Risk Analysis 

Marek's Disease Vaccine, 

Serotype 1, Live Virus 

USDA Product Code: 16L1.RO (Unlicensed) 

Updated 

Merial, Inc. 

U.S. Veterinary License Number 298 

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Serotype 1, Live Virus, Code: 16L 1.RO (Unlicensed) 

Table of Contents 

I. Introduction .......................................................................................................................................................... 6 

A. Objective ......................................................................................................................................................... 6 

B. Proposal ........................................................................................................................................................... 6 

II. Description of the Regulated Biological Agent Construction ................................................................................ 7 

A. The Backbone Biological Agent ........................................................................................................................ ? 

B. Donor Biological Agents and Donor DNA or Genes ....................................................................................... 1 0 

C. Construction and Characterization of the Regulated Biological Agent ........................................................... 13 

Ill. Biological Properties or Virulence for the Regulated Biological Agent used for Master Seed ............................. 21 

A. What are the known phenotypic characteristics or effects of the Regulated Biological Agent identified as 

Master Seed? (e.g., expresses envelope gene/sequence, lacks thymidine kinase activity, lower titer in 

MA 105 cells, etc.) ............................................................................................................................................ 21 

B. What are the virulence characteristics of the Regulated Biological Agent used for the Master Seed? .......... 21 

C. Is the Regulated Biological Agent used for the Master Seed virulent for target animals, non-target animals? .. 

........................................................................................................................................................................ 21 

D. Do the Donor DNA sequences enhance the virulence or the ability of the Master Seed to survive in the target 

animals, non-target animals? .......................................................................................................................... 23 

E. Discuss the potential for horizontal gene transfer or recombination of the Regulated Biological Agent.. ....... 24 

F. Describe the shed/spread capabilities of the Regulated Biological Agen\... ................................................... 26 

G. Discuss the expected environmental impact or survivability of the Regulated Biological Agent. and provide 

available scientific evidence ........................................... ............................................................................ ... 28 

IV. Risk Assessment. ........................................... ..................................................................................... . ...29 

A. Procedure ............................................. . . ................................................................... 29 

B. Hazard identification ...................................................................................................................................... 30 

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Serotype 1, Live Virus, Code: 16L 1.RO (Unlicensed) 

1. Animal safety .............................................................................................................................................. 30 

2. Public health safety .................................................................................................................................... 34 

3. Environmental Safety ................................................................................................................................. 34 

C. Release Assessment Prior to a Proposed Field Safety Trial .......................................................................... 36 

D. Risk characterization ..................................................................................................................................... .40 

1. Likelihood ratings ....................................................................................................................................... 40 

2. Consequence ratings ................................................................................................................................. 40 

3. Degree of certainty rating .......................................................................................................................... .40 

4. Calculating the expected risk ..................................................................................................................... .40 

5. Risk ratings ....................................................................................... , ....................................................... .41 

V. Risk Management Recommendations ............................................................................................................... 44 

A. Procedure .................................................................................................................................................... .44 

B. Recommendations .......................................................................................................................................... 44 

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Page4 of74 

Tables Page 

Table 1: Sequence of the primers used in PCR reactions and expected size (in bp) obtained after PCR 15 

performed with different primer pairs on the- candidate, the-parental 

strain and a mixture of- and Rispens. 

Table 2: Calculating the Expected Risk 45 

Table 3: Risk Ratings 46 

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Appendices 

I 

Risk Analysis 

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Page 5 of74 

2. Purchase, H.G. and R.L. Witter(\986),. "Public health concerns from human exposure to oncogenic avian 

herpes viruses: A revieW' J. Am. Vet Med Ass.\89:1430-6 

References 

1. Afonso et al., J. Viral., 74:3815-31, 2000 

2. Borenstein and Davidson, J Viral Methods, 82:119-127, 1999 

3. Bublot M and Sharma J, Marek's Disease. An Evolving Problem, p.\68-185, 2004 

4. F. Davison & V. Nair (Eds.), Marek's Disease. An Evolving Problem, p. 17-31, 2004 

5. H. G. Purchase et al., Am. J. Vet. Res., 32:1811-1823, 1971 

6. Hertig et al., Virology 235:367-76, 1997 

7. lsfort R.J. et al., Virology, 302:125-33, 1994 

8. Jarosinski KW et al, 84:7911-7916, 2010 

9. Jarosinski KW, et al, J. Viral. 81:10575-10587, 2007 

10. Jones ei al., J. Viral., 70:2460-2467, 1996 

11. Jones et al., Proc Natl Acad Sci, 90:3855-9, 1993 

12. Kaleta, E. F. and Neumann, Diseases of Poultry, 12th Edition, p. 461, 2008 

13. Kung et al., Microbiollmmunol., 171:1-25, 1991 

14. Laidlaw and Skinner, J. Gen. Viral., 85:305-22, 2004 

15. Moore et al, Avian Dis., 44:827-41, 2000 

16. Morgan et al, Avian Dis. 36:858-870, 1992 

17. Okamuraa H. et al, Vaccine, 20: 483-489, 2002 

18. Osterrieder and Vautherot, Marek's Disease. An Evolving Problem, p.\7-31, 2004 

19. Purchase, H.G. and R.L. Witter, J. Am. Vet Med Ass. 189:1430-6, 1986 

20. Reddy et al., PNAS, 99:7054-9, 2002 

21. Rispens et al, Avian Dis., 16:108-25, 1972 

22. Schat & Nair, Diseases of Poultry, 12th Edition, p. 452-514, 2008 

23. Singh et al., J. Viral, 77:5855-62, 2003 

24. Spatz SJ and Schat K.A., Virus Gene, 42:331-8, 2011 

25. Spatz SJ, Petherbridge L, Zhao Y and Nair V., J Gen Viral, 88:1080-1096, 2007 

26. Sun et al., Virology, 397:270-6, 2010 

27. Thiryetal., Rev. Med. Virol.\5:89-103, 2005 

28. Thiry et al., Vet. Microbial., 113:171-177,2006 

29. Witter et al., Avian Dis., 41:407-21, 1997 

30. Zhang and Cui, Sci China C Life Sci, 48:81-8, 2005 

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I. Introduction 

A. Objective 

Risk Analysis 

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May 19, 2014 

Page 6 of74 

1. Identify where the Regulated Biological Agent was constructed and where the product will be made, 

tested and manufactured. Address the available level of containment. 

The parental virus is an RN strain of a Marek's Disease Serotype 1 vaccine with the insertion of the long 

terminal repeat (LTR) of the reticuloendotheliosis virus (REV). This construct was developed atThe virus was transferred to 

Merial Select, Gainesville, GA --- - --------

- ------- - -- -- --- ------ --

-

2. Provide a brief (one sentence) description of the Regulated Biological Agent. 

The Regulated Biological Agent contains 

Marek's disease vaccine (RN strain), serotype 1 which comprises a LTR sequence of REVB. Proposal 

1. What is the intended use of the product? 

a. Species: Avian; day-old chickens 

b. Proposed claim: Marek's Disease Vaccine, Serotype 1, Live Virus, Code 16L 1.RO (Unlicensed) is 

indicated for use in healthy day-old chickens as an aid in the prevention of Marek's disease 

caused by very virulent Marek's disease virus 

c. Geographic area: United States (all states), 

d. Route of administration: Subcutaneous 

e. Brief description of the expected safety profile: The Marek's Disease Vaccine is expected to elicit 

an immune response in the chicken to Marek's Disease Virus without causing gross observable 

lesions as directed by 9CFR113.330 (c) (3). 

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11. Description of the Regulated Biological Agent Construction 

A. The Backbone Biological Agent 

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Page 7 of74 

1. What organism was used for the Backbone Biological Agent? Are there any known virulence 

features associated with the Backbone Biological Agent? What happens in the target species? 

What is the previous safe use of the Backbone Biological Agent? 

(i) If available provide history of previous sa_fe use using published literature or internal 

documents. Include the recommended CDC/NIH biosafety level for use of the Backbone 

Biological Agent. 

2. Physical characteristics of the Backbone Agent 

a. Provide a flow diagram or explanation Of the process of how the Backbone Biological Agent was 

constructed. 

- ----- ---------------------------

-The virus was produced by infecting specific pathogen free (SPF) primary cell culture (chicken 

embryo fibroblasts, CEFs) and observing the cytopathic effect (CPE). The virus/cells were then harvested 

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using a trypsin solution and then frozen back in a cell-associated form using a specific freezing solution 

containing DMSO that protects the viability of the cell/virus. Viral DNA was extracted from infected CEFs 

and was used to generate the biological agent. 

(i) Describe the proposed site for Donor DNA insertion . 

. - ------------ - -- -- - - --

The sequence of CVI988 vaccine strain has been deposited into Genbank (Accession 00530348). This is 

our sequence of reference for the CVI988 genome for the present SIF. 

- - - - -

-- ------- -- - -

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(ii) Do the flanking regions of the proposed insertion site Backbone Biological Agent have any known 

regulatory elements that could moderate the expression of the inserted Donor PNA? 

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B. 

Serotype 1, Live Virus, Code: 16L 1.RO (Unlicensed) 

--------- ------ ----- ------- ----- ---

- - - - -- - --------- - -- - -

(IIi) Identify unique restriction endonucleases (not more than five) that will give identifiable digestion 

"patterns useful for characterizing the final Backbone DNA 

Don~r Biological Agents and Dono"; DNA or Gene~ 

1. Whaiare the Donor Biological Agents ilsed to source each Donor DNA sequence inserted into the 

B~ckbone Biological Ag~~t? '· . . . . . . 

j_''' 

. ,, -,._~- -~"" 

tJ:om paren~a-1- MDV ~8¢cine strain Snd 

... we .therefore ·Eansicie"red lh~-- as 

The~ virus wa~oiJI:ai~<>d 

DNA from ii · 

In summary, the Donor Biological Agents used to source Donor DNA sequences are the following: 

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a. Has there been safe use of the Donor Sequences or Donor Genes as well as Safe use of the Donor 

Biological Agent? 

{i) Provide relevant references for safe uSe. 

No liCensed vaCcine h'8s incorporated the donor sequence 

For both examples, the parental MDV1 virus was a virulent strain. In-virus, 

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i' 

the backbone is the non-p'athogenic ....... strain and therefor~, th.\-' virus is expected to , .":·:·: _-. · --" --· ._ - - :r-:- ·_- .-,_T-_-.;:_,-,_,...,,_.,_.,:r,-· - - - -· · 

be further.attenuaJed compared to th~se examples. . · '··· · · •... · . 

- - --- -- --- - ------ --- ---- ------ -

Donor sequence. No identified virulence genes were therefore brought from the ....... Donor 

sequence into the- candidate. 

The Donor sequence had a long homologous sequence with the parentaliiiiiill genome. A sequence 

analysis of the genomic regions flanking the indicated that this sequence is 

identical to the Donor sequence. In particular, the sequenced region contains 

the Donor sequence does not contain genes involved in MDV_virulence . 

. In Sl!mmary, the genomic part that is known to Contain MDV virulence genes is o~!ginat~ng frQ_~ the:-

- in the 11!11111 candidate. The Donor sequence includes · ' · -

--------- - -----

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- a sequence where no gene involved in virulence has been identified. The resulting chimera is 

therefore expected to conseiVe the safety of the- backbone. 

Figure 4: Localization of major genomic differences between attenuated CVI988 strain and oncogen.ic· 

on the Genbank 00530348 CVI988 sequence. 

b. 

-

Are there specific parts of the Donor Gene(s) or Sequences that were used for insertion? Show 

pertinent sequences or restriction endonuclease sites. 

The Donor Sequence -contains MDV sequences flanking- insert that were used for 

targeting insertion of the-into the exact same locus of the- genome by homologous 

recombination. As explained above and shown in figure 3, the MDV flanking sequence encompasses I 

These flanking sequences do not 

contain genes known to play a role in MDV virulence. 

C. Construction and Characterization of the Regulated Biological Agent 

1. Provide a flow diagram on the construction of the Regulated Biological Agent. 

Include the following: 

a. Final Backbone Biological Agent: 

b. Donor DNA or gene 

c. All shuttle vectors 

d. Host cell lines used 

e. Selection techniques and methods used to construct the final Regulated Biological Agent 

See Section liB for flow diagram. 

The flow diagram on the construction of- agent is shown in Figure 3. The Backbone Biological Agent is 

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the- Gallid herpesvirus 2 (Marek's disease virus serotype 1)-strain .••••••• 

- is the Donor sequence. The Host cell line used is primary chicken embryo fibroblasts (CEFs). 

Genomic DNA isolated from purified > ---------- --

-- -------------~-- -~- - ------

Pathogenesis experiments in-chickens showed that, like parental- virus,- was fully 

attenuated and unable to transform T-lymphocytes. Since- virus was avirulent in chickens, its protection 

efficacy against challenge with- a highly pathogenic field strain, was evaluated at- Results showed 

protection compared to. protection by commercial CVI988 vaccine indicating 

A virus suspension of- virus was sent from- to Merial Select for evaluation. The material was 

subjected to plaque purification and 3 clones that contain 

- were selected. 

- . 

--~ -~ - - - - - - - - - -- ---- -- - - - --- -- ------ --

-

2. Describe the laboratory methods or criteria used to evaluate the flanking regions of the. include 

the sequences 

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------------------------------------- ---- -- -

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3. Physical characterization of the Regulated Biological Agent 

May 19, 2014 

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a. Characterize the physical map using unique Donor DNA and Backbone Biological Agent restriction 

endonuclease sites and describe resulting restriction fragments and digestion patterns. 

The restriction map of-is shown in the- genetic stability report (Appendix 1, figure 2). A 

Southern blot analysis was performed on the- DNA di11ge,:ted 

hybridization patterns were identical to the expected 

======Obse~rved~ ones based on the sequence of the Donor DNA. 

b. Devise a PCR or restriction endonuclease test based on the Backbone Biological Agent sequence 

and the Donor Sequence that will identify and characterize the Donor DNA/Backbone Biological 

Agent construct. 

As described above, the PCR using prim"ers. can differentiate the- agent from the parental 

- virus and any other MDV1 isolates. 

Results of the PCR with primers-are shown on Figure 6. This PCR can clearly distinguish- from 

other serotype 1 Marek's disease viruses (Rispens, GA-22 and 

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c. What .will be the criteria for stability and purity of the Regulated Biological Agent Master Seed n and 

n+5? 

-

The genomic (2) stability by Southern was blotting evaluated and by (3) (1)-.:~~~================ by sequencing 

(1) the- master seed virus (MSV), (2) the in vitro passages from the 

MSV (X+5), and (3) the 5~ in vivo back-passage in chickens (BPS). Results were exactly as expected and 

identical for the 3 tested samples confirming identity and showing the genetic stability of thecandidate. Results are described in the- genetic stability report (Appendix 1) 

d. Provide the genetic sequence in electronic format for any new or altered genetic sequences, 

including insertion and flanking sequence. 

Enclosed with this submission is an electronic file containing the- sequences described in the 

-genetic stability report: the sequence from the 

- -- - - ----- -- - - -- - - -

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4. What is the recommended NIH/CDC biosafety level for the Regulated Biological Agent? 

According to NIH/CDC definition, Marek's disease virus is a Risk Group 1 agent since it is not associated 

with disease in healthy adult humans. BSL2 practices and procedures were utilized to ensure proper 

containment of the experimental construct in a research setting. At this time, considering the generated 

safety data, we would consider BSL 1 sufficient for- similar to the parental organism. 

