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Experimental Transmission of Psittacine Proventricular Dilatation Disease (PDD) and Preliminary Characterization of a Virus Recovered From Birds With Naturally Occurring and Experimentally Induced PDD

Christopher R. Gregory, Branson W. Ritchie, Kenneth S. Latimer, W. L. Steffens, Raymond P. Campagnoli, Denise Pesti, and Phil D. Lukert

Psittacine Disease Research Group, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602 (USA)

Abstract. Proventricular dilatation disease (PDD) was experimentally reproduced in five psittacine birds following inoculation with clarified tissue homogenates from a bird with naturally acquired PDD. Enveloped viral particles, measuring 80 nanometers in diameter, were recovered consistently from tissues and/or feces of birds with PDD, whether the infection was obtained naturally or experimentally. Principals inoculated with tissue homogenates from control birds unaffected with PDD and control birds in contact with PDD-affected birds remained clinically normal.

Key Words: Avian, Disease transmission, Gastrointestinal disease, Infectious disease, Natural and experimental disease, Neurological disease, Proventricular dilatation disease, Proventricular dilatation syndrome, Lymphoplasmacytic ganglioneuritis, Virus, Viral infection 

Introduction

Proventricular dilatation disease (PDD) was first described in macaws in the late 1970's.^1-5 Common clinical signs include depression, weight loss (with or without decreased appetite), constant or intermittent regurgitation, and passage of undigested seed in the feces, indicating a maldigestive or malabsorptive disorder.^4-19 Concomitant central nervous system signs may include ataxia, abnormal head movements, seizures, and proprioceptive or motor deficits.^{4,6,8,12,13,16-18} At necropsy, emaciation, pectoral muscle atrophy, and dilation of the gastrointestinal tract, including the proventriculus, are observed.^{4,6-8,10-12,14,17-19}

Although a presumptive diagnosis of PDD may be based on clinical signs and gross pathology, definitive diagnosis requires histologic evaluation of affected tissues. Examination of these tissues by light microscopy reveals lymphoplasmacytic infiltrates in both central and peripheral nervous systems. The myenteric plexuses of the tunica muscularis in the ventriculus or proventriculus are affected most commonly.^{1-4,6-8,10,12,14,15,17,18} Infiltrates also may be observed in plexuses of the crop, duodenum, and esophagus; conduction fibers of the heart and pericardial ganglia; and around vessels in the brain and spinal cord.^{1-4,6-8,11,12,14,15,18} Antemortem evaluation of crop biopsy is diagnostic in approximately 75% of birds with PDD.²⁰

The cause and pathogenesis of PDD are unknown. Some findings suggest an infectious disease.^{4-6,8,11,12,14} Histopathologic lesions suggest an inflammatory response to viral infection; however, virus isolation, detection of serum antibodies to certain viruses (paramyxovirus types 1-4, 6, 7; avian herpesvirus; avian papovaviruses; avian encephalitis virus; and Eastern equine encephalomyelitis virus), and molecular studies (Eastern equine encephalomyelitis virus) have been unsuccessful in establishing a viral etiology.^{1-4,7,10,11,17,18,23}

The lymphoplasmacytic ganglioneuritis that characterizes PDD has been experimentally induced by inoculating adult psittacine birds with a clarified tissue homogenate derived from a PDD-affected bird that contained an enveloped virus approximately 80 nm in diameter.²⁵ A virus with similar ultrastructural characteristics recently has been recovered from the feces of a PDD-affected macaw in Europe.²⁶ 

Materials and Methods

Animals (experimental disease transmission): Eight adult psittacine birds, with behavioral and/or physical problems that rendered them unsuitable as companion or breeding birds, were used to attempt experimental transmission of PDD. These birds included an Eclectus Parrot (Eclectus roratus), three Scarlet Macaws (Ara macao), a Moluccan Cockatoo (Cacatua moluccensis), an Umbrella Cockatoo (Cacatua alba), and two Hispaniolan Amazon Parrots (Amazona ventralis). An additional Eclectus Parrot, a second Umbrella Cockatoo, two African Grey Parrots (Psittacus erithacus), and two Amazon Parrots (Amazona sp.) were housed in the same room as the experimental birds and served as contact controls.

