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Tissue distribution of psittacid herpesviruses in latently infected parrots, repeated sampling of latently infected parrots and prevalence of latency in parrots submitted for necropsy

Correspondence: ¹ Corresponding Author: David N. Phalen, The Faculty of Veterinary Science, The University of Sydney, 425 Werombi Rd., Camden, NSW, Australia, 2570.

	Abstract

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Psittacid herpesvirus-1 (PsHV-1) is the cause of an acute fatal disease in parrots and is implicated as the cause of papillomatous lesions of the digestive tract. Not all infections cause disease and some parrots are infected asymptomatically. Latently infected parrots are potential sources for virus dissemination. Tissues from parrots that died spontaneously with a history of coming from flocks where a PsHV-1 outbreak had occurred were examined for PsHV-1 DNA. Fourteen of 16 parrots examined were infected with at least 1 variant of PsHV-1; of these 13 (93%) had viral DNA in either or both the oral and cloacal mucosa, suggesting that most latently infected parrots could be detected by sampling these sites. Nine of 9 parrots shown to be infected 5 years prior to this study were positive again on repeat sampling and were infected with the same virus genotype. Opportunistic sampling of parrots submitted for diagnostic necropsy indicated that the prevalence of PsHV-1 in parrots in the sampled population was approximately 9.3%. PsHV-1 genotypes 1, 2, and 3 were found in these birds, but genotype 4 was not. Six necropsy specimens were found to be infected with two PsHV-1 genotypes and it was concluded that infection with one serotype did not protect against infection with another. Psittacid herpesvirus 2 (PsHV-2) was identified in 4 African grey parrots and a blue and gold macaw. Prior to this study PsHV-2 had only been found in African grey parrots.

Key Words: Nested PCR • parrots • Psittacid herpesvirus-1 • Psittacid herpesvirus-2

	Introduction

TOP Sources and manufacturers Abstract Introduction Materials and Methods Results Discussion References

 
Pacheco disease is an acute rapidly fatal disease of parrots.7,9,10 There is a single confirmed case of Pacheco disease in a superb Starling (Lamprotornis superbus) and 5 suspected cases of Pacheco disease in 4 other species of passerine birds.14 Outbreaks of this disease have resulted in massive die offs of captive parrots.7,9¹⁹ Pacheco disease is caused by infection with the -herpesvirus, psittacid herpesvirus-1 (PsHV-1). PsHV-1 has 4 distinct genotypes. All 4 of the PsHV-1 genotypes have the potential to cause Pacheco disease, however, the outcome of an infection with one of them will depend on the genotype of the virus, the species of parrot infected, and other undefined factors. For example, all 4 PsHV-1 genotypes have been shown to cause Pacheco disease in Amazon parrots, but only PsHV-1 genotypes 2, 3, and 4 have been identified in African grey parrots with Pacheco disease, and only genotype 4 causes mortality in macaws and conures. Parrots from the Western Pacific distribution, e.g., Australia, Indonesia, the Philippines, and Southeast Asia appear to be somewhat refractory to Pacheco disease, but when they do succumb to PsHV 1 infection, it can be caused by any of the 4 PsHV-1 genotypes. Other unknown factors also play a role in the pathogenesis of Pacheco disease; for example, PsHV-1 genotype 2 is the second leading cause of Pacheco's outbreaks in Europe, but has not been detected in parrots with Pacheco disease in the USA.14

It appears that many infections with the PsHV-1 genotypes are subclinical. Subclinically infected parrots and parrots that survive Pacheco disease, with or without treatment, are believed to develop latent infections.8 Some of the parrots latently infected with PsHV-1 genotypes 1, 2, and 3 will subsequently develop PsHV-1 DNA containing papillomas of the mucous membranes of the digestive tract.11 It is assumed that all parrots latently infected with PsHV-1 shed virus at low levels or shed virus intermittently and are a potential source for infection of naïve parrots and a potential source of Pacheco outbreaks or the spread of mucosal papillomas.2