5. Provide a short summary or description of genetic motifs that may have resulted as a consequence 

of the genetic recombination (II.C.3.d). Are there any known motifs thatmay promote homologous 

recombination, DNA insertion, or gene expression of existing or new .open reading frames? 

inserted at the exactly same location in thegenome. Ttfei Virus has beEm plaque purified and a homogenous viral pOpt.ilati~ri havingConfidential Business Information Redacted 
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~ ------- -----~-------- -----

------- -------- -------- ----- --- -------

- - ------ - - --- - --- - --- -- ------ --

-----------------------------

-- ---------

potentially be transferred to another field MDV isolate by homologous 

recombination. Such genetic transfer requires having both viruses infecting the same cells. MDV vaccines 

are administered by a parenteral route to the birds before (in ovo) or at hatch, whereas the natural infection 

by field MDV1 (wtMDV) isolates occurred by the respiratory tract MD vaccines induce a rapid onset of 

immunity that restricts wild type MDV infection and replication. The likelihood of having a cell co-infected 

with vaccine and wtMDV at the same time is therefore very low. If nevertheless co-infection occurs, different 

chimeras could be generated by single or double crossing-over between- and wtMDV1 genomes 

that could lead to the generation of a Such new virus would 

emerge in a vaccinated host and therefore its replication would be inhibited by the vaccine-induced immune 

response. It may fail to reach and replicate in the feather follicie sufficiently to have enough virus shed that 

could be successfully persist into the environment and transmitted to other chickens. Based on the current 

should not increase MD condemnations 

since it should show attenuated phenotype for body weight loss, mortality and MD lesions compared to the 

parental wtMDV. The risk 

if it occurs, the consequences would be negligible. 

In summary, we consider that the insertion 

recombination or DNA insertion. 

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to a wild type MDV is therefore very low and, even 

candidate does not promote 
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111. Biological Properties or Virulence for the Regulated Biological Agent used for Master Seed 

A. What are the known phenotypic characteristics or effects of the Regulated Biological Agent 

identified as Master Seed? (e.g., expresses envelope gene/sequence, lacks thymidine kinase 

activity, lower titer In MA105 cells, etc.) 

The insertion of the. expression cassette into the serotype 1-strain vector, 

- had no negative effect on the in vitro replication of the vector as determined during 

The cytopathic effect of the MSV to MSV X+S of- showed typical herpesvirus 

infection oi chicken embryo fibroblast distinguished by foci of infection with rounded refractive cells resulting 

in plaque formation. 

B. What are the virulence characteristics of the Regulated Biological Agent used for the Master Seed? 

The- virus used as the Backbone Biological Agent was isolated from 

- No known virulence feature is associated with the Backbone Biological Agent. 

- As described in Section 11.8.1, 

Studies ~ere conducted to demonstrate that the MDV- SR-1 virus was not pathogenic induding a 

reversion to virulence study, safety in mice, atrophy of lymphoid organs such as thymus, spleen and bursa, 

and safety in non-target avian species (See Section Ill C for individual study design and results). No 

difference in virulence was observed between the parental- strain and the MDVSR-1 virus. 

C. Is the Regulated Biological Agent used for the Master Seed virulent for target animals, non-target 

animals? 

The regulated biological agent, MDV SR-1-is avirulent for chickens. The ability of the virus to 

revert to a virulent fonn was evaluated in 

inoculated intraperitoneally With the MDV SR-1- MSV; blood was taken at seven days postinoculation and used to inoculate another group of one-day-old chickens. Four additional passages were 

made. At each passage, narve hatch mates were placed with the inoculated birds to serve as contact 

controls to monitor viral spread. Also at each pa:ss<1ge 

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from each passage were necropsied and examined for lesions typical of Marek's Disease. A final passage 

was conducted using 25 one-day-old chickens. A control group receiving the MSV diluted the same way as 

that given to the initial vaccinates and a second control group receiving a placebo were evaluated with the 

final product passage. Na"ive hatch mates were also placed in contact with birds of each of the three groups: 

final backpass, MSV inoculates and the birds receiving the placebo. These birds were also necropsied at 

seven weeks post-inoculation; lesions were not observed in any of the birds. MDV SR-1- MSV was 

recovered from BP1, BP2, BP4 and BPS. Virus was not recovered at BP3. No virus was recovered from the 

contact controls at any point evaluated in the study. Under the conditions of this study the MDV SR-1, 

- MSV did not revert to virulence nor did it spread to non-vaccinated contacts. The study satisfied 

the recommendations of Veterinary Services Memorandum 800.201 for evaluating the lack of reversion to 

virulence in chickens. This study report was accepted by CVB on 20 December 2011. 

The safety of MDV SR-1- experimental X+5 (final product passage) as evaluated in mice-

-showed no recovery of virus after subcutaneous injection and epidermal scarification of 15 mice. On 

days 3, 7, and 14, the trachea, brain, spleen, heart, liver, lungs, kidneys and skin at the site of inoculation 

were removed from 5 vaccinates and 2 naiVe controls that received a placebo only .••••••• 

- from the tissue homage nates and evaluated for presence of Marek's disease virus on chicken 

embryo fibroblast monolayers. All tissues were negative for virus recovery. This study supports the lack of a 

negative effect of the MDV SR-1 ••• virus in non-target animals. This study report was accepted by 

CVB on 16 April 2012. 

Safety in non-target avian species,-, was evaluated using the following avian species: quall, 

turkey, duck, pheasant and pigeon. Each group of birds was inoculated with 

experimental X+5 (final product passage). A group of na"ive birds of each 

species were inoculated with a-placebo. On study days 46 and 47 birds from each species 

were bled and tested for Marek's disease antibodies. The birds from all the species were necropsied at 

either day 46 or 47 and observed for gross observable lesions of Marek's disease. The MDV SR-1-

experimental X+5 vaccine was shown to be safe for non-target avian species. No lesions were seen and no 

humoral antibody to Marek's disease was induced by the vaccination. This study report was accepted by 

CVB on 19 April 2012. 

A 120 day safety study- was conducted according to 9CFR 113.330 Section (b). Two 

hundred (200) one-day-old, SPF chickens were randomly assigned to one of four groups of fifty and to 

housing in negative pressure isolation units. Fifty chickens were vaccinated SQ with the MDV, SR-1, 

-Master Seed Virus (MSV),- 10Xconcentrated on Study Day 0. The remaining 150 

non-vaccinated chickens were divided into three groups of 50 chickens each in order to serve as challenge 

controls, sham-vaccinated and/or sham-vaccinated contact controls for the SQ vaccinated group. None of 

the chickens vaccinated with the 1 OX dose of MDV SR-1,- MSV, negative controls or contact 

controls had gross lesions of Marek's disease during the study or at the final necropsy on Study Day 120. 

The incidence of Marek's disease in the challenge control group was 100 percent. There were no significant 

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differences in body weights between the MDV SR-1,- MSV 1 OX vaccinates and the negative control 

birds- between the contact controls and the negative controls- or between the MDV 

SR-1,- MSV 10X vaccinates+ the contact controls and the negative controls- One 

hundred percent of the birds in all the groups survived to five-days-of-age. The MDV, SR-1,-

Master Seed Virus (MSV 10X)-proved to be safe throughout the 120 day observation 

period when administered SQ to one-day-old chicks. This study report was accepted by CVB on 08 May 

2012. 

D. Do the Donor DNA sequences enhance the virulence or the ability of the Master Seed to survive in 

the target animals, non-target animals? 

1. What is the tissue tropism of the Regulated Biological Agent in target and non-target animals? 

MDV replicates in chickens and induces a detectable level of viremia. Leukocytes become infected and 

provide the main mechanism of cell-associated virus transport to other tissues through formation of 

intracellular bridges (Schat & Nair (2008) Marek's Disease in Diseases of Poultry, Ed. Y.M. Sail, 121

" 

Edition, page 461-3). 

MDV replicates exclusively in cells of avian origin (chicken, turkey, duck, quail, pheasant, pigeon, or goose) 

(H.G. Purchase eta/., "Responses of cell cultures from various avian species to Marek's disease virus and 

turkey Herpesvirus," 1971 Am. J. Vet. Res. 32, 1811-1823). MDV is typically characterized by proliferation 

in lymphoid cells in various tissues and organs, primarily blood and spleen. The vaccine strain is produced 

in primary cells of chicken embryos. MDV is cell associated, and is present in very small concentration in 

the supernatant of infected cell cultures. 

SPF 

chickens were inoculated with MDV SR-1- experimental X+5 vaccine or a licensed Rispens vaccine. 

An additional group of birds received diluent only to serve as negative controls. On days 7 and 28 postinoculation, the spleen, liver, lung, kidney, and gonads were isolated from five birds from each group of 

vaccinated and the spleen was isolated from three of the negative control birds. from 

each organ were evaluated for virus isolation on chicken embryo fibroblasts monolayers. The presence of 

the virus was identified by the observation of cytopathic effect from one spleen taken from the MDV SR-1 

- vaccinates on study day 28. Rispens virus was isolated from multiple birds and multiple tissues 

tested. The results of this study support the conclusion that the tissue tropism of the vaccine construct and 

the parental virus are similar (spleen isollati•on) 

CVB forreview on 20 December 2013. 

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was conducted to re-evaluate potential to recover virus 

from multiple tissues. On days 7, 14 and 49 post-inoculation, birds vaccinated with either- or a 

licensed Rispens vaccine were tested for virus recovery from the following tissues: spleen, liver, lungs, 

kidneys, and feather tips and from blood. The results of the study showed the same type of virus recovery 

from- with the exception that more of the tissues showed virus recovery of-at day 14. 

No virus was recovered at day 49 from the- vaccinates. This study report was accepted by CVB on 

23 December 2013. 

E. Discuss the potential for horizontal gene transfer or recombination of the Regulated Biological 

Agent. 

1. Is there any reason to believe that the potential for horizontal gene transfer or recombination is 

different in target and non-target animals? 

Gene transfer among herpesviruses may occur by homologous recombination. Since the genomes of the 

three MDV serotypes [serotype 1 (MDV1) or Gallid herpesvirus 2 including the CVI988 vaccine strain, 

serotype 2 (MDV2)) orGallid herpesvirus 3 including SB-1 vaccine strain, and serotype 3 or Meleagrid 

herpesvirus 1 or herpesvirus of turkey (HVT)) share little nucleotide sequence homology (Osterrieder and 

Vautherot, 2004 The genome content of Marek's disease-like viruses. In F. Davison & V. Nair (Eds.) 

Marek's Disease. An Evolving Problem, Elsevier Academic Press, pp 17-31 ), recombination events 

between MDV serotypes is unlikely to occur. It has indeed never been reported despite the wide use of 

combination vaccines such as HVT +881, HVT +CVI988 or HVT +SB1+CVI988. The potential for horizontal 

gene transfer from- is therefore restricted among MDV1 viruses. 

To occur, such potential homologous recombination requires: (1) co-infection of the same cell with 2 

different viruses at the same time and (2) replication in this cell. Since Marek's disease virus infection is 

restricted to chickens and occasionally some related avian species including quails, turkeys, pheasants 

(Schat & Nair (2008) Marek's Disease, in A.M. Fadly, Diseases of Poultry, 12~ Edition, pp452-514), MDV 

recombination can potentially occur in these species only. The results of the non-target safety in avian 

species and mice did not show virus replication in the main tissues targeted by Marek's disease virus 

confirming the very low risk of such event to occur. 

The- virus contains a-DNA sequence inserted into the-strain genome. 

While unlikely, there is potential for the transfer of to the genome of a 

MDV1 isolate by homologous recombination. lithe- vaccine is administered together with the 

-vaccine, there is a potential of-transfer from- into the- genome. Homologous 

recombination will insert the. at the same locus of the- genome as in that of- Since the 

backbone of-is the- genome, this horizontal transfer will not create a new virus with a 

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different phenotype thanMay 19, 2014 

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Horizontal transfer may also occur between- and field MDV1. As stated above, both viruses need to 

be present in the same cell at the same time for a recombination to occur. MDV vaccines are administered 

to the birds before natural infection and the route of vaccination (subcutaneous or in ova administration) 

differs from that of natural infection (respiratory infection by infected danders). The- virus will 

therefore replicate earlier than the field MDV1 and in different tissues. In addition, MD vaccines induce a 

rapid onset of immunity that will restrict wild type MDV infection and replication. It is known also for 

herpesviruses that once a cell is infected with a virus, it subsequently blocks co-infection with another virus 

(Thiry ~tal., 2005 .. Recombination in alphaherpesviruses. Rev. Med. Viral. 15, 89-103; Thiry et al., 2006. 

Recombination in the alphaherpesvirus bovine herpesvirus 1. Vet. Microbial. 113, 171-177). The likelihood 

of having a cell co-infected with vaccine and wtMDV at the same time, and, subsequently, of gene transfer 

is therefore very low. 

In the unlikely event 

three hundred (300) one-day-old SPF chicks were randomized into six different groups and 

inoculated subcutaneously (SO), in the nape of the neck, 0.2 ml per bird, with either: 

Group 1 -MDV SR-1 

Group2- MDVSR-1- (X+5)plusvMDVSR-1 GA22,-

Group3-vMDVSR-1 GA22,-

Group 4- MDV SR-1- (X+5) plus MDV SR-3 HVT,-

Group 5- MDV SR-3 HVT, 

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(See Section 5 Table: 1 of the study report for the inoculum titers) 

After the inoculations, the chicks were placed into their respective units. On study Day 21, ten randomly 

selectedbirds fr?m the-GA22 and-HVT vaccinates were bled for····· 

-- - ---- --------------- - -- from these groups were inoculate•d 

tissue culture and the vir~s recovered was confirmed as being GA22 from ihe-/GAZ2 vaccinates 

and HVT virus from the-/HVT vaccinates. No-was isolated at Day 21. On Study Day 35, all 

the r~maining bi_l-ds in Grou-Ps 1-6 were terminated, sexed, Weighed and obser:,-ed for gross Ma~ek;s 

Dis8~~9 lesioi1:S~ There was no increase in virulence observed in the GA grou_P_ illiillllllll as compared to the GA22 group Th~ birds containing 

·-HVT or HVT only had no gross lesioils associati9d ___ ' . There Was no significant 

group effect on the body weight between the GA 22 and the-GA 22 inoculated groups-

- and HVT and the-/HVT inoculated g~oup.~-- at end of the 35 day 

-------------- --~0-:t)Se~atiail_periOd. this stuaY.~eP6rt was·?ccep~ed by-~he cVB 6n fS JU1Y 201~.- .. -- . ,, ~- -~-- - '";;!· 

F. oeS'~~ib~ ttie shed/spread capabiiitti!s of the Regulated Bioiogicl!l A~ent. 

Viru~'6ou~E.not b:re~d~;r~i:t irom eit~~r cloacal or .. , '' 

the results obtained with the disseihlni:itioh 

;;:21. __ -_~:;·_- _ '><<-.-~.c-,;. _·1 -~--~,--,.:·.c. · ·· · ~·:.;t~'i-·-~·,'~'(-'S;:· 

~ ~ - -- - ------------------- ------

random!y selep.ted vaccinated an~ five non-vaccina1ed conta9~s per ~r.o~p were ,termin~.ted and necropsied, 

Wit~;_siSiee'iis iMdividually naJVesteC!. on the la~t day~~ the stu~\, (D..i9 49), all the remaining birds were 

eUtffaOI2:ed ~hd necropsied fof signs of MDV and th·.~ ~tUdy v#s temiiiiated. Spleens -frpiil fiVe randomly 

~eiecte~ vaccinated b1rd~ and the remafnirlg five .non-VB.ccinat~d contacts per grOuP'-WSre individually 

harvested. Virus isoiation was only attempted on the spleens harvested from the non-vaccinated contacts. 

All tracheal and cloacal swabs were negative for virus recovery. 

All of the featherfollicle samples of the MDV SR·1- vaccinates tested negative for virus recovery. 

