Pacheco's disease (PD) is a common, often fatal, disease of parrots. We cloned a virus isolate from a parrot that had characteristic lesions of PD. Three viral clones were partially sequenced, demonstrating that this virus was an alphaherpesvirus most closely related to the gallid herpesvirus 1. Five primer sets were developed from these sequences. The primer sets were used with PCR to screen tissues or tissue culture media suspected to contain viruses from 54 outbreaks of PD. The primer sets amplified DNA from all but one sample. Ten amplification patterns were detected, indicating that PD is caused by a genetically heterogeneous population of viruses. A single genetic variant (psittacid herpesvirus variant 1) amplified with all primer sets and was the most common virus variant (62.7%). A single primer set (23F) amplified DNA from all of the positive samples, suggesting that PCR could be used as a rapid postmortem assay for these viruses. PCR was found to be significantly more sensitive than tissue culture for the detection of psittacid herpesviruses.

Mucosal papillomas are relatively common lesions in several species of captive neotropical parrots. They cause considerable morbidity and in some cases, result in mortality. Previous efforts to identify papillomavirus DNA and proteins in these lesions have been largely unsuccessful. In contrast, increasing evidence suggests that mucosal papillomas may contain psittacid herpesviruses (PsHVs). In this study, 41 papillomas from 30 neotropical parrots were examined by PCR with PsHV-specific primers. All 41 papillomas were found to contain PsHV DNA. This 100% prevalence of PsHV infection in the papilloma population was found to be significantly higher than PsHV infection prevalence observed in other surveys of captive parrots. PsHV genotypes 1, 2, and 3, but not 4 were found in these lesions. Psittacus erithacus papillomavirus DNA and finch papillomavirus DNA were not found in the papillomas. A papilloma from a hyacinth macaw (Anodorhynchus hyacinthinus) was found to contain cells that had immunoreactivity to antiserum made to the common antigenic region of human papillomavirus (HPV) L1 major capsid protein. However, four other mucosal papillomas were negative for this immunoreactivity, and negative control tissues from a parrot embryo showed a similar staining pattern to that seen in the cloaca papilloma of the hyacinth macaw, strongly suggesting that the staining seen in hyacinth macaw papilloma was nonspecific. Based on these findings, it was concluded that specific genotypes of PsHV play a direct role in the development of mucosal papillomas of neotropical parrots and there is no evidence to suggest the concurrent presence of a papillomavirus in these lesions.

Fragments of 419 bp of the UL16 open reading frame from 73 psittacid herpesviruses (PsHVs) from the United States and Europe were sequenced. All viruses caused Pacheco's disease, and serotypes of the European isolates were known. A phylogenetic tree derived from these sequences demonstrated that the PsHVs that cause Pacheco's disease comprised four major genotypes, with each genotype including between two and four variants. With the exception of two viruses, the serotypes of the virus isolates could be predicted by the genotypes. Genotypes 1 and 4 corresponded to serotype 1 isolates, genotype 2 corresponded to serotype 2 isolates, and genotype 3 corresponded to serotype 3 isolates. The single serotype 4 virus mapped to genotype 4. DNA from a virus with a unique serotype could not be amplified with primers that amplified DNA from all other PsHVs, and its classification remains unknown. Viruses representing all four genotypes were found in both the United States and Europe, and it was therefore predicted that serotypes 1, 2, and 3 were present in the United States. Serotype 4 was represented by a single European isolate that could not be genetically distinguished from serotype 1 viruses; therefore, the presence of serotype 4 in the United States could not be predicted. Viruses of genotype 4 were found to be the most commonly associated with Pacheco's disease in macaws and conures and were least likely to be isolated in chicken embryo fibroblasts in the United States. All four genotypes caused deaths in Amazon parrots, but genotype 4 was associated with Pacheco's disease only in Amazons in Europe. Genotypes 2, 3, and 4, but not 1, were found in African grey parrots. Although parrots from the Pacific distribution represent a relatively small percentage of the total number of birds with Pacheco's disease, all four genotypes were found to cause disease in these species.

An organism commonly referred to as 'megabacterium' colonizes the gastric isthmus of many species of birds. It is weakly Gram-positive and periodic acid-Schiff-positive and stains with silver stains. Previous studies have shown that it has a nucleus and a cell wall similar to those seen in fungi. Calcofluor white M2R staining suggests that the cell wall contains chitin, a eukaryote-specific substance, and rRNA in situ hybridization demonstrates that it is a eukaryote. To characterize this organism phylogenetically, DNA was extracted from purified cells. rDNA was readily amplified by PCR with pan-fungal DNA primer sets and primer sets derived from the newly determined sequence, but not with bacteria-specific primer sets. Specific primer sets amplified rDNA from isthmus scrapings from an infected bird, but not from a non-infected bird or other control DNA. The sequence was confirmed to derive from the purified organism by in situ rRNA hybridization using a specific probe. Phylogenetic analysis of sequences of the 18S rDNA and domain D1/D2 of 26S rDNA showed the organism to be a previously undescribed anamorphic ascomycetous yeast representing a new genus. et al., 1980;Baker, 1992;Gerlach, 2001). These long, slender organisms (2-3620-80 mm) stain with silver stains and periodic acid-Schiff (PAS) and are weakly Gram-positive. The 'megabacterium' is found in the isthmus between the glandular and grinding stomach, where it grows on the lumenal surface and may penetrate koilin (Dorrestein et al., 1980;van Herck et al., 1984). It is associated with a lymphoplasmacytic gastritis in poultry (Mutlu et al., 1997) and a chronic fatal wasting disease in companion birds (Gerlach, 2001).The organism was originally thought to be a yeast because of its staining characteristics (Dorrestein et al., 1980). Subsequently, van Herck et al. (1984) concluded that it was a bacterium, as they were unable to demonstrate cytoplasmic organelles or a nucleus. They did, however, show nucleus-like structures in Geimsa-stained organisms, but interpreted them to be 'granules'. Scanlan & Graham (1990) reported isolating a bacterium from the stomach of a budgerigar using standard microbiological techniques. The isolated bacterium, however, was smaller than the organism observed in vivo and was not characterized by PAS or silver stains. Attempts by other investigators to grow this organism with standard microbiological isolation techniques have been unsuccessful. However, Gerlach (2001) reported isolation of this organism on MRS medium, but was unable to maintain it past a few passages. Huchzemeyer et al. (1993) also reported isolating an organism from the proventriculus of ostriches using MRS agar. This organism had the same biochemical properties as the one isolated byAbbreviations: D1/D2, domains 1 and 2; PAS, periodic acid-Schiff; PNA, peptide nucleic acid.

DNA from a novel alphaherpesvirus was amplified from a cloacal papilloma, a cutaneous papilloma, and the normal cloacal mucosa of African grey parrots (Psittacus erithacus erithacus). Phylogenetically, the virus was most closely related to the psittacid herpesvirus, but demonstrated sufficient nucleotide and amino acid diversity to be considered a new alphaherpesvirus. It is proposed that the previously described psittacid herpesvirus be designated as psittacid herpesvirus 1 (PsHV-1), and this new species be classified as psittacid herpesvirus 2 (PsHV-2). It is speculated that PsHV-2 co-evolved with the African grey parrot and should therefore be present in these birds in the wild.