quail. Dissemination of the recombinant organism in vivo was also shown to be no different from that of the parental HVT. No transmission of the recombinant organism was seen to occur from vaccinated chickens to other in-contact, unvaccinated chickens. Though no other avian species were included in the shed / spread study, HVT vaccines have previously been shown to spread to in-contact turkeys. Another study was performed which showed that the recombinant organism did not replicate in bovine, monkey, or hamster cell lines in vitro, and was thus unlikely to be able to replicate in mammalian species, including humans.

4.7 Comparison of the Modified Organisms to Parental Properties

The host range, tissue tropism, and shed / spread capabilities of the recombinant organism are expected to be the same as the parental HVT vaccine strain.

4.8 Route of Administration / Transmission

Avian herpesviruses are mainly spread in shed feather dander, but wild-type HVT has been shown to not spread readily between chickens infected early in life (Cho and Kenzy, 1975). Like its parental organism, the recombinant organism did not spread from vaccinated chickens to in-contact unvaccinated chickens. Wild-type HVT is known to persist in the environment. In vitro studies performed by the manufacturer showed environmental persistence of the recombinant organism as a cell-associated virus to be restricted to under 48 hours. The route of administration proposed for the use of this vaccine is subcutaneous (in one-day old chickens).

5. Human Safety

5.1 Previous Safe Use

Though the recombinant organism has not been used previously in Canada, it has been licensed for use in the U.S. since 2007. Parental HVT has been safely used for decades in vaccines to control MD. The ILTV gene insert is not expected to be a safety concern since it consists of only protein-expressing gene sequences of the complete virus. Diluents and preservatives in this product have all been used previously in other products with no safety concerns.

5.2 Probability of Human Exposure

Human exposure to the vaccine itself (as a cell-associated virus) is likely to be limited to veterinarians, poultry operators and animal technicians during vaccination. Consistent with what is observed with other recombinant HVT vaccines, the organism may be shed from vaccinated animals to a minimal degree, thus exposure of these personnel to mature virus in shed feather follicle epithelium is also possible.

Since literature suggests that HVT may replicate in these avian species for extended and possibly indefinite periods, as it is a herpesvirus which is capable of persistent infection (Calnek and Witter, 1991; Cho 1974), there is a chance that individuals working in abattoirs will also be exposed to the recombinant virus.

Exposure to humans through the consumption of meat from vaccinated birds will be reduced by the fact that the recombinant HVT + ITL virus is localized to lymphocytes associated with visceral organs and feather follicles, and not the tissues humans predominantly consume as meat. Moreover, even if trace amounts of the recombinant virus were present in chicken meat, studies have shown that the vast majority of ingested nucleic acid is efficiently degraded in the human digestive tract (Jonas et al., 2001).

Human exposure is thus possible, though it is not expected to have any adverse health effects.

5.3 Possible Outcomes of Human Exposure

There are no reports of HVT or ILTV causing infection in any species other than chickens and turkeys. Even in these species, the virus may replicate, but it does not cause clinical disease. The recombinant organism has been shown to be unable to replicate in mammalian cell lines. Exposure of humans to this vaccine by any route, including inadvertent injection, is expected to cause no serious adverse effects.

5.4 Pathogenicity of Parent Micro-Organisms in Humans

As above, the parental HVT and donor ILTV organisms are non-pathogenic to humans and are unlikely to replicate in mammalian species.

5.5 Effect of Gene Manipulation on Pathogenicity in Humans

The ILTV gene insert is not expected to result in any change in pathogenicity of the HVT backbone.

5.6 Risk Associated with Widespread use of the Vaccine

No significant public heath issues are expected to result from widespread use of the vaccine.

6. Animal Safety

6.1 Previous Safe Use

The recombinant organism was found to be safe in experimental studies in chickens (intended target species) at vaccinal and 10-times vaccinal doses, as well as in several non-target species, including turkeys.

INNOVAX-ILT was administered subcutaneously to more than 200,000 day-old chickens in the field safety trials conducted by the manufacturer, and the vaccine has been licensed in the U.S. since 2007.