Only at day 21 cduld vtrus be recovered from feather follicles of the MDV SR-1- vaccinated birds. 

All of the feather f~_llicle samples tested from_~he sham v~ccinated birds were negative for virus recovery. 

There was no virus recovered from any of the spleens harvested from the sham-vaccinated contacts at 21 

and 49 days using either virus recovery or PCR. There was no evidence of clinical disease in any of the 

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non-vaccinated contact birds. Under the conditions of this study, birds vaccinated SQ with the MDV, SR-1, 

- experimental vaccine showed similar dissemination patterns to birds vaccinated SQ wfth the 

commercially available MDV, SR-1 Rispens vaccine. There was no evidence of shed/spread to nonvaccinated contact birds, demonstrated by the lack of clinical disease and negative virus recovery in the 

contact birds housed with the MDV- vaccinated birds. The study report was accepted by CVB on 

27 December 2011. 

1. Include shed/spread potential in non-target animal species. 

As discussed in Section IIIC, Safety in Non-target Avian Species,-· no lesions and no humoral 

antibody attributable to Marek's disease was induced by the vaccination. 

As discussed in Section IIIC, the safety of MDV SR-1- experimental X+5 (final product passage) as 

evaluated in mice- showed no recovery of virus after subcutaneous injection and epidermal 

scarification of 15 mice. 

2. Include in the discussion any contributions to shed/spread capabilities that might be associated 

with particular attributes of the Backbone Biological Agent and Donor DNA sequence. 

A study perfonned on th ~ - -- - --- - -- - ----

1\lt<Jge•th<lf,, these studies do not indicate an increased risk of horizontal transmission due to the 

insertion of. In agreement with these published data, our studies indicate that the- virus did not 

shed to contacts as discussed in Section Ill. F. 

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G. 

Serotype 1, Llve Virus, Code: 1si..1.RO (uniicensed) 

3. Ref'7rence re~evant ~ciEmtlfic publications. 

Jarosinski twJ, et al. 2007. Horizontal transmission of Marek's disease virus requires US2, the UL 13 protein 

kinase, and gC. J. Viral. 81:10575-10587. 

Jarosinski twJ, Osterrieder N. 201 0. Further analysis of Marek's disease virus horizontal transmission 

confirms that UL44 (gC) and UL 13 protein kinase activity are essential, while US2 is nonessential. J. Viral. 

84:7911-7916. .. 

Jones D., Brunovskis P., Witter R. and Kung H.J. (1996) Retroviral insertional activation in a herpesvirus: 

transcriptional activation of US genes by an_ integratecl_ !eng terminal repeat in a Marek's disease virus 

clone. J. Viral. 70, 2460-2467. · · · · 

Okamuraa H., M. Sakaguchi, K yokoga...,a, E. Jokunaga,S. Abeb, S. Tokiyoshi, K, Mizuno~. L~Ekof 

contact transmission of recombin-ant Marek's-disease Virus type 1 expres-sing the fusion protein--of 

Newcastle disease virus Vaccine 20 (2002) 483-489 

S~n AJ, Xu xv; Pelherbridge L, Zhao YG.'NairV, Cui zz. (2010) Functional evaluation of the role of 

reticuloendotheliosis virus long terminal repeat (L TR) integrated into the genome ,of a·field strain of Marek's 

disease virus. Virology 397(2):270-6. ' · ·· ' 

Witter RL, Li D, Jones D, Lee LF, Kung HJ. Retroviral insertional mutagenesis of a herpesvirus: a Marek's 

disease.virus mutant attenuated for oncogenicity but not for jmmunosuppre_ssion or in vivo replication. Avian 

Dis. 1997 Apr-Jun;41(2)'407-21. · ' ' · ·' · · 

Zhang Z., Cui Z. (2005) Isolation of recombinant field strains of Marek's disease virus integrated with 

reticuloendotheliosis virus genome fragments: Sci China C Life Sci 48, 81-8. 

:(-J. , ' ;-.. ·r p- < . ' 

;u_,_ 

• - - • - ' _., -· :"' "-_· • • -,,.·,,-{~·- _- ·:';, -_ '_"'-'•_: • .>- ,_ - - -- -'_ ---- -'_ -- - __ , ---- - _< 

. biscusi!'ihe ~Jipected environrrleri!ai linpa'ct Ci'i survivabili\Y.~f the Reg1hated Biologi~'al Agent, and ·, ._.'\;~-;:~-·-::··. :~ __ ~;':~_,,..-... '.i'\;_: - _,,--:__ ·_-'_·;·.~~- ;_ :i!;"' __ '.; .. ~_,; ''?-_- ~-,~~~ ··,·-·- ' 

· Provide ·a\i~illibiEt S~il~tific e'vid·ence. · · ·'.t·· ' · · ·' \f ·';Y · 

. -_ -_ ,~.-·)- :;.~~~_. ,-_._·. )~ _:-, __ -. .- ~---~>~· .. _;, __ > .,_· ., .. < • -- .-' ,:.-;-:· ,;,;·_ ' -'<· ' .· -, :; ': 

Chickens are the natural host of MDV SR-1 v~ruses. MDV is shed mto the erivironineht bY feather follicles . ·-.'. - . ,, . . : ·.:• . ·~ .. 

of infected chickens. Prevalence of infection in chickens is dependent upon the control measures used to 

,~ont\?,1 the dis7ase in th,~.field<Th7,.s,urviv.~~]\r ~f MDV SR-1- in the environ mentis flXpected to be 

,·· similar to, or less, th.an.. that of the_ parental- strain Besed on th,e resulls o,t. the dis~eminati9n study . >•.· .. · _ ,_ -" _ . .- ~-"~-- .·--?--·~ . ..,-,·-·~··"'·!_,~--'"- -'•-t ·t·,~=(~ .••. -- _._.':T·-r-·-.-•• ,.-: · . . : • •:-• :;,; ·"' ., , ,. ·.r '! :·--lt·- , .;···- :-: 

, · (Se~ -~ectio~ Ill ~·-th~t sh~~ed nO ,~!~!!~·re,~ovefl! ~rpm ~at~-~r f9.Jiicles: ~9r any _yir'~s 

. -.. ieco~rred frdrn ri_a_rve·~~ptact_~~- -~The Ml?~ SR-:1- V8?d~~--~lil .. be .~r,q~6h ir\~-~fi-~~~of~i9t.~,Q}orm. - -~ > :• 0 -': , ._ -~-•o-.--'-'' ,_ >_"-- __ ~'-~'f''-: r _-.-,.-~_-- :--:· _" , - -~• , --,-- "7 ~--- _-:,----;---=~ --- .,, __ -- :,-,-~- --d,-.o'T- •--:",•{-,. ,,_ •, ,..,.,1 - .c._'----_-. , 00 -'-"'-~'c 

;, ·This, f9itn is poorly resistant iii thoi_enviroriment siriQe.it is .depehdeHI pn the Viabiliiy pf th.e cells. Th~ virus !(~·o,;, :•_-c- _ ,- __ ,. • > ~--,. -- {.; _ ., _ , ,_,, ' , ,., - ----": _ -~t:"~J;7 ,. 'L-:~,:~ ·,;•_,:_ .. '· '-

. may be ~hed ~s cell-frei~ virus pythe feath.~r follicle of the yaccin11ted chic.kens:, blit:?CcOrding to !he results ~-:- i ._,,._ .. ,,_,, __ - -___ .·:.;-, ·. . !( .- ~·· ... ,_. ,f ..T~.;.;-- ,, :·=, .·-"· ;,'-!:. . 

of the dissemination study- and shed/spread study- MDV SR-1.- is not 

\: .transmitted h_orizontally among chick~n·s' indicating that the amount or infectiviiy of virus sheet rs--~bt -~·· ~ ---· ., \._. '· . _,;-_ . .. . '•" . 

sufficient to infect na'fVe chickens. ' "' 

Th~ qosUra~ge specificity bf the experime~tal vaccine was evaiJated with the non-target avia_n species 

safety t~st,- discussed in Section IILC of lhis report. 

A study design that includes the length of time the virus can be recovered from sterile shavings (same as 

those found in commercial chicken houses) saturated with .... was conducted .... The test 

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inoculated shavings were held at room temperature for the duration of the study. 

-- .. ,f 

May 19, 2014 

Page 29 of74 

On days 0, 1, 2, 3, and 7 post-inoculation (or when three consecutive days read no virus recovery),-

-shaving sample were collected aseptically and placed in a sterile sample bag containingThe procedure was repeated with the Rispens shavings 

sample. On Day 0,- showed an average of 3.3 plaques per ml of shaving solution and the licensed 

Rispens vaccine showed an average of 2 plaques per ml of shaving solution. There was no virus recovery 

after day 0. This study was accepted by the CVB on 01 January 2013. 

1. Do the Donor DNA sequences or gene sequences associated with the Backbone Biological Agent 

enhance the ability to survive in the environment or increase resistance to therapeutic agents? 

The insertion of the. should not contribute to the ability of the backbone virus to survive in the 

environment. is not located in a genomic position close 

to MDV1 genes contributing to the horizontal transmission The survivability of 

- virus with the. insert described in Section Ill. G showed no virus recovery after the day of 

inoculation. 

There is no therapeutic agents for MDV and therefore, the potential effect on resistance to therapeutic 

agents is not applicable. 

IV. Risk Assessment 

Risk assessment may be defined as the likelihood of an adverse event occurring and the consequences if 

that adverse event occurs. An adverse event is defined as safety hazards to animals, public health, or the 

environment. A safety hazard is defined as a danger, risk, or peril; absence of predictability associated with 

an event; or an expected or unpredicted event. 

A. Procedure 

The risk assessment is conducted based on safety characteristics of the vaccine. The safety characteristics 

are based on empirical data and established scientific facts. The completion of a risk assessment requires 

that the vaccine microorganism is properly characterized. This infonmation is included in the Veterinary 

Biologics Summary Information Format Section Ill (page 21) that is provided by Merial, Inc. 

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May 19, 2014 

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Haza;8 identification consists of identifying all possible adver~e events to anima_! safety, public health 

safety, and environmental safety relative to the recommended use of this experimental vaccine, Marek's 

Disease Vaccine, ~erptype 1, Live Virus, Product. Code 16l1.RO, unlicensed. 

The vaccine that will be us,ed in this proposal is ve;':Oib"sely i~i,3led to a USDA licensed VOCoC;n~.·-

···Such 

r· modification is not known to increase risk to animal safety, public health safety or environmental safety, '· ,, ' - ' .. :·.-,· .. ,_ ·, 

sine~ si~_il~f _inserts are present in other USDA licensed vaccin_es lillilliililii!.. '" . tJerial has submitted c~nsiderablio in vivo \md in vit?o data on·- These data shqwed· that the ,,-,~~--- '-- _.,:;; .... _. >/ ' ._:. -- -,._\;;.: - - ~. '. --~·'•' - _ _.. ·'·:: • -- - ' 

:-c9hstruct was safe in mice, chickens, ducks, quail, pi§~~ri!i, turkeys arid pheasabts_.,Jhe safety 

prOfile of-in mice and the other non-target avi_an sp'edes is tHe same as the com;;,erC-i81 Rispens 

va~cine. 

1. Animal safety 

a. sUmmary 

No ariimal safety problems have been identified with this experimental vaccine. The vaccine was safe when 

tested in one-day-old chicks. The vaccine was also safe when tested in several non-target species and one 

mammalian species suggesting that the va·C9in8 Vfbuld be safe if o~her_n9n-target species were exposed. 

The vaccine d_id not revert to virulence and was genetically stable through five backpassages in chickens 

(See Section Ill C). Studies in cell culture indicated that the vaccine is genetically stable at n, n+5, and 

backpass 5 (bp5) passages (See Sections II.C.3.c)}. 

b. Target animal safety: 

(i) Vaccination of chickens 

The Master Seed Virus did not demonstrate any virulent properties when inoculated into one-dayold chicks at 10 times the recommended field dose as written in 9CFR 113.330. A test was carried 

out to study the pOssibility of reversion to virulence of the vaccine strain. Five passages were 

carried out. Infected leukocytes were fourid in each passage. No abnormal signs were noted in 

the birds during the passages. Naive hatchmates were placed in contact with each group of birds 

receiving the backpassed virus. No· virus was recovered from those contacts, nor any lesions seen 

at necropsy (See Section Ill C). 

{ii) Vaccination/challenge 

No <=!dverse immune interactions are expected with the use of this vaccine. In 

vaccination/challenge studies, susceptible chicks vaccinated by -- -- ------

- vaccine protected against 1!!11 when challenged- at 5 days of 

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Serotype 1, Live Virus, Code: 16L 1.RO (Unlicensed) 

age- accepted by CVB on 20 December 2011 ). These results support the product 

claim to "aid in the prevention of Marek's disease caused by very virulent Marek's disease virus". 

(iii) Reversion to virulence 

Backpassage studies demonstrated that the- virus does not revert to virulence when 

administered to chicks. Supportive data include: 

• -Reversion to virulence -five passages on chickens (See Section Ill C) 

• Recombinant virus in vivo genetic stability analysis by Southern Blot and PCR, n, n+5 and 

bp5 (See Section II.C.3.c). 

These studies also demonstrate the genetic and phenotypic stability of the- MSV in vivo 

[See Sections II.C.3.c and Sections III.A and Ill. B) 

(iv) Purity testing 

The MSV was tested by the applicant and found to meet all standard requirements of 

9CFR113.300. 

(v) Identity testing 

(vi) 

The presence of the Ill, has been confirmed by the use of conventional PCR as described in 

Section II.C.3.b. It is proposed to test each serial for identity using-to confinm the 

presence 

Effect of gene manipulation on pathogenicity . 

The recipient- virus Was derived 

recommended for use in day of age chickens. The strain is 8.Virulent for chickens. 

which is 

The donor insert,. has no known pathogenic or toxic properties and it does not code for any 

polypeptide. No pathogenic or toxic properties have been identified for- when present in 

the experimental- vaccine (see Section Ill). 

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Serotype 1, Live Virus, Code: 16L 1.RO (Unlicensed) 

(a) Phenotypic effect of the gene modification(s) 

The- virus had no clear phenotypic difference compared to parental virus. In vitro 

replication and safety were similar. The level of-in the blood and in different 

tissues was lower and no infectious virus could be isolated from the feather follicles. In 

contrast, the MD protection level induced by-was at least equivalent to that 

induced by-

(b) Pr<>vi<lUS use o.f dqnor genes and _re.pipient mi~roorga[lism 

•. ~·~ii-~·~~~iili'~=·~·~i'~liil~ll ................................................ ... 

---- -- -- ------------ --- -~------ -

-- ------------- - -

RecipientMicroorqa~is~: the par~ntal strain of .... :irus is thE;IIIIil vaccine strain -· • -.-: - ,.- »-- .• 1·. - _.c. ·--."---·"·-- ,_._ ... ,., .-:o-«-- --.-. 

which is - - -=---=---~-- --- - - -- =---- - ---=-=---

- - - - -

(c) Phenotypic effect of any genetic marker(s) 

The- MSV does not contain any genetic markers. 

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Serotype 1, Live Virus, Code: 16L 1.RO (Unlicensed) 

(vii) Genetic stability 

-is genetically stable in vitro {see Section II.C.3.c}. 

The genetic stability of-in vivo was demonstrated through five backpassages in reversion 

to virulence studies in chickens (see Sections Ill. C). The presence of 

conserved throughout the five backpassages. 

(viii) Phenotypic stability 

Experiments conducted by Merial, Inc. indicate that-is phenotypically stable in cell culture 

(see Section Ill. A.). The phenotypic stability of the- MSV in vivo was studied in a reversion 

to virulence study through five backpassages in chickens (see Section III.C). No reversion to 

virulence was observed after backpassages. 