Two potential means of preventing PsHV-1 infections include the use of immunization or, alternately, testing for and isolation of infected parrots. Currently there is only one commercial PsHV-1 vaccine and it is derived from a single, unreported, serotype.^a Therefore, it is not known how much protection this vaccination provides. Screening parrots for PsHV-1 infection, however, may prove to be useful. Studies in parrots with mucosal papillomas suggest that potential sites for PsHV-1 latency are the mucosa of the oral cavity and the cloaca and analyzing samples of these tissues might be a way to detect latently infected parrots.11 This hypothesis was tested in a preliminary study that used a PCR assay to examine combined oral and cloacal swabs from parrots believed to be latently infected with PsHV-1. In that study, a population of parrots was found that were positive on multiple samples over a period of 12 months. The sensitivity of this assay was not 100% and parrots infected with genotype 3 were infrequently detected. Subsequently, a comparison of the primer sets used in that study with the sequences of the 4 PsHV-1 genotypes suggested that lack of sensitivity was the result of multiple mismatches in the primer sets particularly with genotype 3.8 The ability to detect all PsHV-1 genotypes by PCR has been further improved with the development of a single semi-nested set of PCR primers that can detect all known PsHV-1 variants.12

This development led to the unexpected detection of a novel -herpesvirus, psittacid herpesvirus-2 (PsHV-2). The sequence of the amplified DNA from PsHV-2 differs from the most closely related PsHV-1 by more than 20%. Little is know about the prevalence and pathogenicity of PsHV-2. It has been identified only in 3 African grey parrots. Virus was detected in a cutaneous papilloma from one parrot, a mucosal papilloma in another, and the healthy cloacal mucosa of the third.11

The overall approach to controlling Pacheco disease in an aviary will also depend on the prevalence of infection in populations of parrots and the species that are found to be infected. For example, a serologic survey of aviary parrots in Europe found 6 of 7 aviaries to have positive birds with an overall seroprevalence of 37.3%.4 Surveys of breeding parrots and professional aviaries in the USA have not been done.

The first 2 objectives of this study were to determine the tissue distribution of PsHV-1 in latently infected parrots and determine if PsHV-1 DNA could consistently be detected in their oral mucosa and/or cloacal mucosa. The third objective of this study was to determine if parrots that were positive for PsHV-1 infection in a previous study remained positive 5 years later. The final objective of this study was to estimate the prevalence of PsHV-1 and PsHV-2 infections in the general population of parrots submitted for diagnostic necropsy.

	Materials and Methods

TOP Sources and manufacturers Abstract Introduction Materials and Methods Results Discussion References

 
Tissue Distribution of Pshv-1 in Latently Infected Parrots
Eleven of the parrots included in this section of the study were housed at the Schubot Exotic Bird Health Center, Texas A&M University, College Station, TX. They were donated as research animals prior to 1986 because they had survived outbreaks of Pacheco disease and were thought to be latently infected with one or more genotypes of PsHV-1. Five additional parrots were submitted as diagnostic specimens (Table 1 and 2). Of these latter 5, the hawk-headed parrot had a history of surviving an outbreak of Pacheco disease, the blue and gold macaw had an unknown history, and the remaining 3 parrots (a yellow-streaked lory, a lilac-crowned Amazon parrot, and yellow-collared macaw) were from an aviary where Pacheco disease had occurred more than 10 years prior to submission. With the exception of hawk-headed parrot, which was euthanized at the request of the owner, all parrots in this study died of naturally occurring disease that was unrelated to their herpesvirus infections. Oral and cloacal mucosal tissue samples and additional tissue samples (approximately 4 x 4 x 4 mm cubes) ranging in number from 2 to 17 were collected from each parrot (Table 2). Each sample was collected with separate sets of scissors and forceps that were cleaned, soaked in formalin, and then soaked in bleach between uses to prevent DNA carryover.