Miscellaneous specimens: Fresh necropsy tissue specimens were obtained from three additional birds that were submitted to the University of Georgia College of Veterinary Medicine for postmortem examination. A Golden Conure (Aratinga guarouba), an African Grey Parrot, and a Severe Macaw (Ara severa) had clinical histories and gross lesions suggestive of PDD. A complete set of tissues including proventriculus, ventriculus, crop, adrenal glands, heart, intestines, and brain was collected and fixed in 10% neutral-buffered formalin. Tissue sections were processed routinely for histologic evaluation. After sample collection for histologic examination, the remainder of the gastrointestinal tract was collected and frozen at -70°C. Tissues from two birds, an Eclectus Parrot and a Severe Macaw, with no clinical history or gross lesions suggestive of PDD were similarly processed for histologic evaluation and virus recovery. Approximately one gram of fresh feces was collected from the experimental birds and from birds donated for research, including PDD-affected and PDD negative birds.

Experimental transmission of PDD: Approximately 1 gram each of proventriculus, ventriculus, crop, and brain from a necropsied bird (Umbrella Cockatoo) with histologically confirmed PDD was homogenized in 10 milliliters of sterile PBS. The homogenate was clarified by centrifugation and shown to contain an 80 nm virus by electron microscopy. An Eclectus Parrot, Scarlet Macaw, and Moluccan Cockatoo were inoculated by combined intramuscular, subcutaneous, intraocular, oral, and intranasal routes. One milliliter of the clarified supernatant was used for each inoculation site. Two Hispaniolan Amazon Parrots and an Umbrella Cockatoo were inoculated with clarified tissue homogenates from an eclectus parrot which did not have histologic lesions suggestive of PDD and did not contain the 80 nm virus. Approximately three months after the initial trail, two additional scarlet macaws were inoculated by similar routes. This inoculum was clarified supernatant from homogenized necropsy tissues obtained from the inoculated Scarlet Macaw of the original experimental group. All birds were observed daily for clinical signs of PDD including central nervous system disease or the passage of undigested food in their feces. Feces which contained undigested food was collected for virus recovery. Necropsies were performed on experimentally infected birds which died or were euthanized. Various tissues subsequently were collected for routine histologic evaluation and virus recovery. 

 

Results

Clinical changes in the experimentally infected birds included acute onset of central nervous system and/or gastrointestinal signs. Deaths ranged from 11 days to three months post-inoculation. Specifically, the Eclectus Parrot exhibited a peracute onset of depression, ataxia, and tremors accompanied by the passage of loose stool containing undigested pellets. These clinical changes were first noted one week after inoculation and the bird died four days later. The only abnormal necropsy findings in this Eclectus Parrot were pectoral muscle atrophy and a moderately dilated proventriculus. Histologically, severe multifocal lymphoplasmacytic infiltrates were noted within the ganglia of the proventriculus and ventriculus. Multifocal mild perivascular lymphoplasmacytic infiltrates and focal gliosis were observed in the cerebrum. Severe multifocal perivascular lymphocytic infiltrates also were present in the myocardial conduction fibers and pericardial ganglia. The Moluccan Cockatoo remained clinically normal until one month after inoculation. This bird then exhibited anorexia following acute onset of severe neurologic signs characterized by torticollis, nystagmus, ataxia, and tremors of the head and extremities. Necropsy examination revealed a moderately dilated proventriculus, ventriculus, and intestinal tract. All organs were devoid of food. In addition, approximately three ml of transudate were removed from the cranial vault. Histologically, severe multifocal lymphoplasmacytic infiltrates were noted within ganglia of the crop, proventriculus (Fig. 1), and ventriculus. The cerebrum and brainstem contained moderate multifocal perivascular lymphoplasmacytic infiltrates (Fig. 2). 


 

Fig. 1. Moluccan Cockatoo, proventriculus, H&E stain.  Severe lymphoplasmacytic ganglioneuritis in experimental PDD.	Fig. 2. Moluccan Cockatoo, brain, H&E stain.  Mild perivascular lymphoplasmacytic infiltrates in experimental PDD.

The Scarlet Macaw remained clinically normal for approximately three months after inoculation. This bird then began to pass increasing concentrations of undigested pellets in its feces. Histologic evaluation of a crop biopsy showed the presence of severe multifocal lymphoplasmacytic infiltrates within ganglia. The bird begin to rapidly lose weight despite a ravenous appetite and became increasingly depressed and ataxic. This bird was euthanized approximately ten days after the onset of clinical signs. At necropsy, the proventriculus was severely distended and undigested food was present in the crop, proventriculus, ventriculus, and intestines. Severe multifocal perivascular lymphoplasmacytic infiltrates were noted in the cerebrum, cerebellum , and midbrain. Severe multifocal lymphoplasmacytic infiltrates also were observed within the ganglia of the proventriculus, ventriculus, crop, and duodenum (Fig. 3). Similar infiltrates also were noted within pericardial ganglia (Fig. 4) and cardiac conduction fibers. 