(ix) Alteration in tissue tropism 

The results of transmissibility studies indicate that the proliferation/tissue tropism of the 

experimental v'accine virus is similar to its parental- virus (Section Ill. D). 

(x) Effect of overdosing 

Target host animals were inoculated with 10 times more virus than the recommended field dose 

with no adverse effects observed (see Sections Ill. C). 

c. Non-target animal safety 

(i) Susceptible non-target animals 

The safety of-has been confirmed in non-target species. The data indicate that thevaccine strain was safe in these species (see Section Ill. C.). 

(ii) Virulence in non-target animals 

The results of the safety studies indicate that the hosUrange specificity of the- MSV has not 

been altered from that ofthe parental- strain, and thatthe vaccine is safe in non-target 

avian species (see-in Section Ill. C). It was also shown to be safe in a non-target 

mammal species (mouse). In virus proliferation studies, no virus recovery was detected from tissue 

samples of mice inoculated with-(see Section Ill. C.). 

(iii) Possible outcomes of non-target animal exposure 

- is not expected to pose a safety risk in non-target species. The parental virus is derived 

strain. The studies discussed above suggest that these 

safety characteristics have not been altered in-(see Section III.C.). 

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2. Public health safety 

a. Summary 

Risk Analysis 

Marek's Disease Vaccine, 

' ·J • 

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May 19, ·2014 

Page 34 of74 

Hazards to public health are not expected. There are no reports of Marek's disease causing productive 

infection in any species other than avian species, and no infection of humans has been reported despite 

accidental inoculation occurring during vaccine administration. The parental serotype 1 vaccine strain from 

which this experimental vaccine was derived has been used as a commercial vaccine for chickens in 

Europe/Asia/US/Latin American without any adverse effects or hazards to public health. 

b. Probability of human exposure 

Human exposure will be limited to personnel administering the vaccine and conducting the field studies. 

c. Pathogenicity of parent microorganism in humans 

There are no reports of Serotype 1 Marek's disease causing productive infection in any species other than 

avian species and no infection of humans has been reported (Schat & Nair (2008) Marek's Disease in .-. -· .~ .. ,-, 

Diseases of F'9)Jitry, Ed, 2~ Edition, page, 452). 

d. Virulence of the vaccine microorganism in humans 

-has never been inoculated into humans. Thus ttie actual outcome of an accidental human inoculation 

is unknown. Based on the current knowledge and wide use of Serotype 1 Rispens vaccines and thesafety data, the-is likely non-virulent in humans. 

-e. Possible outcome of human exposure 

There are no expected safety concerns associated with human expoSure based on the biological properties 

ofwildtype Serotype 1 Marek's disease viruses. It is the opinion of experts in the human and avian cancer 

field that Marek's disease and vaccines against MD pose no human health hazards [Purchase, H. G. and 

R.L. Witt~~·(1986), "Public health concerns from human exposure to oncogenic avian herpes viruses: A 

review" J. Am. Vet Med Ass.18g:1430:6 (Appendix 2)]. 

3. Environmental Safety 

a. Summary 

The safety risks to the environment are low. The shed/spread capabilities of the experimentalvaccine are limited. Some- virus may potentially be spread to the environment through the danders 

of vaccinated chickens but the shed virus unlikely to infect other chickens indicating that theinsertion did not provide an increased spreading ability to the virus. The hosUrahge specificity of the 

experimental vaccine does not appear to have been altered from the Serotype 1 Marek's disease virus, 

which is restricted to avian species. The safety characteristics of the experimental vaccine in non-target 

avian species do not appear to have been altered from that of the recipient virus which was derived from a 

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No adverse effects have been attributed to the use of 

licensed Serotype 1, Rispens vaccines in the field. There are no expected adverse ecological effects 

associated with the proposed field trials with this experimental vaccine. 

b. Environmental distribution 

There are multiple Serotype 1 vaccines currently licensed and being distributed globally. To our knowledge 

the- experimental vaccine should not behave differently in terms of environmental distribution than 

currently licensed vaccines. 

c. Shed/spread capabilities 

The results of studies indicate that the- vaccine microorganism was limited in its shed/spread 

capabilities since viable virus could not be isolated from feather follicles and the vaccine did not spread to 

non-vaccinated contact birds. This study was part of the reversion to virulence- CVB accepted 

on 20 December 2011 and the dissemination/shed/spread, -CVB accepted on 27 December 

2011. The contact controls placed with each group of chickens receiving the backpassed virus and the birds 

placed with the vaccinates in the dissemination study did not show any virus recovered from spleens nor 

were any lesions noted at necropsy (see Section II_I.C). In the non-target avian species studies described in 

Section III.C no adverse reactions attributable to- were report~d for. any of the vaccinates or contact 

control birds. All-recovery attempts from tracheal and cloacal swabs from the vaccinates and 

contacts were negative, as were the feather follicle epithelial cells. 

d. Horizontal gene transmission/recombination potential 

The potential for in vivo recombination with field and other vaccine viruses was evaluated in-

(see Section III.E.). There was no increase in pathogenicity seen when combined with a virulent GA22 

Marek's disease virus. The PCR results of the recovered virus showed only GA22 virus present with no. 

insert. 

e. Potential for transmission to invertebrates: 

The potential for transmission of-is expected to be minimal. The limited shed/spread properties of 

- should reduce the probability of mechanical transmission for the- vaccine microorganisms 

(see Section Ill. F.}. Even if transmission did occur, the attenuated properties of- are not expected to 

be altered (see Section Ill. C.}. 

f. Host I range specificity: 

There are no reports of-or any Serotype 1 Marek's disease virus causing productive infection in any 

species other than the avian species and no infection of humans has been reported (Schat & Nair (2008} 

Marek's Disease in Diseases of Poultry, Ed. Y.M. Sail, 121

h Edition, page 452}. Our non-target studies 

showed that the insertion of the- did not modify the host range specificity of the vector. 

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U.S. Vet. Lie. No. 298 

· Risk Analysis 

Marek's Disease Vaccine, 

Serotype 1, Live Virus, Code:.16L1.RO (Unlicensed) 

g. Survivability of the microorganism in the environment: -. -·J..c: 

May 19, 2014 

Page 36 of74 

The very limited amount of virus shed by the feather follicles via dander may potentially survive for months in 

the environment, similar to the parental type virus and other licensed Serotype 1 vaccines. However, the 

amount and/or infectivity of .. shed from vaccinated chickens appear very low since the vaccine did not 

spread to nalve chickensh. Physical and/or chemical factors affecting dispersal in the environment: 

No physical and/or chemical factors have been identified that would enhance the dispersal of !hein the field. 

i. Adverse ecolOgical effects 

The demonstrated safety a~d lack of proliferation of-in other non-target avian and mammalian 

species (see S~ction Ill. C), .. and the esta~li;shed limited h~st range of-reduces the risk of spread to 

non-target animal species: .... -

c. Release Assessment Pri~r to a Proposed Field Safety Trial 

Environmental releaSe propOsal 

Summarv 

The safety characteristic of the vaccine microorganism must be evaluated within the context of the target 

environment. Thus, the release assessment consists of a comprehensive evaluation of the proposed 

release so as to detenmine 1) the location and characteristics of the release site; 2) the test dose and total 

amount of the _experimental biologic to be used in the proposed study; 3) the frequency and duration of 

response to the test material; 4) potential escapes into occupational, residential, or outdoor environments; 

and 5) the individuals, populations, or ecosystems that will be, or may be, exposed to the experimental 

biologic. 

For the purpose of risk asses_sments, proposed studies with li~~ experimental vaccine microorganisms are 

classified as e"fther contairied stu-dies or environnlental rele~ISeS. A Contain~d StUdy is define~ as!an 

experiment in lab~i-a:tory contained or physically corlt~ined animal facilities. An EnviryJnmentaf Release 

refers to the release of the vaccine microorganism into ttie accessible enviro~me_nt. Planned introduction 

into the environment refers to the deliberate release of live microorganisms from contained facilities to the 

environment. Differences between small-scale field tests and commercial releases are considered in the 

release assessme~t. VB-APHIS recommends and encourages the use of small-scale field tests as 

appropriate for the experimental veterinary _biologic. 

Contained release 

Not applicable. 

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U.S. Vet. Lie. No. 298 

Risk Analysis 

Marek's Disease Vaccine, 

Serotype 1, Live Virus, Code: 16L1.RO (Unlicensed) 

1. Environmental release 

Summarv 

May 19, 2014 · 

Page 37 of74 

Merial, Inc. proposes to conduct safety field tests to determine the safety of the Marek's Disease Vaccine, 

Serotype 1, Live Virus, unlicensed, USDA product code 16L 1.RO in the target animal species, chickens, 

with the objective of licensing and commercial distribution. The Field Safety Studies will be conducted in at 

least three regions of the USA. A list of tentative recipient states is included below. These studies will be 

conducted in chicken houses that are typical of the commercial poultry industry. The potential for escape 

and dispersal from the release sites is low. There are no known biological organisms, physiochemical 

factors, or toxic chemicals that would contribute to the establishment of-in the environment. 

Personnel experienced in poultry health management will be conducting the proposed study. 

2. Tentative location of test sites 

Arkansas 

Georgia 

Kentucky 

North Carolina 

Tennessee 

Texas 

3. Characteristics of test sites. 

The test sites include commercial poultry houses, multiple houses per site. Each house will hold 

approximately 17,000-22,000 birds. The chicks will be inoculated at the cooperators hatcheries and then 

trucked to the house(s). The details of each test site will be included in the 9 CFR 103.3 authorization 

request for the Field Safety Study-. 

4. Personnel 

Personnel administering the vaccine will consist of hatcher personnel under the direction of the participating 

cooperator. Merial veterinary staff, and/or qualified Merial R&D staff will be present for the vaccination and 

placement of the chickens. Me rial personnel will be involved in data management and in study follow-up. 

The Veterinary Scientist in charge of this Field Safety Study will be Dr 

5. Experimental design 

Number of animals 

At least 20,000 birds will be vaccinated with the test vaccine. 

Descriotion of animals 

One-day-old commercial layer/breeder/broiler chicks of mixed breeds will be used for this study. 

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Risk Analysis 

Marek's Disease· vaccine, 

Serotype 1, Live Virus, Code: 16L 1.RO (Unlicensed) 

Route of administration 

Subcutaneous inoculation into the nape of the neck of the chicks. 

Dosage 

May 19, 2014 

Page 38 of74 

The vaccine for subcutaneous injection will be prepared for use in the mixing room of the hatchery. The ., vaccine container will be a sealed plastic IV bag, that will be fitted to a Marek's disease vaccinating 

machine using sterile plastic IV tubing. The serials of product that will be used in this stu~y will have been 

satisfactorily tested for" purity before releasing into the environment. 

Total amount of test material 

The vaccine is frozen in 2.0 ml glass vials each containing 1,000 doses. The vaccine will be reconstituted 

and added to sterile diluent at the ratio of one (1) ampule per 200 ml of sterile diluent for subcutaneous 

injection of 0.2 ml per chick. With the exception of Marek's products, the chicks can receive all other 

vaccines according to each company's vaccination program. Approximately 100,000 doses will be required 

for the trial. 

Protocol 

The Field Safety Protocol- was submitted to CVB for approval. 

Frequencv and duration of exposure 

Direct exposure of people to vaccine could potentially occur at the hatchery. The animal caretaker/grower 

responsible for the management of the test flocks will be exposed to the inoculated chickens, but not the 

vaccine, for the length of the field test period, approximately 60 days or less. The employees at the 

slaughter house are not expected to be exposed to the vaccine virus. No evidence exists to show that 

- can be transmitted to humans and therefore will not cause disease in h~mans. The duration of 

exposure is not expected beyond the day of vacCihation. 

The method of disposing of waste 

All vaccine containers, diluent containers, tubing, needles, and gloves will be placed in waste containers 

(i.e., biohazard bags) and autoclaved or incinerated, or destroyed ori site by industry accepted methods. 

The vaccinating IT)achines will be disinfected according to standard hatchery procedures. Dead birds and 

litter will be treated acCording to each cooperating company's program. 

Decontamination of test site 

Following vaccination, the vaccination area at the hatchery will be disinfected and washed according to 

standard procedures. Crates used to transport the chickens will be returned to the hatchery where they will 

be washed and disinfected. 

The broiler/layer houses will be cleaned and made ready for the placement of the next flock of chicks 

Confidential Business Information Redacted 
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U.S. Vet. Lie. No. 298 

Risk Analysis 

Marek's Disease Vaccine, 

Serotype 1, Live Virus, Code: 16L1.RO (Unlicensed) 

according to each company's program. 

6. Potential for escape and dispersal 

May 19, 2014 

Page 39 of74 

Spread transmission studies were negative. Therefore, no escape or dispersal into the environment is 

anticipated. 

7. Potential for establishment in the environment 

Marek's diseas~ virus already exists in the environment as a poultry pathogen. Several live Marek's 

disease vaccines including Serotype 1 vaccines are used commercially in the United States under USDA 

license. The vector organism is derived from a licensed vaccine strain of- The use of 

subcutaneous administration will minimize the chance of escape into the environment. It is not anticipated 

that conditions will exist outside the laboratory that will support perpetuation of the recombinant virus. No 

toxic chemicals or metabolites are known to be produced by the virus, which could endanger the 

environment. 

8. Monitoring 

Monitoring methods 

The monitoring of the field test will consist of maintaining the records associated with the production of 

commercial chickens. Mortality and morbidity will be recorded by the grower at each test .location. The 

flocks will be observed daily for seven weeks for adverse reactions to the vaccine by the grower or field 

personnel of the cooperating company. Meriel's veterinary staff and/or research department staff will visit at 

least once during this period. These potential adverse reactions may include, but are not limited to, unusual 

post-inoculation reactions and/or Marek's disease. 

Any birds dying with clinical signs or lesions of Marek's disease will be necropsied and appropriate tissues 

examined for histopathology and/or retained for viral isolation. Any adverse clinical signs associated with 

the vaccination will be immediately reported to the veterinarian in charge. 

Frequency of monitoring 

Each flock will be observed daily by the grower or caretaker during the growout, which is expected to last no 

more than 60 days for broiler type birds (layers/breeders will growout for a longer period of time). Birds will 

be observed at least once by the Veterinary or Research staff and more frequently if notified that adverse 

events have been observed in the flock. Birds will be observed for clinical signs of Marek's disease. The 

safety of the vaccines will be reported in the field safety study report after seven weeks of observation. 

9. Contingency plans in case of adverse events. 

No adverse events are expected but if adverse events attributable to the Marek's disease vaccine are 

observed, the primary investigator will be notified immediately and the event documented in the study log. 

The grower, the cooperator's veterinarian and Me rial veterinary staff will work jointly to determine a plan of 

action, if necessary. 

Confidential Business Information Redacted 
Merial, Inc. 

U.S. Vet. Lie. No. 298 

Risk Analysis 

Marek's Disease Vaccine, 

Serotype 1, live'Virus, Code: 16L 1.RO (Unlicensed) 

D. Risk characterization 

May 19, 2014 

Page 40 of74 

Risk characterization integrates the results of hazard identification and the release assessment into a risk 

statemen!that includes: 1) a likelihood rating; 2) a consequence rating; 3) a risk rating; and 4) a discussion 

of risk. 

Each likelihood and consequence rating is qualified by a degree of certainty rating and includes a 

justification for the rating. The justification for the ratings consists of identifying the ·applicable sections in 

the hazard identification and. the release assessment that support··ihe assigned rating. The risk rating is ' -i• . • . i ·~ . ·, ·• 

based upon the likelihcioa, consequence, and degree of certainty ratings. 

