Vet Patho l 28:300-304 (199 1)
A Novel DN A Virus Associated with Feather Inclusions in Psittacine Beak and Feather Disease K. S. LATIMER,
B. W.
P. M.
RITCHIE,
RAKI CH,
F. D.
W. L.
STEFFENS,
N IAGRO, AND
P. D.
I. M.
KIRCHER ,
L UKERT
Departments of Veter inary Path ology, Anato my an d Radi ology, Small Anima l Medicine, Med ical Microb iology, and the Athens Diagnostic and Investigati onal Laboratory, College of Veterinary Medicine, Univers ity of Geo rgia, Athens, GA Abstract. Th e nature of feather inclu sion s was characterized in 32 psittacin e birds (30 cockatoo s, one peachfaced lovebird (Agapornis roseicollis), and one red-Ior ed Ama zon parrot (Amazona autum nalis autum nalis )) with natu rally-acquired psittacine beak and feath er disease. Intranuclear inclusion s within feath er epithelial cells and intr acytop lasm ic inclusions within macrophages in the feath er epithelium an d pulp cav ity contained psittacine beak and feath er disease viral antigen when stained by the avidin-biot in complex imm uno peroxi dase technique. Ultrastructurally, inclu sion s were observed prim ar ily within macrophages and to a lesser extent within epithelial cell nuclei. Macroph age inclu sion s app eared as paracrystalline arrays of vira l particl es. Intranuclear inclu sion s were less well defin ed , alth ough scatte red viral particl es were present. Int racytoplasmi c and intranuclear part icles in ultrast ructura l preparations were identifi ed by colloida l gold labeling as psittacine beak and feath er disease virus . Feather epithelium was more frequ entl y and sever ely involved in the disease process than was adjacent follicular epithelium. Plucked feath ers with an int act epiderma l collar and feath er epithelium were preferr ed to follicular bio psies for histopath ologic examination. Key words: Avian species; psittacine beak and feath er disease; DNA virus; viral inclusion s; immunoperoxidase stai ning; ultrastructure; colloida l gold labelin g.
Psittacine beak and feather disease wa s describ ed in South Pacifi c psittacine birds in th e mid 1970s. The d isease is cha racterize d by relati vel y symmetric feather d ystrophy and loss. Additionally, beak abnormalities suc h as abnormal elongation, palatine necrosis, and tran sverse to longitudinal fra ctures or delaminations al so may b e observed." Ev id e nce o f a v ira l et io logy has been supported by natural and ex pe rimen ta l tra ns m iss io n of th e disea se, 6.9.1 1 intranuclear and intracytoplasmic basophilic inclusions in histologic sections of feathers and folli cular epithelium .v-? and paracrystall ine arrays of putative viral particles in ultrastruct ural sections of diseased feath ers.v-? A novel DNA virus recently has been reco vered and cha racte rize d from feather follicle tract s of b irds with psittacine beak and feath er d isease.' ? The v iru s is 14 to 17 nm in diameter, ico sa hedral , and nonen veloped. The viral DNA is 1.7 to 2 .0 kb , single stranded, and circular.' ? Virus obtained from cutaneous tissues of diverse genera of diseased psittacine birds is antigenically sim ila r, indicating that thi s novel DNA v irus probably is associated with clinica l d isease in a large a v ian host range." The purpo se of this st udy was to in vestigate feather inclusions in b irds with ps ittacine beak and feather
disease. Specifically, we wished to determine whether inclusions previously observed by light microscopy and paracrystalline arrays o f v irus-like particles pre v iously observed by electron microscopy co n ta ined v ira l antigen associated with the newl y cha racte rized p sittacine beak and feather disease virus. In addition , we wis hed to review various feather lesio n s and determine whethe r feather or folli cular ephithelium was affe ct ed more frequentl y or se verel y.
Materials and Methods Birds T hirty-two bird s with clinical signs suggestive of psitta cine beak and feather disease were don ated to the Unive rsity of Geo rgia Veterinar y Medical Teac hing Hospital for furt her testin g and observatio n. Clinical signs included varia ble degrees of feath er dystroph y and loss, occasio nally accompanied by mild to severe beak invo lvement. Th ese bird s includ ed 30 cockatoos (ten umbrella, Cacatua alba ; nin e Moluccan , Cacatua m oluccensis; two Major Mitc hell, Cacatua leadbea teri; two citron, Caca tua sulphurea cintrinocristata ; two Philippine red-vent ed, Cacatua haemat uropygia haematuropygius; two lesser sulphur-crested, Cacatua sulph urea sulphure a ; one goffi n, Cacatua goffini; one triton , Cacatua galerita triton; and one black palm, Probosciger ater-
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Psitt acin e Bea k and Fea ther Disease
rimusr : one peac h-face d lo ve bird, Agapornis roseicollis: a nd o ne rcd-lore d A mazo n pa rrot , Ama zona autu mna lis autumn alis .