Prevalence of Pshv Infected Parrots in Diagnostic Submissions and Opportunistic Sampling of Parrots
Parrots included in this study were submitted for diagnostic necropsy to Texas Veterinary Medical Diagnostic Laboratory (TVMDL) (College Station, TX) or Texas A&M College of Veterinary Medicine during the period of July 2000 to July 2003 (Table 1). Submitted parrots were a mixture of pets, aviary parrots used for breeding, and parrots from pet stores. Parrots were selected opportunistically for inclusion in this study. Lung, liver, oral mucosa, and cloacal mucosa were collected from each parrot. Each sample was collected with separate DNA-free instruments. Necropsy specimens that were shown to have died from Pacheco disease were excluded from the study. Two parrots that were submitted that had grossly visible papillomas were also excluded from the study. Oral and cloacal scrapings from 3 pairs of wild-caught hyacinth macaws, originating from 3 sources, were tested by PCR for PsHV DNA at the request of the owner.

Dna Isolation, Pcr Amplification of Viral Dna and Sequencing of Amplification Products
DNA was isolated from 4 x 4 x 4-mm tissue samples using a commercial DNA isolation kit^b following the manufacturer's protocols, rehydrated in DEPC-treated, DNase-free water, and stored at –20°C until use. Approximately 200 ng of DNA was used in each PCR reaction. DNA concentration was determined by UV absorption at 260 nm. Oral and cloacal scrapings were vortexed in 400 µl of phosphate buffered saline (pH 7.2) and the cells were pelleted by centrifugation. The supernatant was poured off and the cells resuspended in 50 µl of cell lysis solution,^b containing proteinase K at a final concentration of 0.2 mg/ml. The solution was incubated at 56°C for 1 h and the proteinase K was inactivated by incubating at 95°C for 5 min. Undigested material was pelleted by centrifugation and 6 µl of the supernatant was used in each PCR reaction. PCR was performed as described using PsHV UL16/17 forward primer, 5'-TGCGTGGGGTTAAACTCGGAAC-3', and reverse primer, 5'-CGACTACACGAGCCTAACATC-3'.12 The original reaction was followed by a second, seminested PCR reaction using 2 µl of the first reaction mix, an internal primer (5'-CGACTTCTCAACGACGTC-3') and the reverse primer and identical reagent volumes and concentrations. Final primer concentrations were 25 µM for both PCR reactions. Reactions conditions were also identical with the exception that the annealing temperature was lowered from 60°C to 58°C for the seminested reaction.14 Amplified products were separated by electrophoresis on a 2% agarose gel containing ethidium bromide and were visualized with ultraviolet light. Results were recorded as negative, positive, if a visual band could be detected with the first set of primers, and low positive if an amplification product could only be visualized after the seminested reaction. Using known concentrations of PsHV-1 DNA it was shown that amplicons could be detected from as few as 10 target DNA molecules (data not shown). Amplicons generated from one or more PCR reactions from all positive tissues from the same parrot were combined and purified with QIAquick PCR Purification Kit^c prior to sequencing.

Both positive and negative controls were included with each set of tissues from a single bird and for each set of 20 samples when tissues or scrapings from multiple birds were examined in a single batch. Negative controls included DNA-free water and DNA from a chicken embryo. The positive control was diluted DNA from a parrot's liver that died of Pacheco disease.

PCR amplicons were sequenced directly with Big Dye^TM Terminator DNA Sequencing Kit and ABI 377 DNA sequencer^d using the amplification primers. Sequences were aligned with Sequencher^TM 3.1.2,^e then imported to Paup^* 4.0^f along with representative sequences of known PsHV-1 genotypes (JV) for determination of genotype. To determine the genotype of each virus, phylogenetic trees were constructed using bootstrap resampling (1,000 replicates) with neighbor joining. Parsimony with heuristic search was used to construct trees determining phenotypic relationships.