1. Likelihood ratings 

•Likelihood ratings are assigned for animal safety, public health safety, and environmental safety 

based on the following criteria: 

Low= 

Medium= 

High= 

2. Consequence ratings 

An adverse event is unlikely to occur 

An adverse event could possibly occur 

An adverse event will most probably occur 

Consequence ratings are also assigned for animal safety, public health safety, and environmental.safety 

based on the following criteria: 

Low=. 

Medium= 

High= 

The consequences if the adverse event occurs wiJI not be severe (the adverse 

event is self-limiting and would have negligible impact). 

The consequences if the adverse event occurs is moderately severe (the 

adverse event will have an impact, but it is not permanent, and can be treated). 

The consequences if the adverse event occurs is severe {the adverse event will 

have an impact, is permanent, and cannot be treated). 

3. Degree of certainty rating 

Each likelihood and consequence rating is qualified by a degree of certainlY rating that is base9 on the 

following criteria: 

Certain= 

Moderately certain = 

Uncertain= 

4. Calculating the expected risk 

The rating is supported by direct sqientific evidence. 

The rating is supported by indirect scientific evidence. 

the rating is not supported by scientific evidence. 

Numerical values have been assign~d to the likelihood, conSequence, and degre~ of certainty ratings 

described above (refer to Table 2). Each numerical value rating was derived from the importance placed on 

the rating of each category. The assigned numerical values are weighed to place emphasis on the severity 

of expected risk. These values reflect the professional judgment of the applica-nt (Merial, Inc.). 

Confidential Business Information Redacted 
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U.S. Vet. Lie. No. 298 

Risk Analysis 

Marek's Disease Vaccine, 

May 19, 2014 

Page 41 of74 

Serotype 1, Live Virus, Code: 16L 1.RO (Unlicensed) 

5. Risk ratings 

The risk ratings are based upon the likelihood, consequences and degrees of certainty ratings, and the 

expected risk for each category. A total of 81 rating combinations are possible; e.g., likelihood lowmoderately certain, consequences low- moderately certain (see Table 3). Each combination has been 

assigned a risk rating of low, medium, or high. The assigned risk ratings were weighed to place emphasis 

on the severity of the expected risk. Again the severity of the risk reflects the judgment of the applicant. 

The low, medium, and high risk ratings are defined for the purpose of decision-making as follows: 

Low= Acceptable risk; very few concerns are associated with the proposal (does not 

justify denying the proposal). 

Medium= 

High= 

a. Animal safety 

Unacceptable risk; moderate concerns are associated with the proposal (either 

identify valid mitigative procedures or deny the proposal). 

Unacceptable risk; major concerns are associated with the proposal (deny the 

proposal). 

Likelihood rating: Low (LL) 

Degree of certainty rating: Certain (C) 

Justification for ratings: 

IV. 8.1. b. i. 

IV. 8.1. b. ii. 

IV. B. 1. b. iii. 

IV. 8.1. b. iv. 

IV. B. 1. b. v. 

IV. B. 1. b. Vi. 

IV. B. 1. b. vii. 

IV. B. 1. b. ix. 

IV. B.1. c. i. 

IV. B. 3. b. 

IV. B. 3. d. 

Consequence rating: Low (Cl) 

Degree of certainty rating: Moderately certain (MC) 

Justification for ratings: 

IV. B.1. b. ii. 

IV. B.1. b. iii. 

IV. B. 3. b. 

IV. B. 3. h. 

Expected Risk Rating: 0. 7500 

Risk Characterization: LL.C.CL.MC 

Confidential Business Information Redacted 
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U.S. Vet. Lie. No. 298 

Risk Analysis 

Marek's Disease Vaccine, 

May 19, 2014 

Page 42 of74 

Serotype 1, Live Virus, Code: 16L1.RO (Unlicensed) 

Risk rating: Low 

The safety risks to the animals and environment are low. The host/range specificity of 

- does not appear to have been altered from the parental type virus, which is restricted 

to avian species. Although the protocol of the field safety study does not include procedures 

for monitoring the shed or spread o-into the environment during the course of study, 

studies previously conducted by the applicant indicate that the shedding capabilities of the 

experimental vaccine are limited, and the virus is unable to spread to other chickens. 

Furthermore, the proposed release will be confined to poultry houses. Thus, exposure of nontarget animals and the dissemination of the. experimental vaccine virus in the environment 

during the course of the field tests will be restricted by its use in poultry houses. 

This assessment indicates that one-day-old chickens will be vaccinated in commercial 

hatcheries, and subsequently transported to commercial chicken houses for the· duration of 

the field trials. All the waste material from the vaccination procedure will be decontaminated 

prior to disposal. The vaccination area at the hatchery and the crates used to transport the 

chickens from the hatchery to the houses will also ~e decon~aminated. 

The- vaccine will be used in combination with other Marek's disease vaccines 

(Serotypes 3 and 2). Our recombination safety study has shown that co-administration of 

these vaccines did not generate a more virulent virus. 

Conclusions and discussion of risk 

The risk to animal safety associated with this proposal to conduct a field trial with experimental 

Marek's Disease Vaccine, Serotype 1, Live Virus, is low. The recipient virus used to construct 

the- MSV was derived from a licensed vaccine strain that has been used as a 

commercial poultry vaccine in the United States and Europe, as well as Asia and Latin 

America. Safety studies conducted by the applicant demonstrate the safety of the 

experimental vaccine in chickens and in several non-target avian and mammalian species. 

The proposed study will be conducted by personnel that have extensive experience in poultry 

health management. 

b. Public health safety 

Likelihood rating: Low (LL) 

Degree of certaintv ratina: Moderately certain (MC) 

Justification for ratings: 

IV. B. 1. b. iv. 

IV. B. 2. b. 

IV. B. 2. c. 

IV. B. 2.d. 

IV. B. 3. b. 

Confidential Business Information Redacted 
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Risk Analysis 

Marek's Disease Vaccine, 

May 19, 2014 

Page 43 of74 

Serotype 1, Live Virus, Code: 16L1.RO (Unlicensed) 

IV. B. 3. d. 

Consequences rating: Low (CL) 

Degree of certainty rating: Moderately certain (MC) 

Justification for ratings: 

IV. B. 2. c. 

IV. B. 2. d. 

IV. B. 2. e. 

Expected Risk Rating: 0.5625 

Risk Characterization: LL.MC.CL.MC 

Risk rating: Low 

Conclusion and discussion of risk 

There are no reports of productive infections cau.sed by Marek's disease in any species other 

than avian. The recipient- virus was derived from a vaccine strain that has been used 

as a commercial poultry vaccine the United States and Europe, as well as, Asia and Latin 

America, with no adverse effects on public heallh. Studies conducted by the applicant 

indicate that the safety characteristics of the parental type vaccine strain have been preserved 

in the recombinant- MSV. Only lhe personnel administering the vaccine will be 

expose.d to the experimental vaccine virus. The other personnel will be exposed only to the 

inoculated chickens. Studies conducted by the applicant demonstrate that the shed/spread 

property of the experimental vaccine from vaccinated chickens is limited. 

c. Environmental safety 

Likelihood rating: Low (LL) 

Degree of certainty raling: Moderately certain (MC) 

Justification for rating: 

IV. B. 1. b. v. (b) 

IV. B. 1. b. vii. 

IV. B. 1. c. i. 

IV. B. 1. c. ii. 

IV. B. 3. b. 

IV. B. 3. d. 

IV. B. 3. e. 

IV. B. 3. g. 

IV. C. 

Consequences if adverse event occurs: Low (CL) 

Degree of certainty rating: Moderately certain (MC) 

Justification for ratings: 

IV. B. 1. c. iii. 

Confidential Business Information Redacted 
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Risk Analysis 

Marek's Disease Vaccine, 

May 19, 2014 

Page 44 of 74 

Serotype 1, Live Virus, Code: 16L 1.RO (Unlicensed) 

IV. B. 3. e. 

IV. B. 3. h. 

IV. C. 

Expected Risk Rating: 0.5625 

Risk Characterization: LL.MC.CL.MC 

Risk rating: Low 

Conclusion and discussion of risk 

The safety risks to the environment are low. The host range specificity of-does not 

appear to have been altered from the parental type vaccine strain, which is restricted to 

certain avian species. Although the Field Safety Study protocol will not include procedures for 

monitoring shed or spread of-into the en~ironment during the course of the study, 

studies conducted by the applicant indicate that the shedlsprea'd capabilities of the 

experimental vaccine are limited. Furthermore, the proposed release will be confined to 

pOultry houses. Thus, the exposure Of non-target animals and the dissemination of 

experimental vaCcine virus ii"l the environment during the course of the field tests will be 

restricted by its use in poultry houses. 

V. Risk Management Recommendations 

A. Procedure 

Risk management uses information from the previous section to determine means of reducing or 

eliminating safety risks to animals, public health, or the environment. 

B. Recommendations 

The safety risks to animals, public health, and the environment are low. A risk rating of low is acceptable 

risk, with very little concern associated with the proposal. The applicant recommends the approval of the 

proposed field test by VB, APHIS provided the study is conducted in accordance with the requirements of 

9CFR 103.3, and the filed protocol of the study. 

Confidential Business Information Redacted 
Merial, Inc. 

U.S. Vet. Lie. No. 298 

Risk Analysis 

Marek's Disease Vaccine, 

Serotype 1, Live Virus, Code: 16L 1.RO (Unlicensed) 

Table 2. Calculating the Expected Risk 

VALUE RATINGS 

Likelihood (L) 

Low (L) 

Medium (M) 

High (H) 

Consequence (C) 

Low (L) 

Medium (M) 

High (H) 

LL = 1.00 

LM = 0.50 

LH = 0.10 

CL = 1.00 

CM = 0.10 

CH = 0.01 

May 19, 2014 

Page45 of74 

*If the Likelihood rating is Medium or High and the Consequence rating is also Medium or High 

use Degree of Certainty Ratings I; for all other combinations use Degree of Certainty Ratings II. 

Degree of Certainty Ratings I 

Certain (C) 

Moderately Certain (MC) 

Uncertain (U) 

Degree of Certainty Ratings II 

Certain (C) 

Moderately Certain (MC) 

Uncertain (U) 

EXPECTED RISK 

c = 0.50 

MC = 0.75 

u = 1.00 

c = 1.00 

MC = 0.75 

u = 0.50 

[(likelihood) x (degree of certainty)] x [(consequence) x (degree of certainty)] = Risk Rating 

Confidential Business Information Redacted 
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U.S. Vet. Lie. No. 298 

Risk Analysis 

Marek's Disease Vaccine, 

Serotype 1, Live Virus, Code: 16L1.RO (Unlicensed) 

Table 3. 

Risk ratings 

Risk Expected Risk Risk Expected 

Characterization Risk Rating Characterization Risk 

LL.C.CL.C. 1.0000 L LM.U.CM.C. .0250 

LL.C.CL.MC. .7500 L LM.C.CM.MC. .0188 

LL.MC.CL.C. .7500 L LM.MC.CM.C. .0188 

LL.MC.CL.MC. .5625 L LM.C.CM.C. .0125 

LL.C.CL.U. .5000 L LH.U.CM.U. .0100 

LL.U.CL.C. .5000 L LL.C.CH.C. .0100 

LM.C.CL.C. .5000 L LH.MC.CM.U. .0075 

LL.MC.CL.U. .3750 L LH.U.CM.MC. .0075 

LL.U.CL.MC. .3750 L LL.C.CH.MC. .0075 

LM.C.CL.MC. .3750 L LL.MC.CH.C. .0075 

LM.MC.CL.C. .3750 L LH.MC.CM.MC. .0056 

LM.MC.CL.MC. .2813 L LL.MC.CH.MC. .0056 

LL.U.CL.U. .2500 L LH.C.CM.U. .0050 

LM.C.CL.U. .25QO L LH.U.CM.C. .0050 

LM.U.CL.C. .2500 L LL.C.CH.U; .0050 

LM.MC.CL.U. .1875 L LL.U.CH.C. .0050 

LM.U.CL.MC. .1875 L LM.U.CH.U. .0050 

LM.U.CL.U. .1250 L LH.C.CM.MC. .0038 

LH.C.CL.C. .1000 L LH.MC.CM.C. .0038 

LL.C.CM.C. .1000 L LL.MC.CH.U. .0038 

LH.C.CL.MC. .0750 L LL.U.CH.MC. .0038 

LH.MC.CL.C. .0750 L LM.MC.CH.U. .0038 

LL.C.CM.MC. .0750 L LM.U.CH.MC. .0038 

LL.MC.CM.C. .0750 L LM.MC.CH.MC. .0028 

LH.MC.CL.MC. .0563 M LH.C.CM.C. .0025 

LL.MC.CM.MC. .0563 M LL.U.CH.U. .0025 

LH.C.CL.U. .0500 M LM.C.CH.U. .0025 

LH.U.CL.C. .0500 M LM.U.CH.C. .0025 

LL.C.CM.U. .0500 M LM.C.CH.MC. .0019 

LL.U.CM.C. .0500 M LM.MC.CH.C. .0019 

LM.U.CM.U. .0500 M LM.C.CH.C. .0013 

LH.MC.CL.U. .0375 M LH.U.CH.U. .0010 

LH.U.CL.MC. .0375 M LH.MC.CH.U. .0008 

LL.MC.CM.U. .0375 M LH.U.CH.MC. .0008 

LL.U.CM.MC. .0375 M LH.MC.CH.MC. .0006 

LM.MC.CM.U. .0375 M LH.C.CH.U. .0005 

LM.U.CM.MC. .0375 M LH.U.CH.C. .0005 

LM.MC.CM.MC. .0281 M LH.C.CH.MC. .0004 

LH.U.CL.U. .0250 M LH.MC.CH.C. .0004 

LL.U.CM.U. . 0250 M LH.C.CH.C . .0003 

Confidential Business Information Redacted 

May 19, 2014 

Page 46 of74 

Risk 

Rating 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

M 

H 

H 

H 

H 

H 

H 

H 

H 

H 

H 

H 

H 
Merial, Inc. 

U.S. Vet. Lie. No. 298 

Risk Analysis 

Marek's Disease Vaccine, 

Serotype 1, Live Virus, Code: 16L 1.RO (Unlicensed) 

Appendix 1 

May 19, 2014 

Page47 of74 

Pages 47-73 redacted due to confidential business information 

Confidential Business Information Redacted 
Merial, Inc. 

U.S. Vet. Lie. No. 298 

Risk Analysis 

Marek's ·Disease Vaccine, 

Seri:ityp'e 1, Live Virus, Code: 16L 1.RO (Unlicensed) 

Appendix 2 

May 19, 2014 

Page 74 of74 

Purchase, H.G. and R.L. Witter (1986), "Public health concerns from human 

exposure to oncogenic avian herpes viruses: A review" J. Am. Vet Med Ass. 

189:1430-6 

Confidential Business Information Redacted 
l'!JBLIC !lMLTH CON'CF;RNS 

FROM HUM<\N EXPOSURE TO ONCOGENIC AVIAN 

l!ERJ?l::S VlRUSES : A ltEVlEW 

by 

1!. G. PURCHASE 

R. L. WITTER 

DEC 12 1985 

1 FrQlll the U.S. Department of Agriculture, Agricultural Research Service, 

Beltsville Area, BA.RC-t;rast, lleltsv:l.llt;, Maryland ;W705 and u.s. Depart:Jnent 

of Agriculture; Agricultural Research Service, Regional l'ou1try ll.eaearch 

Laboratory, 3606 East Mt. Hope Road, East Lansing, Michigan 48823 

Presented at the Annual 11eet:l.ng of the A,nterican Veterinary Medical 

Association, taa Vegas, NE. July 23, 1985. 