 

Fig. 3. Scarlet Macaw, duodenum, H&E stain.  Severe lymphoplasmacytic ganglioneuritis in duodenum in experimental PDD.	Fig. 4. Scarlet Macaw, adrenal gland, H&E stain.  Severe lymphoplasmacytic infiltrates in experimental PDD.

Infiltrates also were present around blood vessels and within neural tracts in the muscularis mucosa of the affected gastrointestinal organs, ganglia of the lungs (Fig. 5), and in the medullary areas of the adrenal gland (Fig. 6). Tissues from this bird were used to inoculate two additional Scarlet Macaws. 


 

Fig. 5.  Scarlet Macaw, heart, H&E stain.  Severe lymphoplasmacytic inflammation of the pericardial ganglion in experimental PDD.	Fig. 6.  Scarlet Macaw, lung, H&E stain.  Lymphoplasmacytic infiltrates surround ganglion and pulmonary vessels.

One of these two Scarlet Macaws remained clinically healthy for approximately three months after infection. At this time, the bird began passing undigested pellets in its feces and exhibited progressive pectoral muscle atrophy. The second Scarlet Macaw remained clinically normal; however, both birds had radiographic evidence of proventricular dilatation. Crop biopsies from both birds contained severe multifocal lymphoplasmacytic infiltrates within ganglia. The severely affected scarlet macaw was euthanized approximately two months after clinical signs were first noted (five months post-inoculation). At necropsy, this bird had a severely dilated crop and proventriculus, a flaccid ventriculus, and undigested pellets in the intestines. Severe multifocal lymphoplasmacytic infiltrates were present within ganglia of the crop, proventriculus, ventriculus, small intestine, and large intestine. Similar infiltrates were noted around blood vessels and within nerve tracts of the submucosa in the proventriculus, ventriculus, duodenum, within pericardial ganglia, and cardiac conduction fibers. Mild multifocal perivascular accumulations of lymphocytes and plasma cells were noted in the cerebrum. The second Scarlet Macaw remained clinically stable although undigested pellets consistently were observed in the feces.

Birds that were experimentally inoculated with tissue homogenates derived from a PDD-negative Eclectus Parrot and the contact control birds did not exhibit clinical signs of PDD throughout the study period. Birds with naturally occurring PDD (tissue submissions) had gross lesions including pectoral muscle atrophy and mild to severe proventricular dilatation. These birds also had characteristic lymphoplasmacytic infiltrates within ganglia of the gastrointestinal tract; however, the inflammation was generally less severe than that noted in the experimentally infected birds. Neither gross nor microscopic lesions suggestive of PDD were noted in tissues submitted from birds without PDD.

Virus particles were demonstrated in the tissues and/or feces of both experimentally infected and naturally affected birds with PDD. Ultrastructural analysis of negatively stained preparations collected from these birds revealed a population of spherical, enveloped viral particles with a mean diameter of 80 nm (Fig. 7). The viral preparation consisted primarily of intact particles with an estimated concentration of 1 X 10¹⁰ particles per grid square on a 400-mesh grid. The viral envelope contained three to five nm surface projections and a nucleocapsid with a mean diameter of 40 nm. Virus particles that morphologically resembled those recovered from other PDD positive birds were demonstrated by electron microscopy in the tissue homogenates from the naturally affected Umbrella Cockatoo initially used to experimentally transmit PDD and from the tissues of the Scarlet Macaw used to experimentally infect the remaining birds. Virus representing at least 10⁶ particles/5g could be demonstrated consistently in extracts of feces from naturally affected and experimentally infected birds. In addition, virus has been consistently demonstrated in feces from the live Scarlet Macaw ten months after PDD was confirmed by crop biopsy and 13 months after the bird was experimentally infected. Virus was not observed in tissue and/or fecal extracts of PDD-negative birds, including negative and contact control birds and tissue submissions. Ultrathin sections of tissue homogenates from the original Umbrella Cockatoo contained low numbers of enveloped, polyhedral, 80 nm diameter virus-like particles with a densely staining central core (Fig. 8).
 

 

Fig. 7.  Electron micrograph, feces, phosphotungstic acid stain.  Typical 80 nm diameter enveloped viruses recovered from birds with natural and experimental PDD.	Fig. 8.  Electron micrograph, inoculum, uranyl acetate & lead citrate stain.  Virions in primary experimental inoculum from an Umbrella Cockatoo with naturally acquired PDD.

