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`SC!0014247 2 0.90192
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`5.50 E:RE:COCI<.' [it?
`NFIDIEDN IH 5.3?06
`
`Ill
`Vol 53
`September 1992
`No. 9
`~~~~~~~~=:[)
`
`CEV Exhibit 1006_001
`
`

`

`Antibody response to and maternal immunity
`from an experimental psittacine beak and feather
`disease vaccine
`
`Branson W. Ritchie, DVM, PhD; Frank D. Niagro, PhD; Kenneth S. Latimer, DVM, PhD; W. L. Steffens, PhD;
`Denise Pesti, MS; Raymond P. Campagnoli, MS; Phil D. Lukert, DVM, PhD
`
`SUMMARY
`Adult umbrella cockatoos, Moluccan cockatoos, Mrican
`grey parrots, and a yellow-headed Amazon parrot ~ere
`inoculated IM or sc with 13-propiolactone-treated psitta(cid:173)
`cine beak and feather disease (PBFD) virus. Thirty- to 45-
`day-old African grey parrot, umbrella cockat?o, and s~l­
`phur-crested cockatoo chicks also ;-ver~ va.ccn~.a~~d with
`the same inoculum. The hemagglutmahon mhibitwn (HI)
`and agar-gel diffusion tests wer~ used to. assay f?r P?St(cid:173)
`vaccination development of anh-PBFD virus antibodies.
`All adult vaccinates seroconverted and had increases in
`HI and precipitating antibodies. The vaccinated chicks had
`increased concentrations of HI antibodies, but precipitat(cid:173)
`ing antibodies could not be detected. To demonstrate th~t
`chicks from vaccinated hens are protected from PBFD vi(cid:173)
`rus challenge, 3 African grey parrot chicks and 2 ~m­
`brella cockatoo chicks from vaccinated hens and 1 Afncan
`grey parrot chick and 1 umbrella cock~~oo chick ~rom
`nonvaccinated hens were exposed to punfied PBFD virus.
`Chicks from the vaccinated h ens remained clinically nor(cid:173)
`mal during the 50-day t est period. Chicks from the non(cid:173)
`vaccinated hens developed clinical and histologic lesions
`of PBFD. Infected tissues from these birds were confirmed
`to contain viral antigen, using immunohistochemical
`staining techniques. The PBFD virus was recovered from
`the affected birds. These findings indicate that adult and
`30- to 45-day-old psittacine birds will seroconvert follow(cid:173)
`ing vaccination with 13-propiolactone-tr eated PBFD virus.
`Also h ens inoculated with 13-propiolactone-treated PBFD
`viru~ produce chicks that are, at least temporarily, resis(cid:173)
`tant to virus challenge.
`
`Psittacine beak and feather disease (PBFD) is caused by
`a 14- to 17-nm nonenveloped virus containing single-
`
`Received for publication Oct 17, 1991.
`From the Departments of Small Animal Medicine (Ritchie), Medical Microbi(cid:173)
`ology (Niagro, Lukert, Pesti), Pathology (Latimer, Campagnoli), Anatomy and
`Radiology (Steffens), College of Veterinary Medicine, University of Georgm, Ath-
`ens, GA 30602.
`.
`Supported in part by funds from the International Avian Research F?undatwn,
`Terry Clyne, Richard and Luanne Porter, Charles G?ddard Founda~IO?, . Isabel
`Taylor, Avicultural Society of Greater Cincinnati, B1rd Club~ of V1rg~ma, the
`Association of Avian Veterinarians, Miami Parrot Club, and Ze1gler Brothers Inc.
`The authors thank Mary B .. Ard, Glenn Weatherly, J anice Matthews, a nd the
`staff of the Educational Resource Center for technical assista nce.
`
`0
`
`0
`
`stranded circular DNA. 1· 3 The PBFD virus shares ultra(cid:173)
`structural and nucleic acid similarities with 2 other re(cid:173)
`cently described viruses, the apparently apathogenic
`0 k
`4 7 p 't
`porcine circovirus and the chic en anemia v1rus. ·
`s1 -
`tacine beak and feather disease is characterized by the
`progessive formation of dystrophic feathers. Beak lesions
`may or may not develop. This disease has been reported
`in many species of captive and free-ranging Old World
`psittacine birds.8 -
`12 Viral infections have also been doc(cid:173)
`umented in New World psittaciformes, including Amazon
`parrots and a Scarlet macaw. 2•13 •14 Neither the e?'act_ host
`range of the PBFD virus nor seroprevaleJ?-ce of ;;mti~odies to
`the virus in either captive or free-ranging psittacme pop(cid:173)
`ulations is known.