Diagnosis of psittacine beak and feather disease Diagnosis of psitt acine beak and feather d isease was based upo n cli nica l signs and th e presen ce of ty pica l inclu sion s in he ma tox ylin a nd eos in-stai ned fea ther a nd ski n biop sy section s. If th e bi rd died o r was eutha na tized, a n a tte m pt was mad e to recover psitt acine bea k a nd feather d isease virus from d iseased cuta neo us tissues. The meth od of vi rus recovery was perfo rm ed following publ ished m eth od s as deta iled below. Jt) S kin and feather biopsy Histopathologic sp ecimen s were o btained while th e birds wer e und er isofluranc seda tio n, deli vered by a face m as k. Severa l di seased fea the rs were plucked , and a skin biop sy was tak en from eac h bird. All speci me ns were di vid ed a nd pla ced in 10% neutral buffer ed formalin for ro utine histopathologic eva luat ion a nd in chilled Trump's fixa tive for tra nsm ission electron mi croscop y. T issues for light mi croscopy were processed ro utinel y, em bedde d in par affin , a nd sec tion ed at 4 /-L m . Init ial sec tio ns were stained with hem atoxylin an d eosin. Replicat ed tissue sections were prepared for im m unostai ni ng as detai led below . Recover y of psittacine bea k a nd feather disea se virus and primary a ntibod y production Psitt acin e bea k and feathe r disease virus was recovered from feather follicle tract s o f th e skin of d iseased birds following published meth od s.'? Frozen feather follicle tracts were th a wed , minced , and ho moge nize d . The hom ogen at e was centrifuged a t 1,000 x g for 10 minutes. The supe rna ta nt was collected a nd layered o nto a 45 % suc rose cushio n in TRI S-buffer ed saline solution (T BS, pH 7.6) a nd centrifuged a t 140,000 x g for 2 hours a t 4 C. Cru de vira l pellet s were resu spended in TB S, adjusted to 1.40 g/cc with cesium chl orid e, and centrifuged to eq uilibrium a t 27 0,000 x g for 16 hours a t 20 C. Th e vira l band was identifi ed , collected by side pun cture o f th e tube, di al yzed aga inst TBS , and furth er purifi ed by centrifugatio n at 270 ,000 x g fo r 90 minutes at 4 C th rou gh a 20-60% (w/v) linear sucrose gradie nt. Th e grad ient was co llected usin g a dens ity grad ient frac tio na to r (ISCO , Inc., Linco ln, NE). Virus-co nta ini ng fractio ns were di al yzed agai ns t ph osph at e bu ffered sa line so lutio n (PBS, pH 7.4) an d stored at - 20 C un ti l used. Rab bit a nti-psittacine beak and feather di sease whole virus a ntibody was produced for im munoh istoche m ical sta ini ng of pa ra ffin- a nd resin- em bedded tissues. T his pr im a ry a nti bod y was genera ted by inocu lating a ra bbit (initial imm uniza tion a nd two booste r injections) with puri fied vi rus suspensions o btained from diseased birds during th e ini tia l c ha racteriza tio n of the virus.!" Afte r a n a ppropriate tim e interval , blood was collected from th e rabbit, a nd seru m was sepa rate d. Seru m was heat inac tiva ted at 56 C for 30 minutes to elim ina te co m pleme nt ac tiv ity. Rabbit immunoglobul in was precipitat ed with a m mo nium sulfate , res uspe nde d in PBS (pH 7.2), dial yzed , a nd aliq uo ts were stored at - 20 C
30 1
unt il usc. Prior to immun ostaining, a ntibo dy so lutions fo r usc o n pa ra ffi n tissue sections were th awed a nd adsorbed wit h chicke n liver a nd kid ne y powder to m inim ize non specific stai ning . Antibod y solutio ns for im m unoelectro n microsco py were add itiona lly adsorbed agai nst psitt aci ne skin powder ob ta ined fro m a psitt acine bea k an d feat her d isease virus-free bird . O pti m um di lutions of eac h antibody wer e deter m ined by prior test ing. Immunohistochemistry Immun ohi stoch emical sta ini ng was per formed by the a vid in-b iot in co m plex (A BC) immunop ero xidase technique using a co m mercial kit (Vecto r Labo ra to ries, Burlingam e, CA).' Repli cat e par affin tissue sections were placed on slides coa ted with poly- L-Iysin e a nd dri ed overn ight at 60 C. Ti ssue section s were deparaffiniz ed , rinsed in deionized wa ter , a nd treated for 15 minutes with 3% hydrogen pero xide to qu en ch endoge no us pero xid ase activ ity. Ti ssue sections were rinsed in PBS (pH 7.4). D ilut ed normal goa t seru m was a pplied to th e tissue sections to reduce non speci fic sta ining. Primar y rabbit a ntibody was a pplied at a pre de ter mi ned diluti on ( I : 4,000), an d sectio ns were incuba ted o vernig ht at 4 C. T he slides were rinse d in PBS, goa t an ti-ra bbit biotinylated a nt ibod y ( I : 200) was ap plie d, an d the sec tions were incubat ed for 30 m inut es a t roo m tem pera ture (23 C). Fo llowing a rin se in PBS, A BC reagent was applied a nd sectio ns were incubated at room tem pera ture for I hour. Tissue sectio ns were rinsed in PBSagain , and a freshly prepared chro moge n so lut io n of hyd rogen pero xide and d ia mi nobenz id ine substra te (Sigma Chem ical Co., St. Louis , MO) was a pplied for 6 minutes. After a final rinse in deion ized wa ter , tissue sect ions we re co untersta ined with G ill's hem at oxylin, deh yd rat ed , covers lipped , and exa mined. A negat ive control was included for eac h spec ime n by subs tit uting norm al ra bbit serum (I : 4,000 dilut ion ; Vector Laborato ries, Burlingam e, CAl for th e prima ry antibod y a nd performing subseq uent steps as pre viou sly descr ibed . Feather sectio ns from a bird with typ ical psittacin e beak and feat he r di sea se inclusions were included as positive and negati ve co ntro ls to valida te th e stain ing technique and to pro vid e guide lines for di stingui shin g positi ve from non specific (backgro und) sta in ing. Positi ve sta ini ng was ind icat ed by th e presence of a brown pigm ent. Routine transmi ssion a nd immunoclectron microscop y Eac h skin bio psy or plu cked -feather speci me n preser ved in Tru mp's fixative was divided eq ua lly and prep a red for rou tine tra nsm ission elect ro n m icroscopic a nd im rn unoe lectron m icroscopic exa m inatio ns. T hose spec im ens for the form er (tra nsm issio n electron mi crosco py) were pos t-fixed in I % phos pha te- buffered osm ium tet ro xide (p H 7.2), dehyd rated in grade d etha no l, a nd e m bedded in Spurr low-viscos ity resin. T hi n sec tio ns were placed o n for mva r-ca rbo n coa ted high -tran smi ssion nickel grids , sta ined with mc t ha no lic uran yl ace ta te a nd Reynold 's lead citra te, a nd exa mi ned in an electron mi croscop e a t 80 ke V. Specime ns for immun oelect ron mi croscopi c exa m ina tio n were deh ydr at ed in etha no l and em bedded in L. R. White res in (Lo ndo n Resin Co m pa ny, London , Engla nd). Thin sec-
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Fig. 1. Electro n micrograph. Psittacine beak and feather d isease viru s; citro n cocka too . Virions recovered fro m d iseased cuta neo us tissues. Negatively sta ined with ph osph otungstic acid. Bar = 0. 1 iLm . tion s were placed on form var-carbon coa ted high-transm ission nickel grid s and incuba ted for I hour with saturated sod ium metaperiodate. G rids were then incubated for 10 mi nutes in 0.2 M T BS con tai ni ng 1% glob ulin-free bovine ser um alb umin . Nonspecific staining was m inim ized by incubating th e grids for 30 minutes in d iluted who le goat serum ( I : 10). Th e grids were j et-washed in T BS for I m in ute and incub ated with rabbi t primary antibod y at a predeterm ined di lution ( 1: 500 ) ov ernight at 4 C in a moist cha mber. The grid s were jet- washed thorough ly in TBS and incubated for 30 mi nutes wit h goat anti-rabbi t IgG (I : 20 dilutio n) conj ugated to 10 nm colloida l gold par ticles (Jan ssen Life Sciences Prod ucts, Beerse, Belgium). Followi ng ano ther je twas hing in T BS an d deionized water , grids were stained in aq ueous 2% uranyl acetate for 8 min utes in Reynold 's lead citrate for 12 min utes. Negati ve contro ls were prepared in a similar manner except that diluted normal rabbit seru m (I : 500) was used in place of the primary rabbit antibody. Specimens subseq uent ly were viewed in an electron micro scop e at 60 keV .
