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Sources and manufacturers a. b. c. d. e.

Genosys Biotechnologies, The Woodlands, TX. Pharmacia, Piscataway, NJ. Life Technologies, Gaithersburg, MD. Promega, Madison, WI. Precision Scientific, Chicago, IL.

References 1. Davis VS, Boyle JA: 1990, Adapting the polymerase chain reaction to a double-stranded RNA genome. Anal Biochem 189: 30-34. 2. Diaz-Ruiz JR, Kaper JM: 1978, Isolation of viral doublestranded RNAs using a LiCl fractionation procedure. Prep Biothem 8:1-17. 3. Guatelli JC, Gingeras TR, Richmond DD: 1989, Nucleic acid amplification in vitro: detection of sequences with low copy numbers and application to diagnosis of human immunodeficiency virus type 1 infection. Clin Microbiol Rev 2:217-226. 4. Heine H-G, Haritou M, Failla P, et al.: 1991, Sequence analysis and expression of the host-protective immunogen VP2 of a variant strain of infectious bursal disease virus which can circumvent vaccination with standard type I strains. J Gen Virol 72:1835-1843.

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5. Higuchi R: 1989, Rapid, efficient DNA extraction for PCR from cells or blood. Amplifications 2:1-3. 6. Kibenge FSB, Jackwood DJ, Mercado CC: 1990, Nucleotide sequence analysis of genome segment A of infectious bursal disease virus. J Gen Virol 71:569-577. 7. Kusukawa N, Uemori T, Asada K, Kato I: 1990, Rapid and reliable protocol for direct sequencing of material amplified by the polymerase chain reaction. BioTechniques 9:66-72. 8. Li HH, Gyllensten WB, Cui XF, et al.: 1988, Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 335:414-417. 9. Lukert PD, Saif YM: 1991, Infectious bursal disease. In: Diseases of poultry, ed. Calnek BW, Barnes HJ, Beard CW, et al., 9th ed., pp. 648-663. Iowa State University Press, Ames, IA. 10. Rosenberger JK: 1989, Infectious bursal disease. In: Isolation and identification of avian pathogens, ed. Purchase HG, Arp LH, Domermuth CH, Pearson JE, 3rd ed., pp. 165-166. American Association of Avian Pathologists, Kennett Square, PA. 11. Saiki RK, Gelfand DH, Stoffel S, et al.: 1988, Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. 12. Saiki RK, Scharf S, Faloona F, et al.: 1985, Enzymatic amplification of ß-globin genomic sequences and restriction site analyses for diagnosis of sickle cell anemia. Science 230: 1350-1354.

J Vet Diagn Invest 4:455-458 (1992)

Budgerigar fledgling disease (papovavirus) in pet birds Richard S. Kingston Budgerigar fledgling disease (BFD), a contagious disease causing high mortality in fledgling budgerigars (Melopsittacus undulatus), was first reported in the United States5 and Canada1 and subsequently has been recognized in other countries 11,14,19,22,23 The causative agent was identified as a papovavirus and has been characterized as the first nonmammalian member of the polyomavirus genus.3,16 In addition, papovavirus infection of numerous other bird species has been described.7,10.12,14,20,21,24 This report describes BFD in a small aviary and briefly reviews papovavirus infection in pet birds. Seven budgerigars, 20-30 days old, were presented for diagnostic evaluation. Beginning 6 months earlier, mortality in young budgerigars had markedly increased from < 10% to >90%. The aviary owner reported that young birds most often died between 10 and 14 days of age (range, 10-28 days), and those that survived were stunted and lacked normal feather development. Adult budgerigars, however, all appeared normal and were producing a normal number of fertile eggs. The affected budgerigars were small when compared with

unaffected age-matched nestlings. They lacked filoplumes on the head and neck and down feathers on the back and breast and had underdeveloped primary and secondary flight feathers and tail feathers (Fig. 1). They often had full crops, distended abdomens, mottled tan-red livers, and reddened skin. Multiple tissues were fixed in 10% formalin, tissue homogenates were negatively stained with 2% phosphotungstic acid, and kidney, skin, and brain were fixed in 4% formaldehyde- 1% glutaraldehydel 7 and routinely processed for light and electron microscopy, respectively. In addition, samples were collected for bacterial culture and virus isolation. Although the severity of histological lesions varied among birds, essentially similar changes were present in all. The most consistent finding was cells with karyomegaly. In these cells, the nucleus was enlarged, had marginated chromatin, and often contained a large slightly basophilic to amphophilic inclusion. These cells were most prominent in feather follicles, which often were severely affected, although not all follicles were involved. Focal to confluent ballooning degeneration with karyomegaly was prominent in follicular epithelial cells, barb ridges, and cells within the pulp (Fig. 2). Karyomegalic cells also were in scattered foci in renal From the New Hampshire Veterinary Diagnostic Laboratory, De- tubular epithelium, glomeruli, brain (Fig. 3), spleen, liver, partment of Animal and Nutritional Sciences, University of New myocardium, pancreas, intestine, lung, skeletal muscle, and bone marrow. Hampshire, Durham, NH 03824. In many hepatic periportal zones, there was minimal neReceived for publication November 20, 1991.