`The first sign of PBFD is the appearance of necrotic,
`abnormally formed feathers. The type of feathers that are
`initially involved depends on the stage of molt when the
`clinical signs of disease are first manifested. In young
`birds, all of the feather tracts may be affected during a
`1-week period, whereas in older birds, the disease ~s more
`prolonged, with progressive feather changes durmg en(cid:173)
`suing molts. 3•8· 10•15 •16 If beak lesions develop, they may
`include palatine necrosis, progressive elongation, and
`transverse or longitudinal fractures. 3 •8 •
`11
`15
`•
`Subjectively, PBFD can be diagnosed by the ident~fi~a­
`tion of basophilic intranuclear and intracytoplasmic m(cid:173)
`clusion bodies within infected feather epithelial cells or
`m acrophages, respectively. 2 •8 · 11•13 -1 8 Because other vi(cid:173)
`ruses may induce similar-appearing inclusio_n bodies! a
`confirmatory diagnosis requires the use of viral-spec.lfic
`antibodies or nucleic acid probes to demonstrate PBFD vrral
`antigen or viral DNA, respectivelyY-19 Viral-specific D~A
`probes also can be used to detect PBFD viral nucleic actd
`in WBC of infected birds.19
`The hemagglutination inhibition (HI) test has been u~ed
`to detect anti-PBFD virus antibody titers in birds Wlth
`active viral infections and in clinically normal birds nat(cid:173)
`urally exposed to the virus. Birds with activ~ PBFD v~rus
`infections have been found to have lower anb-PBFD v1rus
`antibody titers than birds that had been ~xpose? to the
`virus, but remained clinically normal. Th1s findmg sug·
`gests that some birds exposed to the virus are able tf
`mount an effective immune r esponse. 20 The purpose ~
`the study reported h ere was to determine whether antJ·
`PBFD virus antibodies could be induced by experimental
`inoculation with 13-propiolactone-treated virus and whether
`
`1512
`
`Am J Vet Res, Vol 53, No. 9, September 1992
`
`CEV Exhibit 1006_002
`
`

`

`vaccinated hens produced chicks that were resistant to
`pBFD virus challenge.
`
`Materials and Methods
`Birds-A group of clinically normal 30- to 45-day-old
`African grey parrots (Psittacus erithacus erithacus), um(cid:173)
`brella cockatoos (Cacatua alba), and sulphur-crested
`cockatoos (C sulphurea), and pairs of adult male and fe(cid:173)
`male umbrella cockatoos, Moluccan cockatoos (C moluc(cid:173)
`censis), African grey parrots, and a yellow-headed Amazon
`parrot (Amazona ochrocephala 01·atrix) were used in this
`study. The adult pairs were housed in drop-through flight
`cages and were fed a mixture of seeds, fresh vegetables,
`and commercial pellets. The chicks were housed in wire(cid:173)
`bottom baskets that were placed in temperature-con(cid:173)
`trolled incubators. Breeding pairs of umbrella cockatoos
`and African grey parrots were inoculated with 13-propio(cid:173)
`lactone-treated, Freund's adjuvanted PBFD virus. Control
`breeding pairs of umbrella cockatoos and African grey
`parrots were not vaccinated. Eggs produced by the cock(cid:173)
`atoos were removed from the nest boxes 5 to 10 days after
`the last egg was laid and placed in an incubator.a One
`clutch of African grey parrots were allowed to incubate
`their eggs and neonates were removed from the nest box
`when they were 2 to 5 days old. Eggs from another pair
`of African grey parrots were removed from the nest sev(cid:173)
`eral days before hatching and were placed in an incuba(cid:173)
`tor.a
`
`Preparation of antigen-Feather follicle tracts from a
`diseased Moluccan cockatoo (vaccine isolate) and anum(cid:173)
`brella cockatoo (challenge isolate) were excised and placed
`in phosphate-buffered saline solution (PBSS, 0.05M phos(cid:173)
`phate buffer, 0.15M NaCl, pH 7.2). Virus was extracted
`by use of a tissue homogenizer.b The suspensions were
`clarified by centrifugation (30,000 x g for 20 minutes, 4
`C) followed by 46,000 x g for 20 minutes, 4 c.c The clar(cid:173)
`ified supernatant fluid was layered over 45% (w/v) su(cid:173)
`crose in PBSS and centrifuged (140,000 x g for 2 hours,
`4 C)c to obtain crude viral pellets. Pellets were resus(cid:173)
`pended in PBSS, adjusted to 1.37 g/ml with cesium chlo(cid:173)
`ride, and centrifuged to equilibrium (270,000 x g for 16
`hours, 20 C).c The gradient was collected with a density
`gradient fractionator.d Fractions containing virus were
`dialyzed against PBSS and were further purified by cen(cid:173)
`trifugation through linear sucrose gradients (20 to 60%
`w/v) in PBSS buffer (270,000 x g for 90 minutes, 4 C).c
`Gradients were collected as described. Fractions contain(cid:173)
`ing purified virus were dialyzed against PBSS and stored
`at -20 C.