Detection of Multiple Pshv-1 Variants in a Single Sample
It was found that amplicons from some of the parrots contained sequences from more than one virus. In these cases, amplicons generated from different tissues were sequenced separately. If a tissue contained more than one variant, the nested primer was replaced with genotype specific primers 5'-AAGACAAGCCCGCCGCCGGCG-3', for genotype 1 (amplicon 300 bp), and 5'-ATGAAGACGATTCGGCTAC-3' for genotype 2 (amplicaon 243 bp) and the virus DNA reamplified and sequenced. In 1 bird, the yellow-collared macaw, 2 virus sequences were present in all positive tissues and one was determined to be genotype 2. To determine the other sequence, the PCR amplicons were directly ligated into the plasmid vector pCR4-TOPO and transformed into TOP One Shot chemically competent Escherichia coli cells following the manufacturer's instructions.^g The transformed colonies were color-selected. Plasmid DNA was purified from overnight cultures of selected clones and the insert DNA was sequenced as above using primers provided with the cloning kit.

	Results

TOP Sources and manufacturers Abstract Introduction Materials and Methods Results Discussion References

 
Tissue Distribution of Pshv 1 in Latently Infected Parrots
The tissues collected from the parrots that died spontaneously in the Schubot Exotic Bird Health Center collection and the 5 diagnostic specimens are shown (Table 2). Fourteen of 16 parrots that were examined were positive for PsHV-1 or PsHV-2 DNA in one or more tissues. The 2 negative parrots came from the aviary that had a history of a Pacheco outbreak 15 years prior to the submission of these parrots. Tissues that were frequently sampled and were positive in 50% or more of the naturally infected parrots included the oral mucosa (9/15), duodenum (5/9), jejunum (7/11), colon (6/9), cloacal mucosa (10/15), and spleen (9/12). Crop was positive 47% (7/15) of the time. There were 13 of 14 (93%) parrots that were positive on either or both oral and cloacal mucosal samples Tissues that were collected infrequently but were positive in multiple parrots included the ventriculus (3/4) and oviduct (2/4 samples). Infrequently sampled tissues that were not included in the table but were collected from positive parrots were sciatic nerve (1/3), spinal cord (0/2), autonomic ganglia (0/3), and adrenal (1/1).

The citron-crested cockatoo had 17/17 tissues positive for viral DNA. In contrast, viral DNA was only found in the cloacal mucosal samples of 2 parrots, a Quaker parrot and a red-shouldered macaw. Of the 85 positive tissue samples, 54 (63%) were found to be positive without using nested PCR. Ten parrots were infected with a single PsHV-1 genotype and 4 parrots were infected with 2 genotypes. One of the African grey parrots was infected with PsHV-2 (Table 2).

Repeat Testing of Previously Positive Parrots
Nine parrots that had tested positive and 16 parrots that had tested negative for PsHV-1 DNA on oral and cloacal swabs in 1999 were retested. All of the previously positive parrots were positive again for PsHV-1 DNA. One of the previously negative parrots, the sun conure, was positive on the repeat sampling (Table 3 Column 3). Six of the 8 patagonian conures were positive and all of them were infected with PsHV-1 genotype 4. The remaining 4 positive parrots, a cherry-headed conure, a sun conure, and 2 Quaker parrots were infected with PsHV1 genotype 1. All of the positive birds in this study, with the exception of the sun conure who was negative in a previous study, were infected with the same genotype that they were infected with 5 years previously.

Origin of the Positive Parrots in this Study
All of positive birds for which multiple organ samples were collected, with the exception of the hawk-headed parrot, were wild caught, as were all of the positive birds that were part of the repeat testing study and the 6 hyacinth macaws tested by request. Of the 13 positive birds submitted for necropsy to the TVMDL, 5 were wild caught, 3 were domestically produced, and the origin of the other 7 was not known.

	Discussion

TOP Sources and manufacturers Abstract Introduction Materials and Methods Results Discussion References

 
In this study we document the tissue distribution of PsHV DNA in latently infected parrots. We also show that parrots that were previously shown to be infected on combined oral and cloacal swabs remained positive 5 years later and were infected with the same PsHV-1 variant. Finally, we report on the prevalence of PsHV-1 and PsHV-2 in parrots submitted for diagnostic necropsy and show that concurrent infection with 2 genotypes of PsHV-1 is possible.