Parental HVT has been safely used for decades in vaccines to control MD in chickens.

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

There are no reports of wild-type HVT causing clinical disease in any species. Back-passage studies in chickens indicate that the recombinant organism is unlikely to acquire virulence.

Studies conducted in target and non-target species demonstrate that the host range and tropism of the recombinant organism vaccine strain were not altered from the parental HVT strain. These studies also show that the recombinant organism has been shown to persist for 35 days (end of experimental period) in chickens, and the literature suggests that it may replicate in chickens for extended and possibly indefinite periods, as it is a herpesvirus vector which is capable of persistent infection (Calnek and Witter, 1991; Cho 1974).

No studies have been conducted to investigate the long-term safety of HVT/ILT-138 in the target species (chickens) or in birds of non-target yet host species (turkeys).

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

In the literature, HVT has been shown to be shed sporadically in the feather dander of vaccinated birds (Zygraich and Huygelan, 1972; Cho, 1974). The recombinant organism did not spread to any in-contact chickens, though it is possible that, as with other HVT vaccines, the organism can be shed and spread to in-contact turkeys, the host species of the parental organism. No studies have been conducted to investigate the transmission of HVT/ILT-138 from chickens to in-contact birds of non-target species.

6.4 Acquisition to Virulence Resulting from Back Passage in Animals

Testing indicated that the master seed virus is genetically and phenotypically stable up to passage MSV+5, and is free of extraneous agents as per 9 CFR 113.300. Backpassage studies in the target species, chickens, have not shown any acquisition of virulence of the recombinant organism.

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

Manufacturer's studies reported no gross lesions or detrimental effects on survivability in one-day old chickens injected with 10 times the vaccinal dose.

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

HVT has a very narrow host range, restricted to turkeys and chickens. The recombinant organism is shed in a manner similar to its parental organism and, while not horizontally transmitted between chickens, it is theoretically possible that it may be spread from vaccinated chickens to in-contact turkeys.

7. Affected Environment

7.1 Extent of Release into the Environment

The recombinant organism is shed in feather dander of vaccinated chickens. 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 barns are cleaned out, or through the vented air. HVT-contaminated litter and air (dust) appear to be capable of infecting turkeys (Witter and Solomon, 1971).

Potential for occasional limited environmental release through accidental spills, unintended syringe aerosols or contamination of the vaccination site also exists during routine use of this vaccine.

7.2 Persistence of the Vector in the Environment / Cumulative Impacts

Studies have shown that HVT virus may be recovered from the environment of vaccinated flocks for over eight weeks post-vaccination and possibly longer (Islam and Walkden-Brown, 2007). MD virus shed in feather follicles has been shown to persist in the environment for eight to 12 months (Schat, 1985).

Manufacturer's studies have shown the recombinant organism (as a cell-associated virus) to persist in the environment for no more than 48 hours. No studies have been conducted to demonstrate the duration (past four weeks) of mature vaccine virus shed in the feather follicles, or the stability/persistence of the virus in the natural environment (e.g. when shed in feather follicle epithelial cells, presence in barn dust and litter, in outdoor manure piles after cleaning of barns, under conditions of sun exposure, etc.).

Herpesviruses are typically inactivated by UV light from the sun (Lytle and Sagripanti, 2005). However, MD viruses in dried feathers and poultry dust have been reported to remain infectious for up to a year (Jurajda and Klimes, 1970; Schat, 1985).

7.3 Extent of Exposure to Non-Target Species

The very limited host range of herpesviruses reduces the risk of spread to non-target mammalian species. Turkeys kept in proximity to chicken flocks vaccinated with this organism are at possible risk for exposure if they are in contact with bedding or exhaust from the chickens' area, or with the chickens themselves. No studies have been conducted to investigate the transmission of HVT/ILT-138 from chickens to in-contact birds of non-target species or the long-term safety of HVT/ILT-138 in non-target species.