`
`:Vaccination-Twenty microliters (10,240 hemagglutin(cid:173)
`ating [HA] units) of virus purified from an infected Mol(cid:173)
`uccan cockatoo was mixed with 13-propiolactone according
`to published procedures. 21 After a 2-hour incubation at
`37 C, 20 J.Ll of virus was mixed with 70 J..Ll ofPBSS and 100
`~l of Freund's complete adjuvant (for the first inocula(cid:173)
`~Ion) or Freund's incomplete adjuvant (for all subsequent
`Inoculations). Test birds were placed under isoflurane
`
`-: ~!Umbach incubator, Swan Creek Supply, Saginaw, Mich.
`
`lssue homogenizer, Tekmar Co, Cincinnati, Ohio.
`,
`, gltr~centrifuge, Beckman Instruments, Palo Alto, Calif.
`ens1ty grad ient fractionator, Isco, Lincoln, Neb.
`
`anesthesia and were vaccinated either IM (1 bird) or sc
`(all other birds) with the 13-propiolactone-treated, adju(cid:173)
`vanted virus preparation. A male Moluccan cockatoo was
`inoculated with adjuvant and 13-propiolactone in PBSS as
`a reagent control. The birds were vaccinated on days 0,
`7, and 14. Blood samples were collected by jugular veni(cid:173)
`puncture on days 0, 7, 14, and 21. The blood samples were
`placed in glass tubes and allowed to clot. The clot was
`separated from the serum by centrifugation, and the serum
`was removed from the tube and stored frozen at - 20 C.
`
`Hemagglutination and HI tests-Blood from a goffin
`cockatoo (C goffini) was collected in Alsever's solution,
`using care to prevent clotting. Cells were stored at 4 C
`for not more than 1 week. Erythrocytes were washed 3
`times in PBSS (pH 7.2) and resuspended in the same buffer
`to produce a 25% stock solution. A 0. 75% working dilu(cid:173)
`tion of erythrocytes was made in PBSS immediately before
`use. Stock solutions were used for up to 7 days, but were
`discarded if hemolysis was evident. Tests were performed
`in 96-well plastic round-bottom microtitration plates.
`The HA test was performed by making serial twofold
`dilutions (starting dilution of 1:50) of purified concen(cid:173)
`trated PBFD virus in PBSS. Cockatoo erythrocytes (50 J..Ll,
`0. 75% suspensions) were added to each well. The tests
`were read when a clear button had formed in the bottom
`of all wells, usually within 30 to 45 minutes.
`Serum from each bird to be tested by HI was heated at
`56 C for 30 minutes. The serum samples were diluted 1:40
`in PBSS, adsorbed with 0.2 volumes of a 25% (v/v) suspen(cid:173)
`sion of the cockatoo erythrocytes, and incubated for at
`least 2 hours at room temperature (25 C) or overnight at
`4 C. Erythrocytes were removed by low-speed centrifu(cid:173)
`gation. Serial dilutions (twofold, 1:40 initial dilution) were
`made in a round-bottom plastic microtitration plate. An
`equal volume (50 J..Ll, 64 HA units as determined by the
`HA test) of PBFD virus was added to each serum dilution.
`After a 1-hour incubation period at 25 C, 100 J..Ll of 0. 75%
`cockatoo erythrocytes was added to each well. The tests
`were read after the control cells formed a clear button,
`and end points were determined as the last serum dilu(cid:173)
`tion that prevented HA.