It is widely assumed that parrots latently infected with PsHV-1shed the virus intermittently or at low levels and are sources of infection for other parrots.2,8,11 Having a screening test that could detect these parrots would prove valuable in controlling outbreaks of Pacheco disease and mucosal papillomas. Two previous studies have suggested that parrots latently infected with PsHV-1 will have viral DNA in their oral and/or cloacal mucosa and that this DNA can be consistently detected in most of these parrots with a PCR-based assay.11 The first study found that many parrots could be detected with each sampling but some parrots were only intermittently positive.8

Subsequent analysis of the primer sets used in this study suggests that the intermittently positive parrots were probably infected with PsHV-1 genotypes that were poorly amplified with the PCR primer sets used.13 In a more recent study PCR primer sets were designed to detect all known PsHV-1 variants and these primer sets were used in this study.

Using the modified primers sets in latently infected parrots we found that the prevalence of PsHV-1 viral DNA in the oral mucosa to be 57% (8 of 14), in the cloacal mucosa was 78% (11/14) and in the combined oral and cloacal mucosae to be 92% (13 of 14). Similar results were found when examining tissues from the positive parrots submitted for diagnostic necropsy. Of the 10 positive cases, the oral mucosa was positive for 5 parrots, the cloacal mucosa was positive for 2, and both the oral and cloacal mucosas were positive for 3 parrots. Why one infected parrot with PsHV-1 DNA in multiple tissues was not positive for either mucosal surface is not known. It is possible that this parrot did not have a mucosal infection. It is also possible that a mucosal infection was present but was missed or that the mucosal samples collected were not infected but adjacent mucosal may have been. The advantage of a swab or scraping technique, such as those used in the live parrots, is that they sample large areas of the mucosa, whereas an analysis of small section of mucosa may be less representative.

These findings suggest that the vast majority of parrots infected with PsHV-1 will have DNA virus in or on the mucous membranes of their oral and cloacal mucosae and that these parrots can be detected by a PCR assay of scrapings or swabs of these surfaces. To consistently detect these parrots, however, it is necessary to submit samples obtained from both the oral mucosa and the cloacal mucosae.

Examination of tissues from necropsy specimens shows that PsHV-1 are present in multiple tissues in addition to the oral and cloacal mucosa. Other sections of the digestive tract including the crop, ventriculus, and portions of the intestine are also commonly infected. Virus can also been found outside of the digestive tract in various organs, the most common one being the spleen. Virus concentrations, in nondigestive tract organs from the necropsy specimens, were commonly detectable with the single PCR reaction yet these birds had no evidence of herpesvirus disease. This may represent a reactivation of viral activity in birds with chronic disease or it may represent typical virus concentrations in some latently infected birds. In either case, it is important for the clinician and pathologist to recognize that the detection of PsHV-1 DNA in a necropsy specimen does not necessarily imply that the virus contributed to the parrot's death.

One tissue that was not examined in this study was the trigeminal nerve ganglia. The trigeminal nerve ganglia is commonly and latently infected by many herpesviruses in their respective hosts, including the infectious laryngotracheitis virus, one of the most closely related viruses to PsHV-1 and PsHV-25,6,¹⁵,¹⁸. While it would have been of interest to examine the trigeminal nerve ganglia in these birds, it was not necessary for this study. Although latently infecting the trigeminal nerve, other herpesvirues can also be found in other tissues, e.g., infectious laryngotracheitis can also consistently infect the trachea1,5,6. This is consistent with the findings in this case where latently infected birds had virus in multiple tissues. We cannot conclusively rule out the possibility that latent infection with PsHV-1 might be confined to the trigeminal nerve ganglia in some cases, but if it does it is rare.

The parrots at the Schubot Exotic Bird Health Center are housed in close proximity to each other. Therefore, it was expected that over time virus transmission from infected birds to naïve birds would occur. However, a newly acquired infection was only found in 1 bird, a sun conure. This sun conure was housed next to the positive cherry-headed conure and may have been infected by it, as both were infected with the same genotype. The relatively low transmission rate in these parrots suggests that virus shedding is infrequent or in low concentrations in latently infected birds, or that the management techniques employed at the Schubot Center that are designed to minimize the transmission of pathogens are effective.