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Figure 1. Affected (left) and normal budgerigars. The affected budgerigar is the same age, is stunted, and lacks normal feather development.

crosis with a minimal to- moderate number of heterophils. Occasional small foci ofcoagulation necrosis with heterophils were randomly scattered in the parenchyma. Splenic white pulp was hypercellular with small foci of karyomegalic cells present. The walls of many small arteries and sheathed capillaries also were prominent because of hypertrophy and karyomegaly of the surrounding cells. In the negatively stained tissue homogenates and thin sections of renal tubular epithelium, virus particles were present in small clusters. On the basis of size and morphology (approximately 40 mm in diameter, nonenveloped icosahedral virions), the particles were identified as papovaviruses. Five live budgerigars (4 adults and 1 fledgling) in the aviary were tested for antibody to BFD virus by fluorescent antibody serum neutralization. All were seropositive ( ≥ 1:10), with titers ranging from 1:10 to 1:320. No bacterial pathogen or Chlamydia were isolated from any of the tissues examined, and virus isolation was unsuccessful. Based on the history, clinical findings, necropsy results, and serology, a diagnosis of BFD was made. In an aviary with BFD, nestling budgerigars (1-3 weeks of age) suffer the highest mortality. A striking finding in these birds is the almost total lack of downy feather development, which is prominent even in budgerigars that survive up to 28 days of age. Normal baby budgerigars are hatched without feathers, but down begins to cover the skin by about 12 days of age; plumage development is complete at a little more than 1 month of age. 15 Although other diseases must be considered, death at 10-28 days of age with a significant lack of feather development should alert the diagnostician to the possibility of BFD. The combination of typical gross necropsy findings (stunted growth, abnormal feather development) and microscopic lesions, including pronounced karyomegaly with intranuclear inclusions, leads to a presumptive

diagnosis of BFD. This diagnosis should be substantiated by virus isolation or by identification of the virus via specific immunofluorescence. DNA amplification via the polymerase chain reaction can also be used to detect BFD virus.18 The transmission and pathogenesis of BFD have not been fully described. Virus particles have been demonstrated in epithelia of feather follicles, esophagus, crop, lung, and kidney, thus implicating several possible routes oftransmission. Feather dust, regurgitated food fed to nestlings, respiratory secretions, and/or urinary excretions could all contain infectious virus and serve as a source of infection. Vertical transmission of the virus through the egg also has been considered. Inclusion bodies have been demonstrated in the internal organs and skin of 1-day-old budgerigars, and eggs from breeding pairs producing affected young will result in diseased birds when fostered by pairs whose own young are normal.1,2 Serologic testing has shown that viral antibodies remain elevated for the lifetime of a persistently infected (carrier) bird. Persistently infected female birds can raise serologically negative uninfected chicks and immunocompetent adult birds can be caged with persistently infected birds and will remain uninfected.8 The development of immunocompetence is crit-

ical. Nestlings unprotected by passive antibody and exposed to papovavirus are susceptible and may die. Those protected may survive but still be contaminated with the virus and serve as a source of infection, which is an important consideration when baby birds are being hand raised and the virus can be easily transmitted. The economic consequences of BFD in a breeding aviary can be devastating. Although adult birds may be unaffected, the mortality in young birds can be extremely high and can essentially eliminate the successful production and sale of young budgerigars. The year before the outbreak, approximately 200 birds from the aviary in this study were fledged,

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Figure 3. Photomicrograph of cerebellum of a budgerigar with BFD containing numerous karyomegalic cells with intranuclear inclusions (arrow) in the molecular layer. HE. Figure 2. Photomicrograph of skin section of a budgerigar with BFD containing a feather follicle (top) with karyomegaly and ballooning degeneration of the epithelium (arrow) and a normal follicle (bottom). HE.