`
`Agar gel diffusion test-Serum samples obtained from
`the test birds at day 0 and 21 following vaccination were
`tested for precipitating antibodies. Noble agar (0. 7%) was
`prepared in PBSS, pH 7.4. In the agar, 6 wells were made
`around a central well. Serum from each bird, undiluted
`and at dilutions of 1:5, 1:10, 1:20, 1:40, and 1:80, was
`placed in the 6 wells. Thirty microliters of purified PBFD
`virus was placed in the central well and the plates were
`incubated overnight at 25 C. With a high-intensity light,
`the plates were examined for formation of precipitin lines.
`Normal rabbit serum was used as a negative control, and
`rabbit serum containing anti-PBFD virus antibodies was
`used as a positive control.
`
`Psittacine beak and feather disease virus challenge(cid:173)
`Three African grey parrot chicks and 2 umbrella cockatoo
`chicks from vaccinated hens and 1 African grey parrot
`chick and 1 umbrella cockatoo chick from a nonvaccina(cid:173)
`ted hen were exposed to purified PBFD virus. The birds
`were given a total of 30 J..Ll of purified virus (1,500,000 HA
`units) at ages 3 to 5 days and 6 to 8 days. For exposure,
`
`Arn J Vet Res, Vol 53, No. 9, September 1992
`
`1513
`
`CEV Exhibit 1006_003
`
`

`

`30 f.Ll of purified virus was mixed in 250 f,Ll of PBSS. One
`hundred microliters of this solution was given orally, an(cid:173)
`other 100 f,Ll sc, and 50 f,Ll intracloacally. All birds were
`examined daily for clinical signs of disease.
`
`Histologic examination and immunohistochemical
`analysis-The African grey parrot chick and umbrella
`cockatoo chick from nonvaccinated hens died and were
`necropsied when they were 44 and 4 7 days old, respec(cid:173)
`tively. A complete set of tissues including liver, spleen,
`kidney, heart, intestines, pancreas, proventriculus, ven(cid:173)
`triculus, thymus, bone marrow, cloaca, and brain were
`fixed in neutral buffered 10% formalin solution. Sections
`were routinely processed for histologic evaluation. Im(cid:173)
`munohistochemical staining was performed by the avi(cid:173)
`din-biotin complex immunoperoxidase technique, using a
`commercial kit.e Tissue sections containing inclusion bodies
`suggestive ofPBFD virus were placed on slides coated with
`poly-L-lysine and dried overnight at 60 C. Tissue sections
`were deparaffinized, rinsed in deionized water, and treated
`for 15 minutes with 3% hydrogen peroxide to quench en(cid:173)
`dogenous peroxidase activity. The tissue sections were
`trypsinized, then rinsed in PBSS. Nonspecific staining was
`reduced by application of normal goat serum. Following
`blotting, the primary immunoglobulins were applied at a
`dilution of 1:2,000. Sections were incubated overnight at
`4 C. The slides were rinsed in PBSS, goat anti-rabbit im(cid:173)
`munoglobulin was applied (1:200 dilution), and the sec(cid:173)
`tions were incubated for 30 minutes at 25 C. After another
`rinse in PBSS, the avidin-biotin complex reagent was ap(cid:173)
`plied and the sections were incubated at 25 C for 1 hour.
`Tissue sections were again rinsed in PBSS, then developed
`for 6 minutes in a freshly prepared solution of hydrogen
`peroxide and diaminobenzidine substrate. Following a fi(cid:173)
`nal rinse in deionized water, the sections were counter(cid:173)
`stained with hematoxylin, dehydrated, coverslipped, and
`examined. Positive staining was indicated by the pres(cid:173)
`ence of a brown pigment. Negative controls for each spec(cid:173)
`imen were run by eliminating application of the primary
`antibody and substituting normal mouse serum at a 1:2,000
`dilution.
`
`Psittacine beak and feather disease virus recovery from
`neonates-Virus purification from the feathers of affected
`chicks was performed as described. Virus suspension (10
`f.Ll) from each bird was adsorbed onto 0.25% formvar-coated
`grids for 5 minutes, negatively stained with 1% phospho(cid:173)
`tungstic acid, and examined at 80 keV.r The virus prep(cid:173)
`aration used for challenge experiments was examined by
`electron microscopy in a similar fashion.