Result s Psittacine beak and feather disease was confirmed in all diseased bird s using a com bination of clinica l signs (32/32 birds) and histopath ologic findings (32/ 32 birds). During the study, 30/ 32 birds died or were euthanatized becau se of severe disease or debilitati on . Recovery of virus from skin and feather follicle tra cts was attemp ted in 20 bird s. In each instance, typical 14 to 17 nm , icosa hedral, no nenveloped virions were obtained in high conce ntrations (Fig. I). Inclusions were observed in hematoxy lin and eosinsta ined section s of plucked feather and skin biopsies from all diseased bird s. Both intracytoplasmic and intr anuclear inclusions were observed in specimens from 23 bird s. Cyto plasmic incl usions only were presen t in biopsy specimens from seven bird s, and intranuclear inclusion s only were present in cutaneo us tissues from two bird s. In hem at oxylin an d eosin-stained sections, inclu-
sions were basoph ilic to ampho phi lic (Fig. 2). Int ranuclear incl usions were observed only within epithelial cells, and multiple globular intracytoplasmic inclusions were observed only within macrophages. Usi ng im m uno peroxi dase stai ning techn iques, both intranuclear and intracyto plasmic inclusions were strongly positive for psittacine beak an d feather disease vira l antigen. Degenerating and necrotic epit helial cells also contained vira l antigen (Fig. 3). Ultrastructurally, intracy to plasmic inclusions within macrop hages appeared as relatively organized paracrystalline arrays of viral particl es. Infrequ entl y, viral particles also were observed within degenerating epithelial cell nuclei, but paracrystallin e arrays were not apparent. Colloida l gold labeling identified the particles as psittacine beak and feather disease virus. Viral particles appeared slightly larger than 10-nm, colloidal gold pa rticles (Figs. 4, 5). Additional feath er lesion s observed by light microsco py included m ultifocal to diffuse necrosis, especially invo lving the basal layer of the feather epithelium. Occasio nally, necrosis was widespread and invo lved the entire pulp cavi ty. Inflammatory cell infiltra tes were observed in the feather pulp cavity in 30/32 diseased birds. Various feathers from the same bird often had infiltrates that differed in character. Based upon predom inating cell type, they were categorize d as heterophilic (28/30 birds), macrophage (24/30 birds), Iymphoplasmactyic (1 1/30 birds), or granu lomatous (macrophages and giant cells, 8/30 birds). Histologic changes within feat hers were often diffuse and severe. In contras t, lesion s within follicular epithelium , when present , were mu ch less seve re. Scattere d mild epithelial cell necrosis and int racytoplasmic an d intranu clear inclusions were observed within the feather follicle epit heli um of 12/3 2 birds. Necrosis of follicular epithelial cells in the absence of inclusions was observed in specime ns from an addi tiona l six birds. Fo urtee n birds with moderate to severe lesion s within
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Fig. 2. Feather; goffin cockatoo. Necrosis of basal layer of feather epithelium. A few epithelial cells contain intranuclear inclusions (arrowheads). Macrophages contain multiple cytoplasmic inclusions (arrows). HE. Bar = 25 /-tm. Fig. 3. Feather; goffin cockatoo. Intranuclear inclusions within epithelial cells (arrowheads) and cytoplasmic inclusions within macrophages (arrows) stain positively for psittacine beak and feather disease viral antigen . Avidin-biotin complex immunoperoxidase technique, Gill's hematoxylin counterstain. Bar = 25 /-tm.
feathers had no remarkable histologic changes in the follicular epithelium. In addition, four diseased birds had multiple dermal lymphoid aggregates adjacent to feather follicles, similar to descriptions of early skin leukosis in chickens with Marek's disease. I Affected feathers appeared clubbed, constricted, or otherwise deformed. Microscopically, feather sheaths were often mildly to moderately hyperkeratotic. When both plucked feathers and skin biopsies from
the same bird were compared, the diagnosis of psittacine beak and feather disease was made more rapidly and easily by examining specimens ofplucked feathers . With these specimens, all feathers were embedded at a uniform depth within the paraffin block and appeared in the same plane of section in the stained slide. With skin biopsy specimens, feather follicles usually were located at different depths within the paraffin block and repeated sectioning was necessary to examine ad-
Fig. 4. Electron micrograph . Cytoplasmic inclusion in a macrophage demonstrating paracrystalline array. Individual particles are identified as psittacine beak and feather disease virus by colloidal gold labeling (dark dots) . Adjacent cytoplasm (right border) contains minimal background labeling . Colloidal gold label, uranyl acetate and lead citrate. Bar = 0.3 /-tm. Fig. 5. Electron micrograph. Epithelial cell intranuclear inclusion containing viral particles identified as psittacine beak and feather disease virus by colloidal gold labeling. Surrounding cytoplasm (right and lower borders) contains minimal background labeling. Colloidal gold label , uranyl acetate and lead citrate. Bar = 0.2 /-tm. Downloaded from vet.sagepub.com at UNIV OF ILLINOIS LAW LIBRARY on March 17, 2015
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ditional feath er follicle s. Full thi ckn ess skin biopsies usually adhered to glass slides, whereas sections of plucked feathers sometimes were dislodged by vigorous or overzealous washing during immunoperoxidase staining.
at different depths within the paraffin block, serial sectioning is often required for ad equate histologic evaluation of skin biopsies.