Discussion

This study documents experimental transmission of PDD to susceptible birds following inoculation with clarified tissue homogenates from birds with the disease. In contrast, tissue homogenates from birds without PDD did not cause disease in inoculated birds. Many routes of inoculation were used, precluding exact identification of the mode(s) of natural transmission. However, the fecal/oral route is the most likely portal of viral entry into the bird’s body. This speculation is supported by the fact that an 80 nm diameter virus is recovered consistently from feces of birds with naturally acquired or experimentally reproduced PDD. Furthermore, PDD typically is associated with lymphoplasmacytic ganglioneuritis involving the gastrointestinal tract of diseased birds.

Specific-pathogen-free (SPF) birds were not available for this experimental trial and sensitive techniques for confirming the absence of PDD currently are unavailable. Therefore, it is remotely possible that the experimental subjects already had PDD, but did not exhibit clinical signs. However, the observation that experimentally infected birds developed PDD while the negative and contact control birds remained clinically normal indicates that the experimental inoculum induced PDD in the principal group.

These findings demonstrate that PDD is transmissible and that an 80 nm diameter enveloped virus can be recovered consistently from naturally affected and experimentally infected birds. Recovery of this virus from naturally affected and experimentally infected birds with PDD, but inability to recover the virus from birds without PDD, suggests that the observed virus is the causative agent of the disease. However, it is possible that an agent other than the recovered virus was present in the clarified homogenates and the presence of the observed virus was incidental, representing secondary viral infection in immunosuppressed, sick birds. It also is possible that low numbers of viruses were missed in samples from birds without PDD. Further studies, using purified virus preparations and/or ultrafiltered tissue homogenates, are being conducted to explore these possibilities.

The contact controls used in this study also failed to develop clinical signs of PDD, suggesting that the virus is not readily transmissible or requires a specific route of inoculation that was not favored by the experimental conditions. Alternatively, it is possible that these contact controls were already immune to infection. Because all birds in the study were derived from the same initial group of research animals, it is unlikely that only the experimentally infected birds would be susceptible to PDD.

The presence of lymphoplasmacytic ganglioneuritis and variable clinical signs (GI only, CNS only, or GI and CNS ) has led several researchers to speculate that PDD might be caused by more than one etiologic agent. The experimental transmission study reported here demonstrates that the same virus-containing inoculum can cause varying clinical changes, even within birds of the same species. Additionally, a morphologically similar virus has been recovered from the tissues or excrement of birds that were diagnosed with PDD by crop biopsy, even though some of these naturally affected birds had predominately CNS signs, predominately gastrointestinal signs, or a combination of CNS and gastrointestinal signs.

The experimental transmission studies reported here demonstrate that PDD can have an extremely variable incubation period, ranging from one week to three months. Further research is necessary to determine why some birds develop an acute form of the disease and die within two to four weeks, while other birds develop a persistent form of the disease and survive for months to years. Additionally, further research will be necessary to determine why some birds infected with the same viral suspension develop varied clinical signs of disease. Finally, ongoing molecular studies will further characterize the unique virus associated with PDD. 

Acknowledgments

The authors would like to acknowledge the following organizations and individuals for their financial support of this research: the Cowan Avian Health Foundation, the International Avian Research Foundation, Dr. Joe and Sue Still, Loro Parque Foundation, Zoo Atlanta, Riverbanks Zoological Park, Veterinary Medical Experiment Station, Terry Clyne, Dr. Richard and Luanne Porter, Knick Enterprises, The National Aviary in Pittsburgh, International Aviculturist's Society, Midwest Avian Research Exposition, Kathleen Sazbo, Bobbi Brinker, and Zeigler Brothers Inc. The authors also would like to thank Dr. Frank D. Niagro, Department of Clinical Investigation, Eisenhower Army Medical Center, Fort Gordon, GA for his assistance in earlier studies of PDD. 

References

1. Gerlach S. 1991. Macaw wasting disease - a 4-year study on clinical cases, history, epizootiology, analysis of spezies, diagnosis and differential diagnosis, mikrobiological and virological results. Proc Ann Conf European Chap Assoc Avian Vet, pp. 273-281.

2. Graham DL. 1984. Infiltrative splanchnic neuropathy: a component of the wasting macaw complex? Proc Internatl Conf Avian Med, p. 275.

3. Mannl A, Gerlach H, Leipold R. 1987. Neuropathic gastric dilatation in psittaciformes. Avian Dis 31: 214-221.

4. Phalen DN. 1986. An outbreak of psittacine proventricular dilatation syndrome (PPDS) in a private collection of birds and an atypical form of PPDS in a nanday conure. Proc Ann Conf Assoc Avian Vet, pp. 27-34.