`
`Identification of viral DNA-Virus samples used for vac(cid:173)
`cinating adults, for challenging chicks, and recovered from
`the affected chicks were compared by the polymerase chain
`reaction (PCR), using PBFD virus-specific primers. Ten mi(cid:173)
`croliters of each purified virus preparation was mixed with
`90 f.Ll of PCR mixture containing 10 mM tris-HCI, pH 8.3,
`50 mM KCl, 25 mM MgCl, 0.1% gelatin, 200 f,LM each of
`dATP, dGTP, dCTP, and dTTP, 100 nM each of the for(cid:173)
`ward and reverse PCR primers, and 2.5 U of Taq DNA
`polymerase. The mixture was covered with mineral oil.
`Samples were subjected to 40 cycles of heating and cool-
`
`• Vector Laboratories, Burlingame, Calif.
`r JEOL JEM-100S, Peabody, Mass.
`
`1514
`
`ing, using a thermocycler.g Aliquots (10 f.Ll) of the PCR
`mixtures were subjected to electrophoresis on 2% agarose
`minigels in tris-borate-EDTA buffer according to estab.
`lished methods.22 Gels were stained with ethidium bro.
`mide, illuminated with short-wave uv light, and ph oto.
`graphed.
`
`Dot blots - Five microliters of PCR products was diluted
`to 50 f.Ll with tris-EDTA buffer, pH 8. Five microliters of
`4M N aOH was added to the mixture and was incubated
`at 72 C for 1 hour. The samples were cooled to 25 C and
`22 f.Ll of 1M ammonium acetate, pH 7.0 was added. Sam.
`pies were applied to neutral nylon membranesh in a dot.
`blot manifold under low vacuum and uv cross-linked be.
`fore probing. Membranes were probed and developed by
`use of a chemiluminescence detection systemh according
`to manufacturer's instructions.
`
`Results
`!3-Propiolactone-treated PBFD virus was used to inoc·
`ulate a group of 30- to 45-day-old and adult psittacine
`birds. Local tissue reactions to sc injections were mini·
`mal, with a few birds developing mild granuloma for(cid:173)
`mation at the site of the injection. Neither the HI nor
`precipitating antibody titers changed in a control bird
`inoculated with !3-propiolactone-treated PBSS.
`All the adult and neonate vaccinates had increased
`concentrations of HI antibodies by day 21 (Fig 1). Before
`vaccination, HI antibody titers in the adults ranged from
`1:80 to 1:640, with a geometric mean titer (GMT) of 201.
`In chicks, initial titers ranged from 1:40 to 1:640, with a
`GMT of 160. One week after the third vaccination, HI an·
`tibodies in the adults ranged from 1:1,280 to 1:5,120, with
`a GMT of 2,560; and titers in the chicks ranged from 1:1,280
`to 1:5,120, with a GMT of 2,986 (Table 1).
`Precipitating antibodies to the PBFD virus were de·
`tectable by the agar-gel diffusion test in serum from ex·
`perimentally vaccinated adult birds (Fig 2). A line of
`identity was evident when rabbit anti-PBFD antibodies or
`
`• Techne PHC-1 Programmable Dri-Block, Princeton, NJ.
`h Tropix Inc, Bedford, Mass.
`
`Titer
`
`pre 1wk 2wk 3wk pre 1wk 2wk 3wk pre 1wk 2wk 3wk
`
`Bird 1
`
`Bi rd 2
`
`Bird 3
`
`Figure 1-Microtitration plates of typical changes in hemagglutination in·
`hibition antibody titers of 3 adult psittacine birds after vaccination with ~·
`propiolactone-treated psittacine beak and feather disease (PBFD) virus.
`
`Am J Vet Res, Vol 53, No. 9, September 1992
`
`CEV Exhibit 1006_004
`
`

`

`T ble 1- Hemagglutination inhibition (HI) and precipitating antibody titers
`a:intervals a~er vacc_ination with 13-propiolactone-treated psittacine beak
`and feath er d1sease wus
`
`Postvaccination days
`0
`7
`14
`21
`640/10
`640 2,560 5,120/80
`160/1 1,280 2,560 5,120/80
`320/0
`640 5,120 5,120/80
`160/1
`320 640 1,280/10
`80/0 1,280 1,280 1,280/80
`320/1 1,280 2,560 2,560/80
`80/1
`160 2,560 2,560/10
`320/0
`160 2,560 2,560/10
`160/0 5,120 5,120 5,120/80
`< 40/0
`160 640 1,280/0
`< 40/0
`320 640 1,280/0
`
`Age and sex
`Species
`Adult male
`Moluccan cockatoo
`Adult male
`Umbrella cockatoo
`African grey parrot Adult male
`Adult female
`Moluccan cockatoo
`Adult male
`Umbrella cockatoo
`Adult female
`Umbrella cockatoo
`Adult female
`Umbrella cockatoo
`Adult male
`Amazon parrot
`African grey parrot Adult female
`45 d (sex unknown)
`Umbrella cockatoo
`45 d (sex unknown)
`Umbrella cockatoo
`Sulphur-crested
`45 d (sex unknown)
`80/0 2,560 2,560 2,560/0
`cockatoo
`45 d (sex unknown)
`160/0 2,560 2,560 5,120/0
`African grey parrot
`45 d (sex unknown)
`80/0
`320 640 2,560/0
`African grey parrot
`30 d (sex unknown)
`640/0 2,560 2,560 5,120/0
`African grey parrot
`30 d (sex unknown)
`640/0
`640 2,560 2,560/0
`African grey parrot
`30 d (sex unknown)
`640 .. .. 5,120/0
`640/0
`African grey parrot
`Titers on days 0 and 21 are listed as HI/precipitating.