Discussion
Thi s stud y was fund ed by a grant from the University of Geo rgia Co mpanion Animal Research Fund (10-2 1-RR208 083 ; CA-036) and by nu merous private do nations. Major privat e contributio ns were mad e by Dr. J. Vaughn, T. Clyne, G. Ohashi, American Federation of Aviculture, Bird Club s of Virginia , and Zeigler Brothers, Inc. Tec hnical assistance in the prepar at ion of histologic sections was provid ed by R. Bailey, K. Davis, G. Kerce, C. Player, and K. T owson . Ultrastructu ral specimens were prepar ed by M. Ard.
Both intranuclear and intracytoplasmic inclusions det ected by light microscopy were demonstrated by im m unohistochem istry to contain psittacine beak and feather disease viral antigen . Intracytoplasmic paracrystalline arrays of viral particles in macrophages were identified ultrastructurally by colloidal gold labeling as psittacine beak and feather disease virus. Although viral particles were recogni zed and labeled within som e nuclei, paracrystalline arrays were not observed, possibl y because of the ad vanced state of degeneration of the se cells or lack of marked viral replication within nucl ei. Histologic evidenc e suggests that psittacine beak and feath er disease virus may be epitheliotropic in feathers and follicles, targeting replicating cells within the basal layer of th e epithelium . The me chanism of infection of macrophages by thi s virus is uncl ear. Th ese cells may be primarily infected by viru s, because viral inclusions may be observed within these cells in various tissues of th e body, including bone marrow. ' Alternati vely, macrophages ma y become infect ed during phagocyti c removal of virus-containing epithelial detritus. Inclusion-laden macrophages may be obs erv ed within the feather pulp cavity or within feather epithelium. Infrequently, these inclusion-laden macrophages are present in th e inner feather sheath, probably as a consequenc e of continued feather growth and repair despite viral infection. Pr eviously, avian pathologists have suggested that examination of the feather follicle epithelium is essential to diagnose psittacine beak and feath er disease.v-" This study demonstrates th at follicular lesions ar e less severe and less frequent than feather lesions. Often, th e follicular epithelium may appear normal when feathers are moderately to severely affected . In summary, psittacine beak and feather disease can be diagnosed by examining plucked feathers (provided th e feather epithelium and epidermal collar remain intact). " This technique is not only rapid , economical, and noninvasive, but the feather is easily embedded in a single plane within the paraffin block , which facilitates longitudinal tissue sectioning and histopathologic examination. Skin biopsies ar e more time consuming, exp ensive, and invasive and the sites also require suturing. Because feather follicles are located
Acknowledgements
References
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Calnek BW, Witter RL: Mar ek' s disease. In: Diseases of Poult ry, ed. Hofstad MS, Barn es HJ , Calnek BW, Reid WM , and Yoder HW , 8th ed ., pp. 325-360. Iowa State Unive rsity Press, Ames, IA, 1984 Gaskin JM: Psittacine vi ral diseases: a perspective. J Zoo Wildl Dis 20:24 9-264, 1989 Graham DL: An upd ate on selected pet bird virus infectio ns, pp. 264-280. Proc Assoc Avia n Vet, Toronto, ON T, Canada, 1984 Jacob son ER, Clubb S, Simpson C, Walsh M, Lothrop CD, Gaskin J, Bauer J, Hines S, Kollias GV , Poulos P, Harri son G: Feather and beak dystrophy and necrosis in cocka toos: clinicopathologic evaluations. J Am Vet Med Assoc 189:999- 1005, 1986 Latimer KS, Raki ch PM , Kircher 1M, Rit chie BW, Niagro FD , Steffen s WL, Lukert PD: Extracut an eous vira l inclusion s in psittacine beak and feather disease. J Vet Diagn Invest 2:204-207, 1990 McOr ist S, Black DG, Pass DA, Scott PC, Marshall J: Beak and feath er dystroph y in wild sulphur-crested cockatoos (Cacatua galerita). J Wildl Dis 20: 120-124 , 1984 Pass DA, Perry RA: Th e pathology of psittacine beak and feath er disease. Aust Vet J 61:69-74, 1984 Ritchie BW, Niagro FD , Latimer KS, Lukert PD , Steffens WL, Rak ich PM, Pritchard N: Ultrastructura l, protein com position, and antigenic comparison of psittacine beak and feath er disease virus purified from four genera of psittacine bird s. J Wildl Dis 26: 196-203 , 1990 Ritchi e BW, Niagro FD , Lukert PD , Latimer KS, Steffens WL, Pritchard N: A review of psittacin e beak and feath er disease. Characteristics of the PBFD virus. J Assoc Avian Vet 3:143-149,1 989 Rit chie BW, Nia gro FD , Lukert PD, Steffens WL, Lat imer KS: Characterizatio n ofa new virus from cockat oos with psittacine beak and feather disease. Virology 171: 83-88 , 1989 Wylie SL, Pass DA: Experime ntal rep rodu ction of psittacine beak and feather disease/French moult. Avian Pathol 16:26 9-281 , 1987
Requ est reprints from Dr. K. S. Latim er, Department of Veterinary Path ology, College of Veterinary Med icine, U nive rsity of Georg ia, Ath ens, GA 30602 (USA).