Even though studies performed by the manufacturer indicate that the recombinant vaccine virus is non-pathogenic to turkeys, precautionary measures should be followed to reduce the potential for spread of the virus to turkey populations. For this reason, the manufacturer has agreed to add a special warning to the labelling of INNOVAX-ILT indicating that the product should not be administered to chickens with probable direct or indirect exposure to turkeys.

7.4 Behaviour of Parent Micro-Organisms and Vector in Non-Target Species

A study conducted by the manufacturer has suggested that the recombinant organism is safe in non-target species, including turkeys, when administered directly. No practical method exists to determine with certainty the behaviour of the organism over a long period of time, should it become established in a natural turkey population.

8. Environmental Consequences

8.1 Risks and Benefits

Most importantly, the recombinant HVT/ILT-138 virus does not appear to cause the side effects or induction of clinical signs associated with some of the very immunogenic yet virulent live ILT vaccines. The vaccine appears to be effective at inducing immunity even when administered to maternal antibody positive chickens, thus allowing early vaccination (at one day of age) and protection against field challenge.

The primary risk identified for INNOVAX-ILT, as with any vaccine, can be attributed to potential adverse reactions. Manufacturer's safety studies have demonstrated the product to be generally safe in field safety studies conducted in chickens.

An additional, yet theoretical, risk relates to its potential to spread to turkey populations (and potentially other Galliformes) and consequently persist in the environment. It is important to note, however, that no pathogenicity in turkeys is expected for the recombinant HVT/ILT-138 virus, even if it does spread to turkeys. In an experiment performed by the manufacturer, turkeys infected with recombinant HVT/ILT-138 virus (through either direct inoculation or contact with vaccinated chickens) appeared healthy throughout the duration of the study (up to approximately four weeks of age), akin to turkeys infected with wild-type HVT. The Canadian labelling for INNOVAX-ILT also carries a precautionary statement that measures should be taken to avoid contact between vaccinated chickens and turkeys, which should further mitigate the proposed risk of spread to turkeys.

In conclusion, there is a hypothetical risk that turkeys might inadvertently become infected with the vaccine virus, but since there is no evidence to indicate that such an occurrence would be detrimental to turkeys, the benefits of the vaccine in promoting chicken animal health are believed to outweigh the proposed risk of vaccine virus spread to turkeys.

8.2 Relative Safety Compared to other Vaccines

The vaccine is comprised of an avirulent live MD virus and gene sequences of a fowl laryngotracheitis virus, both of which are non-pathogenic. The vaccine has been shown to be as safe as conventional MD vaccines in target and non-target species. The lack of potential for acquisition of virulence and the absence of adjuvants typical of killed virus vaccines are additional positive safety features of this vaccine.

9. Mitigative Measures

9.1 Worker Safety

Veterinarians, poultry operators, and animal technicians could be exposed to the live recombinant organism during vaccination. As was discussed in Section 5 above on human safety, such exposure is not expected to be a safety concern. Moreover, since the vaccine does not contain any adjuvant, the risk of clinical problems due to accidental self-injection of adjuvant (oil) is removed. Nonetheless, measures should be taken to protect personnel from exposure, as stated on the product insert.

9.2 Handling Vaccinated or Exposed Animals

Since chicks reared in a biosecure facility are not often handled directly by humans, and poultry workers typically employ precautionary biosafety measures, exposure through handling vaccinated chicks is not expected to be great. However, poultry workers could become exposed to the vaccine virus through dust and air inside barns that might be contaminated with virus shed through feather dander. Again, the recombinant virus is not believed to be pathogenic to humans.

10. Monitoring

10.1 General

The vaccine licensing regulations in Canada require manufacturers to report all serious, significant 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 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 / client, no further action is usually requested by CCVB. However, if the outcome of the investigation is not satisfactory, CCVB may initiate regulatory action depending on the case, which may include further safety testing, temporarily stopping sales of the product, or product withdrawal from the market.