`
`Figure 2-Agar-gel diffusion test of serum from an adult psittacine bird 3
`weeks after Initial vaccination. Precipitin lines are evident to a dilution of 1/
`80,
`
`inoculated bird serum was used. Precipitin lines were not
`evident when normal rabbit serum was used. Prior to
`vaccination, the precipitating antibody titers in the adults
`ranged from 0 to 1:10. All the adult birds produced pre(cid:173)
`cipitating antibodies by postvaccination day 21. The range
`of titers in the adults was 1:10 to 1:80, with a GMT of 40.
`Vaccinated chicks did not develop detectable concentra(cid:173)
`tions of precipitating antibodies (Table 1).
`. An African grey parrot and umbrella cockatoo hen vac(cid:173)
`Cinated with [3-propiolactone-treated PBFD virus serocon(cid:173)
`verted and had increases in HI and precipitating antibody
`co11:centrations (Table 2). By comparison, HI and precipi(cid:173)
`tating antibody titers in a nonvaccinated African grey
`Parrot and umbrella cockatoo hen were low (Table 2).
`Chicks from the vaccinated and nonvaccinated hens were
`~xposed to purified PBFD virus by the combined oral, in-
`racloacal, and sc routes. Chicks from vaccinated hens
`re~ained clinically normal throughout the 50-day test
`Perro d.
`
`Table 2-Hemagglutination inhibition and precipitating antibody titers in
`vaccinated and unvaccinated psittacine hens
`
`Vaccination
`Species
`status
`Vaccinated
`Umbrella cockatoo
`Vaccinated
`African grey parrot
`Unvaccinated
`Umbrella cockatoo
`African grey parrot
`Unvaccinated
`Titers on days 0 and 21 are listed as HI/precipitating.
`
`0
`160/1
`160/0
`
`Postvaccination days
`14
`7
`1,280
`2,560
`5,120
`5,120
`
`21
`5,120/80
`5,120/80
`160/0
`320/1
`
`The African grey parrot chick from the nonvaccinated
`hen developed normally until it was 30 days old, at which
`time the bird seemed depressed and began to regurgitate
`intermittently. Three days later, the bird started to lose
`powder-down and contour feathers. Several of the pri(cid:173)
`mary and secondary feather shafts appeared dark red(cid:173)
`brown and were loose in their follicles. Over the next 5
`days the bird continued to lose contour, flight, tail and
`crest feathers. The bird seemed increasingly depressed,
`and crop emptying slowed. The bird was euthanatized
`when it was 44 days old.
`The umbrella cockatoo chick from the nonvaccinated
`hen developed normally until it was 40 days old, at which
`time the bird seemed depressed and anorectic. Two days
`later, several primary and secondary feathers were loose
`in the follicles and contained dark-colored pulp cavities.
`Over the next 5 days, all of this bird's flight, tail, crest,
`and contour feathers begin to appear dystrophic. The bird
`died when it was 4 7 days old.
`Histologic evaluation of tissues from both affected chicks
`revealed basophilic intranuclear and intracytoplasmic in(cid:173)
`clusion bodies in feather epithelium, thymus, and bursa.
`Intranuclear inclusion bodies were located within epithe(cid:173)
`lial cells and intracytoplasmic inclusion bodies were in
`macrophages (Fig 3). Other histologic findings included
`necrosis and edema in the feathers.