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A Novel DN A Virus Associated with Feather Inclusions in Psittacine Beak and Feather Disease K. S. LATIMER,
B. W.
P. M.
RITCHIE,
RAKI CH,
F. D.
W. L.
STEFFENS,
N IAGRO, AND
P. D.
I. M.
KIRCHER ,
L UKERT
Departments of Veter inary Path ology, Anato my an d Radi ology, Small Anima l Medicine, Med ical Microb iology, and the Athens Diagnostic and Investigati onal Laboratory, College of Veterinary Medicine, Univers ity of Geo rgia, Athens, GA Abstract. Th e nature of feather inclu sion s was characterized in 32 psittacin e birds (30 cockatoo s, one peachfaced lovebird (Agapornis roseicollis), and one red-Ior ed Ama zon parrot (Amazona autum nalis autum nalis )) with natu rally-acquired psittacine beak and feath er disease. Intranuclear inclusion s within feath er epithelial cells and intr acytop lasm ic inclusions within macrophages in the feath er epithelium an d pulp cav ity contained psittacine beak and feath er disease viral antigen when stained by the avidin-biot in complex imm uno peroxi dase technique. Ultrastructurally, inclu sion s were observed prim ar ily within macrophages and to a lesser extent within epithelial cell nuclei. Macroph age inclu sion s app eared as paracrystalline arrays of vira l particl es. Intranuclear inclu sion s were less well defin ed , alth ough scatte red viral particl es were present. Int racytoplasmi c and intranuclear part icles in ultrast ructura l preparations were identifi ed by colloida l gold labeling as psittacine beak and feath er disease virus . Feather epithelium was more frequ entl y and sever ely involved in the disease process than was adjacent follicular epithelium. Plucked feath ers with an int act epiderma l collar and feath er epithelium were preferr ed to follicular bio psies for histopath ologic examination. Key words: Avian species; psittacine beak and feath er disease; DNA virus; viral inclusion s; immunoperoxidase stai ning; ultrastructure; colloida l gold labelin g.
Psittacine beak and feather disease wa s describ ed in South Pacifi c psittacine birds in th e mid 1970s. The d isease is cha racterize d by relati vel y symmetric feather d ystrophy and loss. Additionally, beak abnormalities suc h as abnormal elongation, palatine necrosis, and tran sverse to longitudinal fra ctures or delaminations al so may b e observed." Ev id e nce o f a v ira l et io logy has been supported by natural and ex pe rimen ta l tra ns m iss io n of th e disea se, 6.9.1 1 intranuclear and intracytoplasmic basophilic inclusions in histologic sections of feathers and folli cular epithelium .v-? and paracrystall ine arrays of putative viral particles in ultrastruct ural sections of diseased feath ers.v-? A novel DNA virus recently has been reco vered and cha racte rize d from feather follicle tract s of b irds with psittacine beak and feath er d isease.' ? The v iru s is 14 to 17 nm in diameter, ico sa hedral , and nonen veloped. The viral DNA is 1.7 to 2 .0 kb , single stranded, and circular.' ? Virus obtained from cutaneous tissues of diverse genera of diseased psittacine birds is antigenically sim ila r, indicating that thi s novel DNA v irus probably is associated with clinica l d isease in a large a v ian host range." The purpo se of this st udy was to in vestigate feather inclusions in b irds with ps ittacine beak and feather
disease. Specifically, we wished to determine whether inclusions previously observed by light microscopy and paracrystalline arrays o f v irus-like particles pre v iously observed by electron microscopy co n ta ined v ira l antigen associated with the newl y cha racte rized p sittacine beak and feather disease virus. In addition , we wis hed to review various feather lesio n s and determine whethe r feather or folli cular ephithelium was affe ct ed more frequentl y or se verel y.
Materials and Methods Birds T hirty-two bird s with clinical signs suggestive of psitta cine beak and feather disease were don ated to the Unive rsity of Geo rgia Veterinar y Medical Teac hing Hospital for furt her testin g and observatio n. Clinical signs included varia ble degrees of feath er dystroph y and loss, occasio nally accompanied by mild to severe beak invo lvement. Th ese bird s includ ed 30 cockatoos (ten umbrella, Cacatua alba ; nin e Moluccan , Cacatua m oluccensis; two Major Mitc hell, Cacatua leadbea teri; two citron, Caca tua sulphurea cintrinocristata ; two Philippine red-vent ed, Cacatua haemat uropygia haematuropygius; two lesser sulphur-crested, Cacatua sulph urea sulphure a ; one goffi n, Cacatua goffini; one triton , Cacatua galerita triton; and one black palm, Probosciger ater-
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Psitt acin e Bea k and Fea ther Disease
rimusr : one peac h-face d lo ve bird, Agapornis roseicollis: a nd o ne rcd-lore d A mazo n pa rrot , Ama zona autu mna lis autumn alis .