'• 
2 

Summs.ry 

Ever sio.ce tbe ident.l.f:lc:ation of a herpesVirus as !:he 10<1.11se o~ Marek • a 

disease (liD) lind the isol.atioll, and lille o~ live vaccines against MD, th~re was 

concern among some of the lay public and some scienti.sts ;:hat there may have 

b!!en a qauaal sssociatiPn betve;~n thes!ll vi):llses ~,tnd b,WDan cancer, 'l.'llis. 

concern was projected as an inorditlllte n1l!llber of terwrts, Pro and con, in the 

scientific l:l.teratute over the past two deca!le"· ltesolut:ion o£ this qtie!ltiPt\ 

was needed to Jnak·e irifotmed decision,s about lJ:censing bi()J.pgf,cs, to lll!itkE\t 

safe and whol.e!!Qlllii! foQd prpdqcts ill, the Udted Statell and tor ~Qrt' I!,Q,d to 

proVide information :for consllllle·rs. Results from sta.Ustical epidemiology, 

serological epid!!ndology, eo1!1pariso:n of etloloJ!ic a$ent!l, laboratocy lnMUJ~Sl 

and pri~~U~te inoculation, culture i:n mall!lllalian c!!lls, and eJ:l!llli:ttst~c;on of 

!1llPQt:t1miatic 11U'ect~o:n a:nd drll.1!ma.tantia1 evidence W:!ll:!l reView!!d .l.n this 

pa}1er. The data iljail:a~!!d that no clear !!vidence of ail li'tio1.:>g1c rel<\tiO)lshtp 

existed and, although addi1;1ona1 st11dies were poas:tbJ.!!, there was en()UJlh 

evide.nc!! to support a i!irm .conclnslon. On this basis expert co!IUJiittees 

sponsored by the government con:cluded that MD was n.ot a pul;lltc health proPlem. 

··., 
Clln<~el;' is one of tM most dreaded diseases in modern times because of its 

high frequency, unpredictabU:i.ty, inevitable occurrence, prolonged duration, 

and chronic and painful co11rse Qften leading to death. Tl\ere has been 

extensive research· in this century to try to identify the causes of cancer, 

dhemica.l ca;;Cinogens, such liS coll.l tar which iu<hlcell cance:J: in chimney sweeps, 

have been elearly showll. to ·be one of the causes of cancer. Similarl]l', 

ionizing radiation csn cause cancer in persona t:nl'l:posely or :Lnedve:rte1ltl]1 

<!;r;posed to it. Agriculturll.l chemicals lilte chlorinated bydroc11rbon 

insecticides, ;Lndustrial chendcais like polychlorinated biphenyls (l.'C8s) and 

chemical contaminsnta lllte dioxins have become widely dist1;ibuted in the 

environment and sometimes in the food chain before they were shown to. c~use 

cancer in e.>;perimental anima.ls. Wi<l,e:t.y used fdods like grains and oilse<~d.s 

have become ·contaminated with mycoto:dn.s, some of the most powerful 

carc.inogenl! k.now. l!ven nat)lral constituents of the diets ox most people such 

as 111eata are irrcrtminated with caus;Lng humau ceucer, A berp<!svirus b 

presumed to cause Burkitt's lymphoma, first descrlbed in Africa, and 

ll~sopharyngeal cat.cinoil!B., first described in Chins. M:>re recently RNA viruses 

such as hum.au T-c<!ll leuket~~ia virus have been it;criminated as the cause of 

acquired immunodeficiency syndrome in ~n. 1 Enormous efforts have been 
dtr!lcted t(.> understanding at the nu>lec\llar level the sequence oJ: events that 

results in cancer. UO;til these events are fully underqtooil. by sdedt::l.sts and 

the infomation is disseminated to the general public there will be a c.ollc,;,:rn 

by the gen~:i:a;l. .pqblic tJml: items :l:n their food and enviroli.mellt to11ld ind11ce 

A review of the public health aspects of aniDlal t\Uilo:r viruses was 

publi&hed in. 19(io, 2 !n ;l.:t, ~:telen. examined thi! rela.tionsh:tp bel:W!!en a 

var:l,ety uf viruses knOW!\ at ~hat time and humim disease including canc.er .lind 

iJmnquqsuppressiQn. He concluded tha.t "over 100 anii!i<ll tilmor viruses ha:ve 'Peen 

iaqla.te<l, and ()illY l;lt;e {Yaba .Tillllqr Virus) has ~inor public significance. • 

However, the heJ:pesvirus of l'fll.rek' s duea~e (t!D) W<I,Jo n_ot isoA;~.'t;eq u.n.t:J:lr 

J.:967l' 4 and the herpe:sv:t:r\lll of tl;rkeys (HVT) was p_at isolate:i:l \Ill til :!.~$9. 5 

W;ith lihe ident1!ieadon of a v,t:rqs as the callll~> <:>f MD l!.nd publicity 

~urroundi~g the development; of vac.niues against 110, 6 there has beau .concern 

among soine ot iiha la;y publ.ic and cSOille. silietitists "that exppaqte to the 110 virti>! 

an.d J:Oil.ated Jl;patho$eli..lc Yl!ccine viruses in th<> l!nv.trot11ll,e:nt, or eat::l.ng po!l.l.f!'tr 

infected w:l,th ·these viruses, could .contribute to the cause .o£ .ca.nc~r in man. 

The question ()f wh.etller thet!! 'l.s l! relationship betw!!en taalle infectototis of 

poultry and diaease in man needs to be resolved so tha.t :l.nfonei:l decisions can 

"• 

! .i 

l 

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5 

be made about licensing of biologics for domestic animals and man and about 

the ~af<i!tY and wholesom<i!Uess of poultry and poultry product$, Also, the 

pre.,<i!nce of; .,uch lt1ruse" in meat e>;n be used as a non-tariff trade barrier to 

prevent the export of poultry and poultry producta. ~stly; such infoP»ation 

i" needed to assur<i! poultry termers and processing plant workers of 

occupational aa:f.'ety and to assure conslllllers that p!ltdtry and p!luitry products 

a:re safe and wholelfome to eat. 

Many sc:l.ent:i.ats hsve attempted to examine th<i! association between canci!r 

viruses in animals and cancer in man using diverse approaches. the MD virus 

is ubiquitous and vaccines against MD are used woddwide (appro:dma.tely 

3 billion do!!es of live vaccine ar~ Use<;! in the 11. S. alotte) on !l!eat ... producing 

(bro!lers) and egg-producing anitlla~s. Humi.ns haV<i! been exposed to _MD virus 

ilirice dilmestication of the chicken and to vaccines agdnilt MD for al!i!ost 

2. decades. There has been atnpl<i! opportunity for hnma.ns to cont~ct: the 

virusea. thus an asl!lociation between these vi_ruses and human cancer could be 

revealed in epidemiologic studies, There hss also been extensive 

experimentation with these viruses in chickens, ~aboratory animals, primates 

an'd humans • 
The voluminous lite~atut:e rdating MD virus and vacciMs againat MP to 

human disease is often contradictory and has neve;r been reviewed in depth, 

'l'he 1aet: ~:eview focused almost exclusively ,on RNA t:lllllOr Viruaes', .part:tcu1a,-1y 

feUne leukemia virus. 2 l'lrl& comprehensive raview of the. literature will 

proVide :re'levani: data to guide scientists and resea:rc.h l!dm;l.nist;r:ato:rs ;l.n 

deciding where additional t-eaeat'eh is needed and nfult res.earcih app:roaehea to 

take. It Wi.l.l pltoVids !l<:bl:l.nlstrato;t's o:f federal. and state !lction and 

regul.al:~lli'Y agencies With the most complete compilation tlf informstion 

publiahed so fat to detetm:!.ne whether a hilllllln health haza'l:li exiBts, the natu;re 

and extent of the ba:r<al:d and whether actiCI!ls are necessary. 'mlb 'revlew w:tU 

be Qf !ntexest to the p:oult;r:y :tndt~stty, wh:teh would suffe;r severe ~co;~;omie 

111 this review we hllV<a :SillljJnat'iZlld the da1:a, developed Mnclus:tons that 

follow logically from the data, 11111d discussed ph:l.losophically!!(l!tte of the 

dilell!!!Uls :in the scientific process. Mention is made of efforts by tbe 

Government to deal with the 'issue by e~tablishlng committees and co!!illiissions 

td study t.he llVsilable data. We Will conf:tne our ol>servat:f.(lne and r-enurrl.ts to 

the public heal;:.!\ B;!!peeta ol: !;he avian hei:peaviruslls nf MO and the 1:1.Ve Vitt~s 

vaccines, including HVT, that prewmt MO. 

'• 

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' 

i 

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I 
7 

Avian ()neogenic and Related Viruses. 

Marek's disease is a lympho-proliferative disease of chickens caused by a 

berpesvirus3•4 and· clla:racter:l.zed by a mononuclear -cell infiltration and 

proliferation in peripheral nerves, gonad, iri!l, via.cera, muscle and skin. 6 

It :J.s ptobabl.t the most colilmonly occurring neoplasm l)f anY anilll4l. tn 1!1~7 it 

cauaed annual lo!lses in the United States in ex~;ess 9£ $1.50 mill;!.on ami 

silllilar losses w•u:e incurred in other countdes of the wotld. Losses ate d.ue 

to dectt~ased agg '{lroduction, mortality from MD., incl;eJi&ed mottlility fro111 otbe~r 

causes, and condemnstions of carcasses in the poultry processing plant. '.the 

diSease and losses from it oc.cur worldwide. 7 

The herpes virus t:hal: causes MD is ndt transmitt.ed vertically fro•l parent 

to oft'sp1iing. 6 There£ote eggs are free of the virus. It is transmitted 

hoi'ii'iontally in the dust and dander that sloughs of£ from the feather follicle 

epitheliWII :l.n which the virus matures to its infectious form. The virus 

'shrvives ·in the environment for several months at room temperature, Moat 

f1deks become infected With the virus within 2 to 3 weeks after ha1:ching and 
the virus spreads to most birda within the flock by a:ppr!):o;~m!lte;Ly 1 weeks of 

age--the aae wnen mQet 1n:oilers are 9laughtered for human consumption. ln 

11 ve birds, the virull persists in infected cells in the blood for .Bever!il 

years and pr<ibably :tn,deUnitely, Virus can be isolated J;rom virtually all 

body tissues, inCluding muscle (meat), for ptolonged ll.BdQds, However, egg!! 

are considered f1:ee of v:l.ruG• Tlua v~rus is h:l,gbly ceu~sssuciated in a.ll ... 

infectious. Beeause ths virus 'la found worldwicle in chickens anll is found ill 

jungle £owl in theil' nat11rs'l ~!Yl,tat:, :l.t is 11\t\'!ly tha:t manldn4 l:las eonsumed 

virus-infl'lcted chicken meat- since long before domeatication of 'the C.hiclten, 

Vaccines are .uaed to control •MD" in all {'!onn:tr:l,"e of the worii!. tha:t P;l,ve. 

intensive poultry produet:l.on systems. Vaccines a::re composed by ehe eeil~free 

lyophUi;~:ed v:l.!;us Ill: ceU-iaSsociated virus in !!ell cultures #hich haVe bl!eu 

frozen in liquid. nitrogen. The virus most co!m!lonly used. ia HVT, which is a 

ncln~pathogenic vi.~s salliSen:l.eally r!Ola.t:ed t.o MD vims w'hleb. w;;~~ :Lsolated from 

5 turkeYil• Other vaccinea include attellUll.ted MD virnl!es llntl lllltura.lly 

apathogenic MD viruses; The natunily apathogenic virus is gaining more 

'' 
9 

acceptance as a vaccine worldwide. A l;lival!!n~ vaccine incorporating HV'l' and a 

naturally apathogeuic virus was licensed in the United States in October 1983. 

All vaccinee are i~:~.jeei:ed in~o chiclt!! on the day they hatch, The vacct'lle 

VirulleS replicate in the chickelll! and persist in thl:!lll for life. Of the three 

vaccfue viruses only the na..turally a.pathogeuie one spruds rudily 

horhontally from ch:i.clten to chicken thOugh aome of the attenuated viruses 

spread poorly. 'l'he vaceil!.es are highly effective in prevl!nting i:be lesions 

and economic las~es from MD; however, they do nat prevent infection with MD 

Virus nor the :spread of MP vUus. hodzontaUy. !>bat of the· egg-type chickens 

throughout the world and moat of the mest-.type ehickens 1i1 some count:tiea such 

as the United States, Canada, and Ita1y are vaccinated. Thus, most chi.ckens 

at sla:ughter contain both the virulent MD virus and the vaccine virus. 

The pathogenic MP viruses of chickens, the apathogenic similar viruses of 

chickens, and the l!VT are serologically distinct from one a!lother. Because 

wtr and the apathogenic viruses do not induce MD, they eannot be referred to 

as MD Viruses or as avian oncogenic herpesviruses, Also, because HVT occurs 

naturally in turkeys, it ·cannot be referred to as a chicken virus. However, 

' ' 
-.;. 

the Yirus:es ate an.ld.gellic.B:11y closely ~;ela~ed though diatinct from one 

al10l:her. ·'fhey have been called serotype 1; serotype 2, and serotype 3 MD 

10 

vi !;'Uses, reepeeti veiy. Fo-,: .the pu-,:pose of tllis papel:', we wUi tefet to t:he!il 

as avian. (oneogen;to:: or oncogeniaally..,re1a.ted) herpesVirua.es (;AOHV). 

Review by Expert Commit·tees 

In the Unit:e.d Statl!s of ·AII!etit\s (USA), veterinaty bioloS:ie!l &t'e te$UU·te4 

under the authOrity l;>.f t!1@ 'Vi:tus~serum ~o:z;in Act •. The ·u.s, DepaJ;tll!en!: ilf 

Agricul.tu:re (USDA) is resPonsible £or assuring thst snc.h biologies a'j:e puJ:e;, 

safe, poteut ani;i e'fficacio\Ul. With the dtac.ovety <>f the c.auliative he1:pea 

v;l.rtis of M:l :l.n ·1967 anti tlte ia.o1atf9n o.f the herpeaVitus o£ tur~eYII {lM) ;l.n 

191:i~, 0 <l,l!est:tpM were t~lised aboU:t l:l!e aa.fety Pf MD virua~i!lfected chickeilS, 

l)r. ,Jesde t, Steinfeld, SuJ:geon General of the Public: Health Service, 

conv•,med a committee 'l,u 1970 to :review the !lt;at\ls 11n<'! cutrent :relationdhip of 

-... 
u 

viruses of the avian leukosis complex t_o h!llMn dis!lase. a Dr. Steinfeld made 

the folloWing apeeif:l,c point!l• 

"Even though there 1s no known association between viruses which are 

common in fowl and. in chicken eggs to human disease or any known hazard to 

hllllllUi haalth, l pel_ieve tile o)l].y ratieinal_ goal we l!hould .strive for is 

eliminat:l,ou of thoae virus&~.~ £rot~~c both eggs and. poultry 1;10 th;li: there can be 

uo poas:i.ble human health hazard associated with t:he:tl' ingestion 411d so the 

fowl pcrpulation will be a more hesl.thy oll!'l." 

A,ttach<!d tO the letter was a report wh:!.ch seated in part "a large number 

of k,ey invel!tigatora who have spent many yl;!ars st_lldying_ the avlan and other 

animal cancer viruses looking for liuks. with human cancer having encouuteted 

M inst11nces wh:l.ch C:Puld re!laOU!Ibly lead to the poat1;1lste that: these. cancer 

virua11s have been naturally transmitted to msn or even, !or that matter, to 

other animal species. Thus the evide!lce sud s<;ient!flc literaturE~, as well as 

the Pp:l.nions of the best sd.entists in this field, all add up to one 

11J. L. Steinfield, Surgeon General of the Public Health Service, Public 

Health Service, Department of Health and Human Services, Washington DC. 