whereas only 10 were fledged in the following year. The BFD source was undetermined; however, new adult budgerigars had been introduced into the aviary several months prior to the outbreak, and 1 or more of these birds were probably carriers of the virus. Several other papers have reported mortality rates ranging from 25% to 100% in fledglings; some of these aviaries had up to approximately 1,200 breeders.1,5,9 One report described a decrease in hatchability from 80% to 40% in an aviary with 90 breeding pairs.9 Once BFD is present in an aviary, successful elimination of the disease, short of depopulation, is very difficult. There is no known treatment, and a vaccine is not commercially available. Preventive measures include maintaining a totally closed flock and prohibiting visitors and visitation to other flocks. Control of the disease via thorough disinfection of the aviary using chlorine or phenolic solutions and fumigation with formaldehyde gas has been recommended and was suc9,12,22 Interruption of breeding (budgerigars cessful in 1 aviary. breed year round), thus removing the supply of susceptible birds, has also been recommended but has met with limited success.9 Successful elimination of BFD from an aviary was achieved via depopulation, decontamination, and restocking with seronegative birds.4 Since the initial descriptions of BFD in budgerigars, similar lesions have been described in a variety of other pet bird species, including conures, macaws, Amazon parrots, other

parrots, cockatoos, cockatiels, lorries, lovebirds, parakeets, that a vaand finches. 7,10,12.13,20,21 Serologic tests have shown. riety of birds can be infected by papovavirus.12,24 Papovavirus infections in budgerigars are clinically and pathologically different from these infections in other birds. In budgerigars, BFD primarily presents as an acutely fatal disease in birds up to 21 days of age or as a more chronic disease with feather anomalies in older birds.2,5 In conures, Amazon parrots, cockatoos, cockatiels, lorries, and macaws, age at the time of death is somewhat older, ranging from nestling to 16 weeks,10,12 although death in splendid parakeets,21 Gouldian finches7 and lovebirds20 occurred at 3 and 8 months, as fledglings to immature adults, and at < 1 year old, respectively. Abnormalities in feather development and structure, lesions typically described in young budgerigars with papovavirus disease, are not commonly seen in other species of pet birds. A variety of gross lesions are described, but all are nonspecific and include pallor of muscles, hepatomegaly with a mottled discoloration, petechia and ecchymoses, splenomegaly, hydropericardium, and ascites. As in budgerigars, the most consistently observed histologic lesion in other birds is karyomegaly, which occurs in a variety of tissues and often is associated with large intranuclear inclusions. Multifocal hepatic coagulative necrosis is also a consistent and significant lesion, although it is variable in extent. In some cases, liver necrosis is limited to scattered microfoci, although in others the necrosis is more confluent. Although the classic feather anomalies typically seen in budgerigars are not seen in other birds, a presumptive diagnosis

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of papovavirus infection can often be made on the basis of clinical signs; postmortem findings, including light and electron microscopic lesions; and serology. Acknowledgement. I thank Dr. R. B. Davis, Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens, for doing the serologic tests.

References 1. Bemier G, Morin M, Marsolais G: 1981, A generalized inclusion body disease in the budgerigar (Melopsittacus undulatus) caused by a papovavirus-like agent. Avian Dis 251083-1092. 2. Bemier G, Morin M, Marsolais G: 1984, Papovavirus induced feather abnormalities and skin lesions in the budgerigar: clinical and pathological findings. Can Vet J 25307-310. 3. Bozeman LH, Davis RB, Gaudry D, et al.: 1981, Characterization of a papovavirus isolated from fledgling budgerigars. Avian Dis 25:972-980. 4. Davis RB: 1983, Budgerigar fledgling disease (BFD). Proc 32nd West Poult Dis Conf, p. 104. 5. Davis, RB, Bozeman LH, Gaudry D, et al.: 1981, A viral disease of fledgling budgerigars. Avian Dis 25: 179-183. 6. Dykstra MJ, Bozeman LH: 1982, A light and electron microscopic examination of budgerigar fledgling disease virus in tissue and cell culture. Avian Path01 11: 11-28. 7. Forshaw D, Wylie SL, Pass DA: 1988, Infection with a virus resembling papovavirus in Gouldian finches (Erythrura gouldiae). Aust Vet J 65:26-28. 8. Gaskin JM: 1989, Psittacine viral diseases: a perspective. Zoo and Wildlife Med 20:249-264. 9. Gough JF: 1989, Outbreaks of budgerigar fledgling disease in three aviaries in Ontario. Can Vet J 30:672-674. 10. Graham DL, Calnek BW: 1987, Papovavirus infection in handfed parrots: virus isolation and pathology. Avian Dis 31:398410. 11. Hirai K, Nonaka H, Fukushi H, et al.: 1984, Isolation of a