Diagnosis of psittacine beak and feather disease Diagnosis of psitt acine beak and feather d isease was based upo n cli nica l signs and th e presen ce of ty pica l inclu sion s in he ma tox ylin a nd eos in-stai ned fea ther a nd ski n biop sy section s. If th e bi rd died o r was eutha na tized, a n a tte m pt was mad e to recover psitt acine bea k a nd feather d isease virus from d iseased cuta neo us tissues. The meth od of vi rus recovery was perfo rm ed following publ ished m eth od s as deta iled below. Jt) S kin and feather biopsy Histopathologic sp ecimen s were o btained while th e birds wer e und er isofluranc seda tio n, deli vered by a face m as k. Severa l di seased fea the rs were plucked , and a skin biop sy was tak en from eac h bird. All speci me ns were di vid ed a nd pla ced in 10% neutral buffer ed formalin for ro utine histopathologic eva luat ion a nd in chilled Trump's fixa tive for tra nsm ission electron mi croscop y. T issues for light mi croscopy were processed ro utinel y, em bedde d in par affin , a nd sec tion ed at 4 /-L m . Init ial sec tio ns were stained with hem atoxylin an d eosin. Replicat ed tissue sections were prepared for im m unostai ni ng as detai led below . Recover y of psittacine bea k a nd feather disea se virus and primary a ntibod y production Psitt acin e bea k and feathe r disease virus was recovered from feather follicle tract s o f th e skin of d iseased birds following published meth od s.'? Frozen feather follicle tracts were th a wed , minced , and ho moge nize d . The hom ogen at e was centrifuged a t 1,000 x g for 10 minutes. The supe rna ta nt was collected a nd layered o nto a 45 % suc rose cushio n in TRI S-buffer ed saline solution (T BS, pH 7.6) a nd centrifuged a t 140,000 x g for 2 hours a t 4 C. Cru de vira l pellet s were resu spended in TB S, adjusted to 1.40 g/cc with cesium chl orid e, and centrifuged to eq uilibrium a t 27 0,000 x g for 16 hours a t 20 C. Th e vira l band was identifi ed , collected by side pun cture o f th e tube, di al yzed aga inst TBS , and furth er purifi ed by centrifugatio n at 270 ,000 x g fo r 90 minutes at 4 C th rou gh a 20-60% (w/v) linear sucrose gradie nt. Th e grad ient was co llected usin g a dens ity grad ient frac tio na to r (ISCO , Inc., Linco ln, NE). Virus-co nta ini ng fractio ns were di al yzed agai ns t ph osph at e bu ffered sa line so lutio n (PBS, pH 7.4) an d stored at - 20 C un ti l used. Rab bit a nti-psittacine beak and feather di sease whole virus a ntibody was produced for im munoh istoche m ical sta ini ng of pa ra ffin- a nd resin- em bedded tissues. T his pr im a ry a nti bod y was genera ted by inocu lating a ra bbit (initial imm uniza tion a nd two booste r injections) with puri fied vi rus suspensions o btained from diseased birds during th e ini tia l c ha racteriza tio n of the virus.!" Afte r a n a ppropriate tim e interval , blood was collected from th e rabbit, a nd seru m was sepa rate d. Seru m was heat inac tiva ted at 56 C for 30 minutes to elim ina te co m pleme nt ac tiv ity. Rabbit immunoglobul in was precipitat ed with a m mo nium sulfate , res uspe nde d in PBS (pH 7.2), dial yzed , a nd aliq uo ts were stored at - 20 C
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unt il usc. Prior to immun ostaining, a ntibo dy so lutions fo r usc o n pa ra ffi n tissue sections were th awed a nd adsorbed wit h chicke n liver a nd kid ne y powder to m inim ize non specific stai ning . Antibod y solutio ns for im m unoelectro n microsco py were add itiona lly adsorbed agai nst psitt aci ne skin powder ob ta ined fro m a psitt acine bea k an d feat her d isease virus-free bird . O pti m um di lutions of eac h antibody wer e deter m ined by prior test ing. Immunohistochemistry Immun ohi stoch emical sta ini ng was per formed by the a vid in-b iot in co m plex (A BC) immunop ero xidase technique using a co m mercial kit (Vecto r Labo ra to ries, Burlingam e, CA).' Repli cat e par affin tissue sections were placed on slides coa ted with poly- L-Iysin e a nd dri ed overn ight at 60 C. Ti ssue section s were deparaffiniz ed , rinsed in deionized wa ter , a nd treated for 15 minutes with 3% hydrogen pero xide to qu en ch endoge no us pero xid ase activ ity. Ti ssue sections were rinsed in PBS (pH 7.4). D ilut ed normal goa t seru m was a pplied to th e tissue sections to reduce non speci fic sta ining. Primar y rabbit a ntibody was a pplied at a pre de ter mi ned diluti on ( I : 4,000), an d sectio ns were incuba ted o vernig ht at 4 C. T he slides were rinse d in PBS, goa t an ti-ra bbit biotinylated a nt ibod y ( I : 200) was ap plie d, an d the sec tions were incubat ed for 30 m inut es a t roo m tem pera ture (23 C). Fo llowing a rin se in PBS, A BC reagent was applied a nd sectio ns were incubated at room tem pera ture for I hour. Tissue sectio ns were rinsed in PBSagain , and a freshly prepared chro moge n so lut io n of hyd rogen pero xide and d ia mi nobenz id ine substra te (Sigma Chem ical Co., St. Louis , MO) was a pplied for 6 minutes. After a final rinse in deion ized wa ter , tissue sect ions we re co untersta ined with G ill's hem at oxylin, deh yd rat ed , covers lipped , and exa mined. A negat ive control was included for eac h spec ime n by subs tit uting norm al ra bbit serum (I : 4,000 dilut ion ; Vector Laborato ries, Burlingam e, CAl for th e prima ry antibod y a nd performing subseq uent steps as pre viou sly descr ibed . Feather sectio ns from a bird with typ ical psittacin e beak and feat he r di sea se inclusions were included as positive and negati ve co ntro ls to valida te th e stain ing technique and to pro vid e guide lines for di stingui shin g positi ve from non specific (backgro und) sta in ing. Positi ve sta ini ng was ind icat ed by th e presence of a brown pigm ent. Routine transmi ssion a nd immunoclectron microscop y Eac h skin bio psy or plu cked -feather speci me n preser ved in Tru mp's fixative was divided eq ua lly and prep a red for rou tine tra nsm ission elect ro n m icroscopic a nd im rn unoe lectron m icroscopic exa m inatio ns. T hose spec im ens for the form er (tra nsm issio n electron mi crosco py) were pos t-fixed in I % phos pha te- buffered osm ium tet ro xide (p H 7.2), dehyd rated in grade d etha no l, a nd e m bedded in Spurr low-viscos ity resin. T hi n sec tio ns were placed o n for mva r-ca rbo n coa ted high -tran smi ssion nickel grids , sta ined with mc t ha no lic uran yl ace ta te a nd Reynold 's lead citra te, a nd exa mi ned in an electron mi croscop e a t 80 ke V. Specime ns for immun oelect ron mi croscopi c exa m ina tio n were deh ydr at ed in etha no l and em bedded in L. R. White res in (Lo ndo n Resin Co m pa ny, London , Engla nd). Thin sec-
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Fig. 1. Electro n micrograph. Psittacine beak and feather d isease viru s; citro n cocka too . Virions recovered fro m d iseased cuta neo us tissues. Negatively sta ined with ph osph otungstic acid. Bar = 0. 1 iLm . tion s were placed on form var-carbon coa ted high-transm ission nickel grid s and incuba ted for I hour with saturated sod ium metaperiodate. G rids were then incubated for 10 mi nutes in 0.2 M T BS con tai ni ng 1% glob ulin-free bovine ser um alb umin . Nonspecific staining was m inim ized by incubating th e grids for 30 minutes in d iluted who le goat serum ( I : 10). Th e grids were j et-washed in T BS for I m in ute and incub ated with rabbi t primary antibod y at a predeterm ined di lution ( 1: 500 ) ov ernight at 4 C in a moist cha mber. The grid s were jet- washed thorough ly in TBS and incubated for 30 mi nutes wit h goat anti-rabbi t IgG (I : 20 dilutio n) conj ugated to 10 nm colloida l gold par ticles (Jan ssen Life Sciences Prod ucts, Beerse, Belgium). Followi ng ano ther je twas hing in T BS an d deionized water , grids were stained in aq ueous 2% uranyl acetate for 8 min utes in Reynold 's lead citrate for 12 min utes. Negati ve contro ls were prepared in a similar manner except that diluted normal rabbit seru m (I : 500) was used in place of the primary rabbit antibody. Specimens subseq uent ly were viewed in an electron micro scop e at 60 keV .