10.2 Human

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

10.3 Animal

Veterinarians should report any suspected adverse reactions to 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

Importer

Intervet Canada Corp.
16750, route Transcanadienne
Kirkland, QC H9H 4M7

Manufacturer

Intervet, Inc.
29160 Intervet Lane, Box 318
Millsboro, Delaware USA 19966-0318

12. Conclusions and Actions

Based on our assessment of the available information, CCVB has concluded that the importation and use of Fowl Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live Marek's Disease 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 of Intervet Canada Corp. may be amended to allow the importation and distribution of the following product in Canada:

  • Fowl Laryngotracheitis-Marek's Disease Vaccine, Serotype 3, Live Marek's Disease Vector (INNOVAX-ILT), USDA Product Code 16J1.R1, CFIA File 800VV/F10.0/I6.2

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

Baigent, S.J., Smith, L.P., Nair, V.K., Currie, R.J. (2006). Vaccinal control of Marek's disease: Current challenges and future strategies to maximize protection. Veterinary Immunology and Immunopathology 112:78-86.

Calnek, B.W., Witter, R.L. (1991). Marek's Disease. In Diseases of Poultry, edited by B.W. Calnek et al., Iowa State University Press, Iowa. pages 342-385.

Cho, B.R. (1974). Horizontal transmission of turkey herpesvirus to chickens IV. Maturation in the feather follicle epithelium. Avian Diseases 19:136-141.

Cho, B.R., Kenzy, S.G. (1975). Horizontal transmission of turkey herpesvirus to chickens. 3. Transmission in three different lines of chickens. Poultry Science 54:109-15.

Fadly, A.M. (2006). Marek's Disease. In The Merck Veterinary Manual, 9th edition, edited by C.M. Kahn, S. Line, S.E. Aiello, Merck & Co., Inc., New Jersey.

Guy, J.S., Barnes, H.J., Smith, L. (1991). Increased virulence of modified-live infectious laryngotracheitis vaccine virus following bird-to-bird passage. Avian Diseases 35:348-355.

Islam, A., Walkden-Brown, S.W. (2007). Quantitative profiling of the shedding rate of the three Marek's disease virus (MDV) serotypes reveals that challenge with virulent MDV markedly increases shedding of vaccinal viruses. The Journal of General Virology 88:2121-2128.

Jonas, D. A., Elmadfa, I., Engel, K.-H., Heller, K. J., Kozianowski, G., König, A., Müller, D., Narbonne, J. F., Wackernagel, W. and Kleiner, J. (2001). Safety considerations of DNA in food. Annals of Nutrition & Metabolism 45(6):235-254.

Jurajda, V. and Klimes, B. (1970). Presence and survival of Marek's disease agent in dust. Avian Diseases 14(1):88-190.

Karaca, G., Anobile, J., Downs, D., Burnside, J., Schmidt, C. (2004). Herpesvirus of turkeys: microarray analysis of host gene responses to infection. Virology 318:102-111.

Lytle, C. D. and Sagripanti, J.-L. (2005). Predicted inactivation of viruses of relevance to biodefense by solar radiation. Journal of Virology 79(22):14244-14252.

Oldoni, I., Garcia, M. (2007). Characterization of infectious laryngotracheitis isolates from the US by polymerase chain reaction and restriction fragment length polymorphism of multiple genome regions. Avian Pathology 36:167-176.

Powell, P.C. (1986). Marek's Disease – A world poultry problem. World Poultry Science Journal 14:205-218.

Schat, K.A. (1985). Characteristics of the virus. In Marek's Disease, edited by L.N. Payne, Martinus Nijhoff Publishing, Boston.

Tripathy, D.N. (2006). Infectious Laryngotracheitis. In The Merck Veterinary Manual, 9th edition, edited by C.M. Kahn, S. Line, S.E. Aiello, Merck & Co., Inc., New Jersey.

Witter, R. L. and Solomon, J. J. (1971). Epidemiology of a herpesvirus of turkeys: Possible sources and spread of infection in turkey flocks. Infection and Immunity 4(4): 356-361.

Zygraich, N., Huygelen, C. (1972). Inoculation of one-day-old chicks with different strains of turkey herpesvirus. II. Virus replication in tissues of inoculated animals. Avian Diseases 16:793-798.