`The affected African grey parrot and umbrella cockatoo
`chicks were confirmed to have PBFD by immunoper(cid:173)
`oxidase staining (Fig 3). Further, PBFD virus was re(cid:173)
`covered from the feathers of these birds, using established
`procedures. Virus recovered from the affected chicks was
`morphologically similar to the virus that was used for
`challenge exposure (Fig 4). Use of PBFD virus-specific DNA
`probes indicated that virus recovered from the infected
`chicks had the same nucleic acid sequence as the virus
`used to vaccinate the adults and challenge expose the
`chicks (Fig 5).
`
`Discussion
`Psittacine beak and feather disease virus has been as(cid:173)
`sociated with acute infections, which are most common
`in young psittacine birds, and chronic infections, which
`are most common in older psittacine birds.3 •8•
`16 The
`12
`15
`•
`•
`HI test has proven to be a rapid method for detecting anti(cid:173)
`PBFD virus antibodies. The presence of high concentra(cid:173)
`tions of HI antibodies in clinically normal birds naturally
`exposed to PBFD virus suggests that some birds encoun(cid:173)
`tering this virus develop subclinical infections that result
`in development of a protective immunologic response. 20
`Whether a bird is resistant to the virus or is fatally in(cid:173)
`fected could depend on the age of the bird at the time of
`infection, the presence and concentrations of maternal
`antibodies, the route of viral exposure, and/or the titer of
`the infecting virus.
`
`Am J Vet Res, Vol 53, No. 9, September 1992
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`1515
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`CEV Exhibit 1006_005
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`1
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`2
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`3
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`4
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`5
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`Figure 3-Photomicrograph of feather epithelium from an African grey par(cid:173)
`rot chick experimentally infected with PBFD virus. Intranuclear and intracy(cid:173)
`toplasmic inclusion bodies are located within infected epithelial cells and
`macrophages, respectively. Presence of PBFD virus antigen in intranuclear
`and intracytoplasmic inclusion bodies (arrows) is confirmed by use of viral(cid:173)
`specific antibodies and the immunoperoxidase technique with hematoxylin
`counterstain. Bar = 25 f!.m.
`
`Figure 4-Transmission electron micrograph of PBFD virus used to exper(cid:173)
`imentally infect psittacine chicks. Inset: Morphologically similar virus re(cid:173)
`covered from the feathers of an affected umbrella cockatoo chick. Virus
`suspensions were negatively stained with 1% phosphotungstic acid and
`photographed at 80 keV. Virus particles are 14 to 17 nm in diameter. Bar
`= 45 nm.
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`1516
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`Figure 5- Detection of PBFD virus nucleic acid by agar-gel electrophoresis
`and ethidium bromide staining. Arrow marks 400-base fragment. Lane 1,
`molecular weight standards; lane 2, nucleic acid from virus isolated from a
`Moluccan cockatoo and used to vaccinate breeding hens; lane 3, nucleic
`acid from virus isolated from an umbrella cockatoo and used to challenge
`expose chicks; lane 4, nucleic acid from virus isolated from an affected
`African grey parrot chick; lane 5, nucleic acid from virus isolated from an
`affected umbrella cockatoo chick.
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`antibodies, the route of viral exposure, and/or the titer of
`the infecting virus.
`Because the manifestation of clinical changes in PBFD
`virus-infected birds can require a prolonged period and
`because latently infected birds may be difficult to detect,
`a vaccination program for this virus would provide the
`most practical solution for PBFD virus control. (3-Propi·
`olactone has been shown to inactivate all viruses against
`which it has been tested. 23 We did not determine whether
`the virus used in these studies was inactivated, but other
`studies have indicated that PBFD virus can be inactivated
`with (3-propiolactone.16
`In this study, HI and precipitating antibodies to the
`PBFD virus could be induced in adult psittacine birds after
`experimental vaccination with (3-propiolactone-treated
`PBFD virus. These findings suggest that a natural im·
`munologic response, which appears to develop in birds
`that are exposed to the PBFD virus, but remain clinicallY
`normal, can be experimentally reproduced. It was deter·
`mined in this study that passively derived HI antibodies
`did not prevent chicks from responding to vaccination by
`the time they were 30 to 45 days old. Titers from the
`individual chicks did not obtain the same level, but all
`birds had an increased HI antibody titer by 1-week after
`the third vaccination.