Result s Psittacine beak and feather disease was confirmed in all diseased bird s using a com bination of clinica l signs (32/32 birds) and histopath ologic findings (32/ 32 birds). During the study, 30/ 32 birds died or were euthanatized becau se of severe disease or debilitati on . Recovery of virus from skin and feather follicle tra cts was attemp ted in 20 bird s. In each instance, typical 14 to 17 nm , icosa hedral, no nenveloped virions were obtained in high conce ntrations (Fig. I). Inclusions were observed in hematoxy lin and eosinsta ined section s of plucked feather and skin biopsies from all diseased bird s. Both intracytoplasmic and intr anuclear inclusions were observed in specimens from 23 bird s. Cyto plasmic incl usions only were presen t in biopsy specimens from seven bird s, and intranuclear inclusion s only were present in cutaneo us tissues from two bird s. In hem at oxylin an d eosin-stained sections, inclu-
sions were basoph ilic to ampho phi lic (Fig. 2). Int ranuclear incl usions were observed only within epithelial cells, and multiple globular intracytoplasmic inclusions were observed only within macrophages. Usi ng im m uno peroxi dase stai ning techn iques, both intranuclear and intracyto plasmic inclusions were strongly positive for psittacine beak an d feather disease vira l antigen. Degenerating and necrotic epit helial cells also contained vira l antigen (Fig. 3). Ultrastructurally, intracy to plasmic inclusions within macrop hages appeared as relatively organized paracrystalline arrays of viral particl es. Infrequ entl y, viral particles also were observed within degenerating epithelial cell nuclei, but paracrystallin e arrays were not apparent. Colloida l gold labeling identified the particles as psittacine beak and feather disease virus. Viral particles appeared slightly larger than 10-nm, colloidal gold pa rticles (Figs. 4, 5). Additional feath er lesion s observed by light microsco py included m ultifocal to diffuse necrosis, especially invo lving the basal layer of the feather epithelium. Occasio nally, necrosis was widespread and invo lved the entire pulp cavi ty. Inflammatory cell infiltra tes were observed in the feather pulp cavity in 30/32 diseased birds. Various feathers from the same bird often had infiltrates that differed in character. Based upon predom inating cell type, they were categorize d as heterophilic (28/30 birds), macrophage (24/30 birds), Iymphoplasmactyic (1 1/30 birds), or granu lomatous (macrophages and giant cells, 8/30 birds). Histologic changes within feat hers were often diffuse and severe. In contras t, lesion s within follicular epithelium , when present , were mu ch less seve re. Scattere d mild epithelial cell necrosis and int racytoplasmic an d intranu clear inclusions were observed within the feather follicle epit heli um of 12/3 2 birds. Necrosis of follicular epithelial cells in the absence of inclusions was observed in specime ns from an addi tiona l six birds. Fo urtee n birds with moderate to severe lesion s within
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Fig. 2. Feather; goffin cockatoo. Necrosis of basal layer of feather epithelium. A few epithelial cells contain intranuclear inclusions (arrowheads). Macrophages contain multiple cytoplasmic inclusions (arrows). HE. Bar = 25 /-tm. Fig. 3. Feather; goffin cockatoo. Intranuclear inclusions within epithelial cells (arrowheads) and cytoplasmic inclusions within macrophages (arrows) stain positively for psittacine beak and feather disease viral antigen . Avidin-biotin complex immunoperoxidase technique, Gill's hematoxylin counterstain. Bar = 25 /-tm.
feathers had no remarkable histologic changes in the follicular epithelium. In addition, four diseased birds had multiple dermal lymphoid aggregates adjacent to feather follicles, similar to descriptions of early skin leukosis in chickens with Marek's disease. I Affected feathers appeared clubbed, constricted, or otherwise deformed. Microscopically, feather sheaths were often mildly to moderately hyperkeratotic. When both plucked feathers and skin biopsies from
the same bird were compared, the diagnosis of psittacine beak and feather disease was made more rapidly and easily by examining specimens ofplucked feathers . With these specimens, all feathers were embedded at a uniform depth within the paraffin block and appeared in the same plane of section in the stained slide. With skin biopsy specimens, feather follicles usually were located at different depths within the paraffin block and repeated sectioning was necessary to examine ad-
Fig. 4. Electron micrograph . Cytoplasmic inclusion in a macrophage demonstrating paracrystalline array. Individual particles are identified as psittacine beak and feather disease virus by colloidal gold labeling (dark dots) . Adjacent cytoplasm (right border) contains minimal background labeling . Colloidal gold label, uranyl acetate and lead citrate. Bar = 0.3 /-tm. Fig. 5. Electron micrograph. Epithelial cell intranuclear inclusion containing viral particles identified as psittacine beak and feather disease virus by colloidal gold labeling. Surrounding cytoplasm (right and lower borders) contains minimal background labeling. Colloidal gold label , uranyl acetate and lead citrate. Bar = 0.2 /-tm. Downloaded from vet.sagepub.com at UNIV OF ILLINOIS LAW LIBRARY on March 17, 2015
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ditional feath er follicle s. Full thi ckn ess skin biopsies usually adhered to glass slides, whereas sections of plucked feathers sometimes were dislodged by vigorous or overzealous washing during immunoperoxidase staining.
at different depths within the paraffin block, serial sectioning is often required for ad equate histologic evaluation of skin biopsies.