papovavirus-like agent from young budgerigars with feather abnormalities. Jpn J Vet Sci 46:577-582. 12. Jacobson ER, Hines SA, Quesenberry K, et al.: 1984, Epomitic of papova-like virus-associated disease in a psittacine nursery. J Am Vet Med Assoc 185:1337-1341. 13. Johnston KM, Riddell C: 1986, Intranuclear inclusion bodies in finches. Can Vet J 27:432-434. 14. Krautwald ME, Kaleta EF: 1985, Relationship(s) of French moult and early virus induced mortality in nestling budgerigars. Proc 8th Int Congr World Vet Poult Assoc Jerusalem, p. 115. 15. Lafeber TJ: 1965, Bird clinic: the budgerigar. Anim Hosp 1: 122-132. 16. Lehn H, Miiller H: 1986, Cloning and characterization of budgerigar fledgling disease virus, an avian polyomavirus. Virology 151:362-370. 17. McDowell EM, Trump BF: 1976, Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med 100:405-414. 18. Niagro FD, Ritchie BW, Latimer KS, et al.: 1990, Polymerase chain reaction detection of PBFD virus and BFD virus in suspect birds. Proc Assoc Avian Vet, pp. 25-37. 19. Pascucci S, Maestrini N, Misciattelli ME, Giovannetti L: 1983, Malattia da virus papova-simile nel pappagallino ondulato (Melopsittacus undulatus). Clin Vet (Milan) 106:38-41. 20. Pass DA: 1985, A papova-like virus infection of lovebirds (Agapornis sp.). Aust Vet J 62:3 18-3 19. 21. Pass DA, Prus SE, Riddell C: 1987, A papova-like virus infection of splendid parakeets (Neophema splendida). Avian Dis 31:680-684. 22. Randall CJ, Lees S, Inglis DM: 1987, Papovavirus-like infection in budgerigars (Melopsittacus undulatus). Avian Pathol 16: 623-633. 23. Sztojko V, Saghy E, Meder M, et al.: 1985, A hullamos papagaj (Melopsittacus undulatus) papovavirus okozta megbetegedesenek hazai megallapitasa. Magy Allatorv Lapja 40:59-63. 24. Wainwright PO, Lukert PD, Davis RB, Villegas P: 1987, Serological evaluation of some Psittaciformes for budgerigar fledgling disease virus. Avian Dis 31:673-676.

J Vet Diagn Invest 4:458-460 (1992)

A comparison of isolation and a commercial ELISA for the diagnosis of chlamydiosis in psittacine birds Thomas R. Haven, Kenneth W. Mills, Elizabeth S. Williams, Michael P. Driscoll, Richard S. Kingston The problem of identifying birds that are persistently or latently infected with Chlamydia and the desire for more rapid diagnosis of diseased animals has resulted in the development of many new test procedures.2,4,5,7,12,14 As advances in technology supply diagnostic laboratories with an increasFrom the Wyoming State Veterinary Laboratory, Department of Veterinary Sciences, University of Wyoming, 1174 Snowy Range Rd., Laramie, WY 82070 (Haven, Mills, Williams), Broadmoore East Veterinary Clinic, 4218 E. Pershing Blvd., Cheyenne, WY 82070 (Driscoll), and the Department of Animal and Nutritional Sciences, Veterinary Diagnostic Laboratory, Kendall Hall, University of New Hampshire, Durham, NH 03824 (Kingston). Received for publication January 30, 1992.

ing variety of tests, each laboratory must choose tests appropriate to their circumstances. Each method attempts to maximize sensitivity and specificity while minimizing cost, time, specialized equipment, and technical expertise required to perform the test. Several recent comparisons of the available techniques for diagnosing Chlamydial infections have not made the choice easy. A commercial enzyme-linked immunosorbent assay (ELISA)a was reported to be as sensitive as culture in diagnosing Chlamydia psittaci infections in birds submitted for necropsy.5 The same test kit was reported to be “ideally suited for office use” in cases of human conjunctivitis; although it was “not quite as sensitive as culture,” it was less costly, faster, and easier to perform.3 Culture or peroxidase-antiperoxidase (PAP) examination of tissues were

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