5. Rosskopf WJ, Woerpel RW, Reed-Blake S. 1986. Pet avian conditions and syndromes - an update. Proc Ann Conf Assoc Avian Vet, pp. 377, 392-393, 399.

6. Cazayoux-Vice CA. 1992. Myocarditis as a component of psittacine proventricular dilatation syndrome in a Patagonian conure. Avian Dis 36: 1117-1119.

7. Clark FD. 1984. Proventricular dilatation syndrome in large psittacine birds. Avian Dis 28: 813-815.

8. Degernes LA, Flammer K, Fisher P. 1991. Proventricular dilatation syndrome in a green-winged macaw. Proc Annl Conf Assoc Avian Vet, pp. 45-49.

9. Graham DL. 1991. Wasting/proventricular dilatation disease: A pathologist’s view. Proc Ann Conf Assoc Avian Vet, pp. 43-44.

10. Hughes PE. 1984. The pathology of myenteric ganglioneuritis, psittacine encephalomyelitis, proventricular dilatation of psittacines, and macaw wasting syndrome. Proc 33rd Western Poult Dis Conf, pp. 85-87.

11. Joyner KI, Kock N, Styles D. 1989. Encephalitis, proventricular and ventricular myositis, and myenteric ganglioneuritis in an umbrella cockatoo. Avian Dis 33: 379-381.

12. Lutz ME, Wilson RB. 1991. Psittacine proventricular dilatation syndrome in an umbrella cockatoo. J Am Vet Med Assoc 198: 1962-1963.

13. Malley DM. 1991. Case report: a case study of a Moluccan cockatoo with proventricular dilatation. Proc Ann Conf European Chap Assoc Avian Vet, pp. 271-272.

14. Rich G. 1992. Classic and atypical cases of proventricular dilatation disease. Proc Ann Conf Assoc Avian Vet, pp. 119-125.

15. Ridgeway RA, Gallerstein GA. 1983. Proventricular dilatation in psittacines. Proc Ann Conf Assoc Avian Vet, pp. 228-230.

16. Spenser EL. 1991. Common infectious diseases of psittacine birds seen in practice. Vet Clin N Am: Small Anim Pract 21: 1227.

17. Suedemeyer WK. 1992. Diagnosis and clinical progression of three cases of proventricular dilatation syndrome. J Assoc Avian Vet 6: 159-163.

18. Turner R. 1984. Macaw fading or wasting syndrome. Proc 33rd Western Poult Dis Conf, pp. 87-88.

19. Woerpel RW, Rosskopf WJ. 1984. Clinical and pathological features of macaw wasting disease (proventricular dilatation syndrome). Proc 33rd Western Poult Dis Conf, pp. 89-90.

20. Gregory CR, Latimer KS, Campagnoli RP, Ritchie BW. 1996. Histologic evaluation of the crop for diagnosis of proventricular dilatation syndrome in psittacine birds. J Vet Diagn Invest 8: 76-80.

21. Bond MW, Downs D, Wolf S. 1993. Screening for psittacine proventricular dilatation syndrome. Proc Ann Conf Assoc Avian Vet, pp. 92-97.

22. Rosskopf WJ, Woerpel RW, Reed-Blake S. 1985. Pet avian disease syndromes. Proc Ann Conf Assoc Avian Vet, pp. 299-317.

23. Gregory CR, Latimer KS, Niagro FD, Roberts AW, Campagnoli RP, Pesti DA, Ritchie BW, Lukert PD. 1997. Investigations of Eastern equine encephalomyelitis virus as the causative agent of psittacine proventricular dilation syndrome. J Avian Med Surg 11: 187-193.

24. Woerpel RJ, Rosskopf WJ, Hughes E. 1984. Proventricular dilatation and wasting syndrome: myenteric ganglioneuritis and encephalomyelitis of psittacines; an update. Proc Internatl Conf Avian Med, pp. 25-28.

25. Gregory CR, Latimer KS, Niagro FD, Campagnoli RP, Ritchie BW, Steffens WL. 1996. Characterization of virus-like particles from tissues of birds diagnosed with proventricular dilatation syndrome (PDS). Proc Animal Dis Res Workers in Southern States, p. 25.

26. Gough RE, Drury SE, Harcourt-Brown NH. 1996. Virus-like particles associated with macaw wasting disease. Vet Rec 139: 24.

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