Personal communication. 
12 

cqncl!la:l.on--not only 1s there no eid.de!lce !:hat "poultry ·product$ .are 11. ll!i~ard 

to mali all a possible cause of cancer, but that the data strongly suppoJ:t: a 

positivi!! l!tatelll=t that t'l."ans,lllil!a!on of cancar f):Qlll chickens to man does not 

occur.~~ 

Priol: to ~he odg;l.na.l licensure of the herpesvirus of tutkeys (Ji,VT) .in 

1!110,6 the iiSliA requested the. De!>art~nt pf Health and !ill!il!ln Serv!c.es to 

:rev:l;ew the pul>li~ b.l!a.:li.l:lt imp':U,alitt<lns <>f su:C:h ·a.n a:tid<lli· 'Experts w~re J:al1ed 

to recview the "U:tersJ;1,1re and all infotmation avaiia:ble. They conc:iuded that 

ltcensing the vac;c:tne woUld pon no public health prohJ.em. b 

In 1983, the USDA raised aim:l.lar concerns regarding the licensure of the 

SB-i MD vacofne. An Ad' Hoc Coriun:l.tt'ee or experts again reviewed the pteaen:t. 

state of l<l>.owleiige, ":Lt w~;~a tile tl11!lmittee'Jl optni!!n th?;t wtthfn the 'Umt~s of 

bPeraonal collllllUII.1cation. D. E • .Eapeae·th, Veterinary Bio:logica 'Sta££. 

Animal and 'l'lant ·Healtll Inf.ecdon Service, I;yat.t!lville, f.ID 20782 .. 
13 

the available data there was up realloll. to anticipatl! any public health 

' -- ""' - .. .c problema from using theal! vacc:ln~>a. 

Review of th~> Literature 

!)tatiatical Epidemiology 

If there were a atatbtic:ally significant correlation between the denScity 

of poultry (numbet produced in a country) and a patticulat type of cancer in 

}\umanSc, oJle·might infer that there was an etiologic relationSchip between the 

.two. Several sCientists have attempted to show such a correlation (Table 1), 

The death certificate files for the state of Washington for 1950 to 196.7 

and t:he 81:ate of Oregon for 1950 to 1964 were examined using .a. case-controlled 

method,8 Among 5368 c:ase•control pair!!, there ·was a statistical:!.y 

significant :association between :fa:rmlng occupations and death :fro!ll leulc.emia 

ll.Personal communication, G. F. V'ande Woude, Laboratory of Moleclilar 

(jncology, National Institutes of liealth, Bethesda, MD. Currently at NCI 

Frederick Cancer Research Facility, P.O. :Box 11, Frederick, MD 21701. 
and nrul.tiple myeloma,. No such association Was seen for Hodgkin's d:l.l;leaae, 

reticulWll cell sarcoma, or 'other lymphomas. PoulJ;ry famers showe4 the 

h:l.gh<1.11t proportionate <;:ase excess, :l..e., ten cl!aes dyina from leUkemia and 

0nty one m4tched control, a difference that was stat:l.sdcally a~gnifl.cant: at 

P< .05, 'Xhere were h:l.!!bet; hequf!ncies !lf leukemia deaths among 

orcbardiats,wheat fatme:t's, dairy tamers, ¢a,t1:1e r@cher!l, >turae~n, ;l;aru!era 

(no explanatl411 given), and farn lahol:'er;~ thOugh the d:l.fJ;ereneee we:re not 

stat:l.sti.ceUy s:l.gniticsnt,, 

Ail ap.a,l.ysis qf ~eath 'in C1.!1Uo~~ tarlll. and non-farDl. re,s:l.d~ts b-et:wl!e~ 

1959 lind X9ll1 snowed a significant <P.£0.05) reduction ·in m:or'taiity :trol!i all 

cancer :l.n farm re:s:l.deuts thl>ugh the toliJ!.l mortal:!. ty was s1mi1ar in f~rm and 

non-fllr!ll residt~nta. 9 <J'fprJ:ality <from leukemia vaa i;l!)r~as~d though no<t 

s1,gt!:l.fieii!ntly sp, '):he proe$dures used invoived caicul,atin~ average 1\!0rtal:l.ty 

ratea Uoilt tl\e l!!li(} ti .s. Census for Calirotuia and using these rateB as 

populations and to calculate standardized mortality ratios between ~ected 

lind actual d!laths. 
1.5 

Th~ posaib),e relationship beti<een h1l!ll411 eane.et d,eatha and exposure to 

pQultry was studied in 1,019 collll.ties in l:en so.utheastern states for 19.50 to 

1969.10 Deaths from uterine cervical and ovarian cancers and from multiple 

myeloma were sign:l..ficantly (1'.5_;.01) higher in counties with high poultry 

poJ?ulations than in counties with low poultry populations. However, the 

sigidfieance for the latter ti<o cancers disappeared when the data were 

compared with data from the total USA. No excess risk l)f lymphomas • Hodgkin's 

disesll&, and lell\(emi!l in Jleople oeeupatiow(ily exposed to poultry was detected. 

In another study, records of death certificates fr0111 Nebraalca for 19.57 to 

1974 wer.e elCallU,n.ed.n Leuk~a deaths (1.084) were each matched with t:wo 

dea.ths (2168) from other causes for age at death, year of death, county of 

residence, race, and sex. There was .an elevated rtsk of leukemia among 

fanners which was statistically signific!lnt (P_50.05) for fanners born - . 

between 1901 and 1943 or who died beti<een 1957 and :!.965. When t;he mortality 

was atrat:l,fied by county of reai.dence and compared with various agricultural 

pJtoductipn factQrs, there was a significantly (P~O,()s) incre;:>.sed i!lcidence 

of .leukemia among farmers from heavy com-producing counties. Also there was 

a higher risk from acute unspecified leUkemia. !ll)lOng farmers from heavy corn, 

hog, and chicken producing and herbicide-using counties. The study using 

similar methods wall conducted in Iowa.12 and invol.ved 1675 leukemic deaths 

-. 
16 

lil:>d .l3,50 c<>ntrola. I.~itikemia. mortality was higher among :famers ( P.:5_ ,-'\)S) • 

Leukemia was also higher among certain age groups o:f .:farmer~> in <;OU!ltiea with 

high soybean, co:i:n and egg p:i;Od!lction and largest li\llllba~ Of a~:res treateit with 

l;erbicides. )t)rtal:t ty from unspecified lymphatic le\ikel!!ia was assoe!e:ted with 

cotn per acre. nlli!lber of !Dilk: cows and nlli!lb.er of egg-laying chickens. 

~not:her ·case-controlled study was conducted 1n Wiaconain uaing mortality 

data from 1968. to :1117!113 invoivin$ All deaths from iiidd.ple myeloma and' 926 

careft.Jll,y lleleeted cQ'Iltrolll. }~lrtality f:rom multiple mYiil.O!!U!, W~J.s 

sigo.lficati.tly (P.:5..0!'i) higher slilbng .fa:t'ml!':ts than non-farmers particularly 

those :tesiding in col,lllties )l.igh in .chicken. 1nven.tcn:y, ~er~iUJ~;I!r us" • ·and 

acrres t.-eii.ted with Jnaeeti<:.fde.l!. 

An intenl!ting J>pidf!!lli!'ilogical !inrvey of e!lophllgeal <;anceJ:;.waa eqnduct;ed 

in No:tth (Jtdna betwe!!!l :1.969 and 1971l4•ti !he mort!liity rat:e 11'1. !:he tw.o 

highest eotiuties waa t~e;rdy 97 t;imes the moJ:tallty rate hom the esophageal 

cancer in· the two lowest rate counties. The io.c:l,dence Of esopb;\j!e&l caJICel!' 

dMiUer RW: .Yira.i <liseases. Childhood Cancer Etiology Newalette:t l977; 

November 15, 044. 

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I 

; 
Qong humans paralleled the incidence of esophageal cancer among chickens, 

there was a higher incide)lce of n:t troaam:l.nea in food samples from the high 

;l,ncidence county than the low incidence county. l{o statistical analyses were 

perforD!ed. 

Fro111 the ab_ove; there are three possible conclusions. The first is that 

the relationship between h1lll)sn cancer and cancer in chickens (including W) is 

-coincidental; the second is that an unknown factor such as a chemical or 

envirolll!lental factor on chicken farms is responsible for both the cancer in 

ch;!.ckens and the cancer in humans; and the third is that there is a ea_usal 

relationship between cancer in chickens and cancer in humans. From these 

data, one C<!litlol; select ~ong these alte:rn,aUves and so n:o conelusio1lS with 

regard to the public health implic<!tions of AOHV can be drawn, 

Serological Epidemiology 

'l'he examination of human sera for antibody to AO!f!i and the examination of 

avian sara for antibody to human putative cancer viruses have also been used 

\ - - - - to show the relationship between cancer in chickens and cancer in hUlllans. Of 

64 healthy human subjects examined by the indirect fluorescent antibody teat 

for antibody to MD virus, all but four, who were young subjects twenty ye,ars 
18 

old or )'Ounger, -were posl.t:lvt1!.15 Converae1y, of <'ileven ·ch:l,cken sera 

~txa!ltined tor l!nt:lbody _to Ell virus-. eight were positive. Ant1-'MD titers ra,nged 

fr= lUO to >1#640 _and and~Ell titer$ va,ry from 1:10 to 1:40, A aubaeq\!a!lt 

:~-J:;ud.y ,from :the .s~ l&bora.tv_i""f exarnine_d seta from 7_2 humans whose work was 

inti!IIStely conne.c:ted V;!.th chlekens aJXd from n. office workers who -did not have 

much contact w:lth chickens. Seta from .70.9% and 16.4% of the workers in 

polil.try fatms 114!1 IU!.ti..,Hl ani! al:lti-tili v:tt'tia .ant:ibPdy t:f.tets ·Of 111601 while 

only 20.8% sud 32.ii:t of the controi sera had -ant:tbody·rel!peetivaly,16 Also, 

of the 48 sera from ·cynomologus monkeys, twelve had antibody to MD virus and 

In a subsequent l;tudy ;tn the USA, ·205 h1llllan se>;a were examine.d foA 

'11 antibody to HVl' and MD virus, l'he sara came fro111 "er~ons lf:tth a Vl!ried 

SlDOUI\t Of eJCVOSUre tO aVi_!ltl harpe!! '(iruses ,_ );rom Bul;k;l, tt '!1 lymphQ)lla p;~,ti!i!JXt$ 

and from students. Sporadic samples had tit-ers ·in the fluorescent autihody 

positive by the fluo-rescent an:tibody test had ·MUt:rali:zing sutibody, Th~l:ti! 

was no pbvious te1ationabi_p beween the extent of contact with .AOHV .and ·the 

' - - . presence of detectable antibody. 
The reasons for the cross reactions between hwnan sera and AOHV and vice 

!!!!!. are un:lqwwn. They could have been caused by exposure o£ the chickens 

and lluma:ns tQ an !'gent or agents with a common antigen. The low incidence and 

low titer of antibody in both chickens and humans against the virus of the 

other species makes it unlikely that diseases in both chickens and humans are 

caused by the aame·viru_s. However, a common antigen between the avian and 

h\llll.!ln v!rus$ is !I pQSsibil:!.ty, 

Compatist,ln o£ ;Etiologic. Agents 

There al:'e common !lntigens detected by different immunologic_al tests among 

many o£ the he:~:pes viruses, Shortly lifter the ;taolation of liVT, it was shown 

to have commoa. antigens with MD virus i1;1 botb the fluo.res·eent antibody and 

5 illlliiWlodiffusion tests. Infectious bovine rhin(ltl:'acheitis, EB virus, and MD 

v:Lrus ~1'1.0 .have common ut:igens ]ly these t!lsta.18•

19 In one set of 

exper.i!nel;lt;8 MD virus, b!it not HVT, cross-reaete!l With_ paeu!;ior<~bies virus by 

. . . . 20 the fluorescent antibody test whel:'eas in other experiments both viruses 

reacted. 21 Some workers were uaable to show a croas reaetion between MD 

vibJs and EB virus. 22 Collllllpn group-apedfie an.d \lllique type-'!pec.ific 

antigens have been demonstrated between Luck' renal adenocarcinoma of frogs, 

. . . . . . . 23 MD and EB v:Lruses, hl!rpes dmplex and cytomegalovirus. It is not 
20 

I!Urpt'ia:!,p.g that viruses tha.t are evollltioilarily :c.lo11e1y :r:elated share common 

antigens. Sollie o£ tllel'!e vil;uses, like MD virus and EB virus share the ability 

to traha:fom cells. However, :i.rrfeo:.tiv:f.ty and replication a,ppaa.r ~o lle !lighl;r 

hol!lt-speu:ific. Thus, these data provide no evidence that the AOHV 'l!!i!Y cause 

TU!liOl" polyll4cclla:ride lltll>l!tancee on MD tumoJ;a and MD l¥tfllicted cells had 

common ·g,I.yccproteiA aut:l.genl! with a variety of htiWln twnor11.24 •

25 Virgin 

fewde guinea pigs· wl!re iliiDluniz ed with tUJDor polysacCharide subs tancea from 

the .avian or hU!lllln tt!lllt!rs. Uterine sepents were then exposed ·to the ·eXtract 

and con'tract;ion of th'e sepen,ts iueaaured. The utet'i we:r:e carefully 

desensitized to !torma1 autlgenl! •prip;; t:o testing, Sti!,t':bticall;y si81i'tfii:ant 

(l',$,01) rea~;.t:tt!M W~~<:te. ill>t;t.ined with a la:!:j!e variety of human cancers 

including leukemia, lYJ!!plioma, I!odskin's dise<>ae, ca:r.ciJ!Plllll of' the P'll).C.!:'eaa, 

stonm()h, colon, kidney ¢.lid lung and sal.'<:olllli. of the bladder lntt not w:i.J;:'b. no.rl!lal 

ch:t.ciketJ,, lli:Pna_e, or I:IU!li8U !;issues. Only 11% o:f; the t;e!Jt i"eslllct:s Jl'are poaid.ve 

when uo:cm111 antigens were u.sed whereaa 20.% of the carcinPmas , ~0% of the 

sarcomas and 85% o:f the lympnontas wei'e positive. Thus there are common 

d:oss-teS.et:tve l!lltigenl.l between MD sud humap, t!lll!ots; the nlltute t!f th!lse 

antigens b uuknown. However, this observation does not necessarily 11.nk MD 

vtrua and h~an eancet. 

I 

' ' i 

f 
21 

Pass:i.ve illllllw.tity could be transferred to guinea p:i.g uterine segments with 

serum from patients with ~81\Cer. However, using this test 63% of subjects 

with diseases other than cancer, 74% of aubjects with. carcinomas a:nd 5.7% of 

subjects 'il'itll !llelanoua and sarcomas reacted t:o MD virus antigens. Only 6% of 

26 these sera reacted with herpes simplex virus or l!:ll: virus. Theile findings 

t:end to bring into question the specificity of the test. 