`Chicks used in this study did not have detectable con·
`centrations of maternally derived precipitating antibod·
`ies at the time of testing, and there was no detectable
`increase in this type of antibody following vaccination·
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`Am J Vet Res, Vol 53, No. 9, September 1992
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`CEV Exhibit 1006_006
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`I poultry, IgG is the cla<>s of immunoglobulin in the serum
`~the tim~ of Jlatchi:r:g. 24 _If the HI antibodies ::rre ~gG and
`the precipitatmg antibodies are lgM, then this might ex(cid:173)
`plain why chicks did not have precipitating antibodies in
`their serum. Also, the agar-gel diffusion test is less sen(cid:173)
`sitive than is.the HI test. It is.possible that precipitating
`antibodies might have been mduced, but m concentra(cid:173)
`tions too low to be detected with this relatively insensi(cid:173)
`tive test system. Isotyping reagents for psittacine
`immunoglobulins, which to our knowledge are not avail(cid:173)
`able would be required to determine which types of im(cid:173)
`mudoglobulins are in the serum of chicks.
`The primary goal of the experiments described in this
`report was to develop a challenge model for testing a re(cid:173)
`combinant PBFD virus vaccine. A small sample size was
`used in this initial study because euthanasia of neonates
`in excess of that required to define the model could not
`be ethically justified. Umbrella cockatoo and African grey
`parrot chicks from vaccinated hens were found to be tem(cid:173)
`porarily resistant to PBFD virus challenge. Although the
`sample size was small, the fact that all of the chicks from
`vaccinated hens were temporarily resistant to infection,
`whereas control chicks from nonvaccinated hens devel(cid:173)
`oped clinical disease, and suggests that maternally trans(cid:173)
`mitted immunologic factors can protect neonates from virus
`challenge.
`Some previous attempts to infect budgerigars with
`feather homogenates containing PBFD virus have been
`unsuccessful. 16
`25 Passively derived protection may have
`•
`contributed to these transmission failures. Determining
`the level and type of antibodies that are needed to protect
`a bird from infection and the critical period when these
`antibodies provide protection from infection will require
`further investigation. Challenge exposing the vaccinated
`adults used in this project was beyond the scope and re(cid:173)
`sources of this study.
`Comparative studies of PBFD virus isolates recovered
`from various genera of psittacine birds have indicated
`that an antigenically similar virus infects a wide range
`of psittacine birds. 2 In the study reported here, virus pur(cid:173)
`ified from an umbrella cockatoo induced PBFD in anum(cid:173)
`brella cockatoo and an African grey parrot chick. Further,
`an umbrella cockatoo and African grey parrot hen vac(cid:173)
`cinated with [3-propiolactone-treated PBFD virus re(cid:173)
`covered from a Moluccan cockatoo produced chicks that
`remained normal after virus challenge. These findings
`strengthen the theory that an antigenically related virus
`causes PBFD in various psittacine genera.
`The experimentally infected psittacine chicks in this
`study were confirmed to have PBFD by use of viral-specific
`antibodies to demonstrate viral antigen in intranuclear
`and intracytoplasmic inclusion bodies. The DNA probe
`~omparison of the virus used for inducing antibody titers
`lU the adults, for challenging the chicks, and recovered
`from the affected chicks indicated that all of the viral
`preparations had a shared nucleic acid sequence. Further,
`PBFD virus could be recovered from the diseased feathers
`of the affected birds.
`r Galah chicks experimentally infected with PBFD virus
`have been reported to develop clinical signs of PBFD ap(cid:173)
`Proximately 4 weeks after infection. 16 In our study, the
`affected African grey parrot seemed depressed when it
`e Was 30 days old and developed progressive feather dys-
`trophy when it was 33 to 44 days old. The affected urn-
`1.
`
`brella cockatoo chick seemed depressed when it was 40
`days old and developed progressive feather dystrophy when
`it was 42 to 4 7 days old. The time variance in developing
`clinical signs associated with PBFD among psittacine chicks
`may be attributed to differences in concentrations of ma(cid:173)
`ternally transmitted antibodies, titer of virus in the in(cid:173)
`oculum, or host responses to the virus.
`Natural exposure to PBFD virus can occur from contact
`with contaminated feather dust, feces, or crop excre(cid:173)
`tions. 26 Because PBFD appears to be restricted in host range
`to psittacine birds and most of these birds are restricted
`to enclosures, it is likely that a widespread and continued
`vaccination program can be used to control this disease
`in companion birds in the United States. As demon(cid:173)
`strated by this study, vaccination of breeding females and
`subsequent transfer of passive immunity to developing
`chicks may be critical in pre