Discussion
Thi s stud y was fund ed by a grant from the University of Geo rgia Co mpanion Animal Research Fund (10-2 1-RR208 083 ; CA-036) and by nu merous private do nations. Major privat e contributio ns were mad e by Dr. J. Vaughn, T. Clyne, G. Ohashi, American Federation of Aviculture, Bird Club s of Virginia , and Zeigler Brothers, Inc. Tec hnical assistance in the prepar at ion of histologic sections was provid ed by R. Bailey, K. Davis, G. Kerce, C. Player, and K. T owson . Ultrastructu ral specimens were prepar ed by M. Ard.
Both intranuclear and intracytoplasmic inclusions det ected by light microscopy were demonstrated by im m unohistochem istry to contain psittacine beak and feather disease viral antigen . Intracytoplasmic paracrystalline arrays of viral particles in macrophages were identified ultrastructurally by colloidal gold labeling as psittacine beak and feather disease virus. Although viral particles were recogni zed and labeled within som e nuclei, paracrystalline arrays were not observed, possibl y because of the ad vanced state of degeneration of the se cells or lack of marked viral replication within nucl ei. Histologic evidenc e suggests that psittacine beak and feath er disease virus may be epitheliotropic in feathers and follicles, targeting replicating cells within the basal layer of th e epithelium . The me chanism of infection of macrophages by thi s virus is uncl ear. Th ese cells may be primarily infected by viru s, because viral inclusions may be observed within these cells in various tissues of th e body, including bone marrow. ' Alternati vely, macrophages ma y become infect ed during phagocyti c removal of virus-containing epithelial detritus. Inclusion-laden macrophages may be obs erv ed within the feather pulp cavity or within feather epithelium. Infrequently, these inclusion-laden macrophages are present in th e inner feather sheath, probably as a consequenc e of continued feather growth and repair despite viral infection. Pr eviously, avian pathologists have suggested that examination of the feather follicle epithelium is essential to diagnose psittacine beak and feath er disease.v-" This study demonstrates th at follicular lesions ar e less severe and less frequent than feather lesions. Often, th e follicular epithelium may appear normal when feathers are moderately to severely affected . In summary, psittacine beak and feather disease can be diagnosed by examining plucked feathers (provided th e feather epithelium and epidermal collar remain intact). " This technique is not only rapid , economical, and noninvasive, but the feather is easily embedded in a single plane within the paraffin block , which facilitates longitudinal tissue sectioning and histopathologic examination. Skin biopsies ar e more time consuming, exp ensive, and invasive and the sites also require suturing. Because feather follicles are located
Acknowledgements
References
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Calnek BW, Witter RL: Mar ek' s disease. In: Diseases of Poult ry, ed. Hofstad MS, Barn es HJ , Calnek BW, Reid WM , and Yoder HW , 8th ed ., pp. 325-360. Iowa State Unive rsity Press, Ames, IA, 1984 Gaskin JM: Psittacine vi ral diseases: a perspective. J Zoo Wildl Dis 20:24 9-264, 1989 Graham DL: An upd ate on selected pet bird virus infectio ns, pp. 264-280. Proc Assoc Avia n Vet, Toronto, ON T, Canada, 1984 Jacob son ER, Clubb S, Simpson C, Walsh M, Lothrop CD, Gaskin J, Bauer J, Hines S, Kollias GV , Poulos P, Harri son G: Feather and beak dystrophy and necrosis in cocka toos: clinicopathologic evaluations. J Am Vet Med Assoc 189:999- 1005, 1986 Latimer KS, Raki ch PM , Kircher 1M, Rit chie BW, Niagro FD , Steffen s WL, Lukert PD: Extracut an eous vira l inclusion s in psittacine beak and feather disease. J Vet Diagn Invest 2:204-207, 1990 McOr ist S, Black DG, Pass DA, Scott PC, Marshall J: Beak and feath er dystroph y in wild sulphur-crested cockatoos (Cacatua galerita). J Wildl Dis 20: 120-124 , 1984 Pass DA, Perry RA: Th e pathology of psittacine beak and feath er disease. Aust Vet J 61:69-74, 1984 Ritchie BW, Niagro FD , Latimer KS, Lukert PD , Steffens WL, Rak ich PM, Pritchard N: Ultrastructura l, protein com position, and antigenic comparison of psittacine beak and feath er disease virus purified from four genera of psittacine bird s. J Wildl Dis 26: 196-203 , 1990 Ritchi e BW, Niagro FD , Lukert PD , Latimer KS, Steffens WL, Pritchard N: A review of psittacin e beak and feath er disease. Characteristics of the PBFD virus. J Assoc Avian Vet 3:143-149,1 989 Rit chie BW, Nia gro FD , Lukert PD, Steffens WL, Lat imer KS: Characterizatio n ofa new virus from cockat oos with psittacine beak and feather disease. Virology 171: 83-88 , 1989 Wylie SL, Pass DA: Experime ntal rep rodu ction of psittacine beak and feather disease/French moult. Avian Pathol 16:26 9-281 , 1987
Requ est reprints from Dr. K. S. Latim er, Department of Veterinary Path ology, College of Veterinary Med icine, U nive rsity of Georg ia, Ath ens, GA 30602 (USA).
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