'When deoxyribosl!-purine fragments from variou.a cancerous tis.sues were use<!. 

to :i.mmani:te guinea !'ig11 and the uteri war!> tested with tumor pol.ysaccharide 

substance as rfl!ieasing anti3en, reactions were obta:l.ned between cancerous 

ti!lsUes and vird antigens at dilutions of 0.8 to 4,9 x 1012 whereas reactions 

with control al\tigen were 0.4 x 1012 and normal iung tissue did not react 

with either viral. Pr epntrol aut;tgen. 26 ~en vir~:~.l ppl.ysaccharide substance 

ant;l.gens fro111 MD virus, Ell virus, herpes simpl:u: vituaes types l and II, and 

mouse hepatitis virus were used as releasing antigens and MD virus and EB 

virus used as immunizing antigens, cr<Jss ~:esctivity was obtained. The highest 

t:!.terll were obtained with EB virus and MD virus and the lowest titers with 

these two viruses all.d herpes simplex and mouse hepatitis virus, 
The above results confir,:n the presence of common antigen~ amou11 ma,ny 

herpesvituse$ from diffetent species of amphibians, mammals and birds. 

f!oweve:r, they do not relat!\1 tp tl!e ptib:Uc l!ealt:h ;tmp!i<::ati<liJJ:J of AOHV, 

AtteQlpts have be-en made t9 sh<;>W that avian herpeeviruses .have 1ir9ader hpf!t 

range th,an just aVian species by inQculating the~~~ :tnto laborl!tory .anin!alli 1 

part:tcU1ai.l;y m!UIIlMls. Succ.essrul induction of malignancy in 111ice :foi1otif.tng 

inocul,ati<ln qf t~o.t ma,1:J>rl<il 'b:o1l! cMekenl! wihlt ·110· w~s iiMedbe!l in A $hart 

abst:raet.e l'AI! mat11tial increasl!i! 'the iuc:idence of lymphomas £tom 3i to 

apptoximately 30%. Additionally, bununi.tation or mice with HV'r protec:ted them 

against challenge with mouse ·tumor JUate:rial induced by MD tumor 

inoculation. e No subsequent studies have been reported, Others have '!:ailed 

eA,tuoa JM, ~;telsen Ail, Morse PA, "Wetder AA: Increased n.umher o'!: ,lympboma·s 

ii1 udce inoculated with Ma.tek'a disease tuinot. In AbstJ:acts of the 71th -.-.. 

atinua! meeting of the Auledcan Socl.ety £01; Ml.crobioJ.ogy, New Orlesna, LA. 

8-13, pp 92, 1971. 

...... ' . I 

l 

l 

t ' 
23 

to induce tumors in Chinese a!ld Syrian hamsters, and. Lewis rats inoculated 

27 when new born and kept far six months. .No viremia or antibody deteetable 

by £1uarescent antibody immunodiffusion tests was present. 

Inoel(l.at;l.a!;! or t!ln cynomolgns, 8 rhesus, and 2 bonnet monkeys with 

cell-associated or. C:ell-'free l!VT or cell-!ls!lociated MDV failed to induce 

. . . . . . . 17 tumors or other diaea.se after ll to 31 months ob~;ervation. Also no 

Viremia was detected. Sera of 3 HVT inoculated monkeys t:eacted With HVT 

antifilll:t iln a fluorescent anUbody test, whereas 8 Ill). virus inoculated monkeys 

reuined negative. No neutralizing sntib!'dy was detacted. In .a f!'ll.ow-up 

st!ldy 14 new born, 25 juvenile, and 24 adult mat!lloaet l!lankeys were :tnjected 

With similar matarial and observed for 12 to 18 months.2

li No Q.Unica.l signs 

o! di!!ealle att:ribut:able to tha inoculum were seen. None of the treated 

monlteya wa.s vmmic when tested approximately 4 weeks post inoculation. Sera 

from o~ .Qf l4 marmoseta inQculated With liV:r and of 2 of 2.9 .mamoset.s given MD 

vi:rusc reacted ,against HVT or .MD vii'liS in a fluorescent antibody test. Howavet:, 

pre:l:noculat:ion s.ers from 2 of 3 reactor marmosets were also positive by this 

test. these results could .t~ot be confitmed by the v:l.rus neutral.itat!.on t~st 

an.d thus the specificity ·of the s~rologic reaction.s remains equivocal. A 

similar absence of tumors and viremia was reported in 5 adult rhesus monkeys 
24 

inoculatll.d w:!:l;h a non-Qnco~tet~ic $train of MD virus. i!? l'r1nllite inoeulation111 

also yielded Mgativ~ resul.tll ~ith an attenuated stratn of MD viruaJ 

.The absence oJ tumtn:- lnduction in pril)l<ltes certainly lends sup!lort .to J;:he 

conclusion that 'ldJ)·· ·vil."lls and IIV'l' are iion~pathogenie in primates l!.nd, by 

extensioU., in bUllll!.'!rs. Hol@Ver, :because of the iiature of the e:r;J)etfme1lts, iio 

definitiVe conolus:l.ons can be iiraw. 'l'ha AOHV' hav~ ·a rkrr61<!' hp11t r.l:n;ge and· 

qnly infect and 'i"l'!pllcate in a few sped.es of birds. 'they do not a.ppear to 

infect and replieate in malll.lllltls., including primates. It is there.fora UiU.ikely 

that l:hey will infect. llnd repli¢ate in hUllll!.ns. 

lf AOHV' coilld prop!lgate read!1.y !n mammaUan cella" then they might in£ect - ' ' 

l!lllllillUtll!, The IIVT produte<l .a eytopatbqgenic. eJ;fect !11 hillill~t;er kid.11ey agd, 

l!iada~r eplthe1ta1 cells:. 410 ·Tl.te vlrus :was paaaage4 successively in hamster 

.ceUs and tQ ¢hieken embcy!l cells, io.t>t:laet\J'!ll p:repa:t!')d :l!l J::hililtena agaJ.nst 

this Virus ·nelit:rldized it but antiserum t:o herpes simplex v:ttus type l. or type 

.fdeBoer G: Central llieregeneeskundug Instituut, Lelystad., The Netherlands. 

Personal communication. 

. 

' ! 

I 

j 

' • l 

~ ' 
25 

2 did nut neutraUze it. The MD virus and HVt produced a cytopathic effect in 

hal!lstet kidney cell.s even after 6 to 7 passages onto newly prepared hall!ster 

31 . . . kidney monolayers. Virus -particles were found in infected cell cultures 

and the cultures contai.ned ext:emaive polykaryocyte.a. 32 the virus prop';igating 

in hamster kidney cells was iaentical to the HVT used to inoculate those cells 

by complement; fiXIltion, fluorescent antibody and serum neutt'alization tests. 

Subsequently, Ml) virus was also shown to replicate in hamst.er kidney cells by 

l!erum nsuttalization, complement fixation, and fluorescent antibOdy testa. 33 

S:!mila:rly, HV'l' and MD viz:us also propasated in p:ri!llary gu:f.nea pig kidney 

11. 34,35 ce s. ln an e;~ttensive and detailed examination of. this phenomenon., MD 

Vi:ttiS or ll'Vt' J.nf.ect;l. Vil:y was fo!.lnd to persist for up 1:0 ten serial passages in 

pt;!.lllary hamster kidnli!Y cultures inocul,ated with cell-assoc;iated MD Virus or 

ltVT. However, infect:iV'ity was only present in these passaged cultures when 

avian cell·s, which were pr~sent ill the inocjllum, persisted :lu the c.,Ut:ure.a. 

tnfectiv:lty could be eliminated by treatment with cytotoxic anti~avian cell 

antibody and .:ell-free virus was unable to infect th<l mallll'l1alian cultures 

aloue. 36 Others have been unable to infect primary hamster kidney 

cultures.27 

MD virus was unable to produce a cytopathic effect in cultures of 15 

m4ID!llalian species including dog, cat, m.ink, swine, bovine, goat, equine, 
namster, mou.se, ·bat, -ra'bbit 1 potprqo, sp()t,teq dolphin, sreen moti.key ·and 

hU!l!8.n.;l7 S;l.l!lilar reaul:ts were obtained with HVT on human, dog, cdi!, 

baboon, rabbit and pig cell~ ,

30 Even after the llvt was p~opa~t..d :i.n 

hamat:er ki<luey cells for aevetal passages, it was unable to inreet these 

tissue cuitures. 

.• 

26 

Neither MD vltil's nor liVl' would repl;i~ate :i.n. cell cilltl.!ril'i! tit u,ttiiol!!!!t 

kidney, Ullll;lnoset ~l<;in and muscle,, hlilnan -embryonic kidneY> and the hUiila..n -cell 

line ·wl~3a.Z8 'l!reat~teut with ililethy:tlll'n:f.tioeth:iiidextran or 6ltrliv;ta1et 

lrradiJ,ated SeMai 'Yit1111 ·or co~.c\llti v!!Hon 'wit:h X"--irl;'adiated iv-i.m"':i.l\feeted 

avian cel'la. <Hii not tesillt :tn teplitation. ln another .stull.y, ceil"'sa.shc:litted 

W ViJ;us and ci!il.l-ftee llVT dtd n()t infect Chinese ha!l!ste't, Syrian :b.SUIIlt~r. 

bovine, do)!, human, rabbit t!r ,!)OtOl"!fU$ ¢<al,ls. 27 Simil.a"!.' "!.'eelllts ,have heel! 

obtained by othet's. Jll,S9 •40 

Once again.mamma,lian caUs were refractory to infection even under 

conditiollil of artlfiC:i.d ce:11 'hyl:f'tidtzati()11 'between v'lrus-infectei! ce1is and 

the lliSIIWUiliali c;ells-With t!lt"!.'aVio,l.et iMC'itiVated send!;i virus. Thuli AO!W 

replicate in hybrid cells composed of'·mammalian ad avian hetel':()~aty.ona • 

. -,,, 
1..7 

Viruses that fail ~o infect ~ammal~sn cell cultures are less likely to infect 

nu1llltnB.ls than are v:irus<aa that infect and grow well iri ma.nunal.ian ceU cultures. 

OpPQ~tunistic. !nfections 

Birds effectii!d with ID and heaJ.thy birds were eL!llllined for the presence of 

bacteria that might pos11 a public health 11<\zard. ~-te_tobacteria were three to 

~ne times more freqne'(ltly encountered in the visl.'lers. of diseased birds than 

in the sue organs of controls. In two cases Saimonella were isolated from 

affected chickens and in three cases enterobacteria were isolated from the 

musc.u1ature o£ affectea birds.41 The b~rds with overt disease, in thl.s case 

MD, would be eXpected to be less re!!~Stant to bacteria,!. infect;ion. J!qwe:ve:r, 

these infection& are likely no more ~ommon a~ong birds affected with MD than 

among b:l,rds with ot]J.er ailments such as injuries. 

Circumstantial Evf,dence 

There have been many reports- of an association between cancer in animals 

and cancer :l,n man. There have also been reports of organisms bolated from 

human and animal nmlipancies which are highly pleo!l!Orphic and variable in 

many characteristic!! and which are capable of developing virul!-lik!'! 
28 

bodies. 42 However, controlled ell:perimental evidence of an etio:lo,gic 

J:elatio1.l!!hip l>etween these prg11nisms and human can.cer is lacUng. S:illlill!ltly 

there b.l!lve 'b~>en many teports o;f cancer vaccines 11nd cures. The ol11y 

info:t111atio11 avai_iable l1as :heen thtQugh the pnblic media, through interv:l.i!ws l>y 

the proj)ommts, or fn :privately printed 1>1ibliaatiQ11s that an ~:~at avail11.hle to 

li'brad.es ¢%' the gen.erd pu:b1~c. T!)e in.fotmatiQn is n.ot publi11hed in the open 

medical Qt !l"cieudlJ.c literature whel:e it can he reviewed and v!llidated )ly 

pther Mied_iJJts. 

Current techniques Qr appJ:oaches for attempHug to relil.olve the public 

health concern~;~ and the -resW.ts of using these technique" s're s~iiiz·ed in. 

Table 2, Unequ!vocallY positive f:l.~dings using e11eh of theae toola coulil ·l;ulve 

incriminated MD o.- itt> vae_c!ne v:I.:Nae!! or both a11 public health concerns. 

'thus a highly s;!,jJliifJ.cant cotre:t.af:ion betwe.en il. pUblic hel\lth (!.Qn(iij:ion ~ lhJh• 

leukemia, and exposure to an AOHV, e.g., MD vi:rua, in the a'baence of a 

correlation between tih"' cond:l.!;lon and other factors, e.g., c<>tnpJ;iJdncdoii: a:nd, 

fertilizer use, coUld have strongly ·suppo-rted the hypothesis that MD v1ru11 ·had 

something to do with the human leukemia. S:f.m:f.lat exa!Dples· o.f findings using 

other techll,iques which would st:tongly support this hypothesis ate in ~able 2 • 

... -, 

! 
29 

Negative findil!gS using each of the techniques currently available, namely 

statistical epidemiology, serological epidemiology, ~1 inoculation, cell 

culture inoculation, and compal:ison of etiologic agents cannqt refute the 

nypothe:sis that the public health condition is caused by exposure to the 

AO.HV. Thus, for example, the absence of a correlation betll'een a public hl!lalth 

condition and expo.sure to AOHV does not confirm that there is no relationship 

between the two. One can conclude only that thb technique failed to f:i.ud a 

relationllhip uuder tile conditions and :l.n the circWIIstances in whi~ it was 

Ui!led, This is the basis for the conclusions of the expert committees and 

eomi1111ioUiil referretl to previ.ousl.y in this paper. 

rrom the above discusslon it appears unlfkely that additional efforta to 

use the t!!.chniquea c\lrrently a'lailable w:l.ll. reeol.v!l the public he.al.th concel;'lls 

of 11D and :Us va~:<:~ines, Certainly research resu.lts of the last 8 years have 

:failed t<> brinl:l: u!! a,ny closer to a resolution of the J;l!aue. 2 A ftitther way 

of ahe!idilll:l: l.ight on the possible rela tionsh:l.p il! ,in the use of the uew 

molecular :biology ·tools to study the .Pathogenesis of cancer in man and 

an:imals. If a unique ::o1e for the AOHV can be found in the molecular 

pathc;>geues1s of htm!an cancer, then the public heal.tl'; hazard of the virus will 

be co.Uined. It is important to note that just finding portions of a viral 

genome in htilllan tumors, e.g., the mye and arc. oncogenes of avian leukosis 
30 

' ' 1 viruses, is inauf:ficien-t it '!:hue gen-es are also presen-t in Ml:!!lal cells. 

Coaversely, if the molecular pathogenesis of all lil.lill<!ll. ¢iltl.cer is ellplain-ed and 

no role is found 'fl:fr any unique AOHV genes or gene products, theu. the abseuce 

of a public health hazard call, be confirmed. 

This comprehetuiiire 'tev:l.ew of the. t:l.terllture 'bdn.gs together, for the ii:t:lit 

time, a laz::ge body o'f :l.nfo:rlllation •relating t.o AQ!{V Sll.d hlllll!ln health hazards., 

patt:l.tl!l4tlY hull!l!'u canc~.r.. 'l'l!'is -review shol!ld be u11e'fl!l J;o scielltiJ!ts and 

adm!q;isnators in public res!!at:ch and action a,geneiell and 1.n private 

industry. 'l'lle rav:::tew Cl.lvera the nUII1eroUII al:hli!pt!! .to resolve the. q,ueation ·of 

tthel:her AORV pose any ptiblie health hazard to htlll!aus. 'fhe large body q£ 

experimental evidence in both .avian and hwar. virolpgy, sero1og;<, pathology • 

and epidellllology nrongly aupp<:>rts .the eouclua:ton 'tMt .P:o et!ol(lgic 

:relatioaship exiata bel:)faen AOHV and human <;llllcer • Thqa, in our optnion 11nd 

in the opinion of experts in the human and avian cancer field, MD all.d vaccines 

posaible, there is suffic:ienl: ev:l;dence reviewed iu this paller to warr;tnt ·thia 

firm oouc1us1on. 

···-· 
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38 

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