Knowing the prevalence of infection of PsHV-1 and -2 and the species commonly infected with them is key to designing recommendations for routine screening for these viruses. Data generated in this study showed that African, Neotropical, and Western Pacific parrots can be latently and asymptomatically infected with PsHV-1 genotypes. It also showed that parrots originating from flocks with a history of a Pacheco disease are likely to have a high prevalence of infection. The survey of parrots submitted for necropsy suggests that the prevalence of PsHV-1 infection is relatively low (9.3%) in the population of parrots submitted to the Texas Veterinary Diagnostic Laboratory as compared to the results of a European study (37.3%).4

Most infected birds were neotropical parrots, particularly Amazon parrots and macaws (Ara species and the hyacinth macaw), but there was a near equal prevalence of infection in African species. Although a cockatoo from the Schubot aviary was positive, no parrots from the Australian and Pacific distribution of parrots were positive in the parrots submitted for necropsy.

Taken together, these results suggest that testing for PsHV-1 in otherwise healthy parrots is indicated if they originate from an aviary with a history of Pacheco disease. Often the disease history of an aviary is not known. However, if the aviary of origin contains birds from multiple sources, it is would seem prudent to test a parrot coming from such a source. A testing program for collections with many birds is also indicated as the probability of eventually acquiring a positive bird increases with the number of birds owned and it may only take one to cause a Pacheco disease outbreak. The data also suggests that domestically raised birds will occasionally be infected with PsHV-1. Testing individual pet birds as part of a new bird exam is recommended at least for the species, such as macaws, Amazon parrots, and conures, that are prone to develop mucosal papillomas. PsHV-1 and PsHV-2 infections were not found in parrots of the Australian and Pacific distribution submitted for necropsy. However, the number of birds submitted was relatively small (n = 18) and additional studies will need to be done before a recommendation can be made that these birds do not need to be tested.

PsHV-2 was originally identified in 3 wild caught African grey parrots. One of these parrots did not have disease. In the other 2 parrots, PsHV-2 was detected in a mucosal and cutaneous papilloma. Papillomas in African grey parrots are rare, so it was uncertain whether they are rare because PsHV-2 infections are rare, or because papillomas are a rare manifestation of PsHV-2 infection12. Three out of 12 African grey parrots examined in this study were infected with PsHV-2, suggesting that infection with this virus is relatively common and that if PsHV-2 does cause papillomas, it is rare for it to do so. Besides these 2 birds with papillomas, PsHV-2 has not been associated any other disease. Therefore, the importance of detecting parrots latently infected with this virus is not known. Perhaps the most important issue relating to PsHV-2 is that it may be detected with primers designed to detect PsHV-1. A PsHV-2 infected bird then might be mistaken for a bird that could be a source of Pacheco disease. A single blue and gold macaw was found to be infected with PsHV-2 in this study. This is the first case of a documented infection in a parrot species that was not an African grey parrot.

All 4 genotypes of the PsHV-1 can cause Pacheco disease. However, PsHV-1 genotype 2 has not been reported to cause Pacheco disease in the USA. Again, it was not known if this meant that the virus was rare in the USA populations of parrots, or if PsHV-1 genotype 2 was a less pathogenic virus that, under the aviculture conditions in the USA, did not cause disease. The later interpretation appears to be more likely. PsHV-1 genotype 2 was found in 4 parrots submitted for necropsy and 3 parrots from the Schubot collection14.

Genotypes 1, 2, and 3 correspond respectively to serotypes 1, 2, and 3, while genotype 4 is also serotype 13,¹⁴. Multiple birds in this study were found to be co-infected with two PsHV-1 genotypes. Combinations of infections including 1 and 2, 1 and 3, 2 and 3, and 3 and 4 were found. This shows that infection with one serotype does not prevent infection with a second serotype. The absence of a dual infection with genotype 1 and 4 may indicate that, because they are the same serotype, infection with one may protect against infection with the other. However, the number of co-infected birds in this study was small and a larger study or an infection trial would have to be done to prove this. The co-infection data also suggests that in order to protect parrots from infection with three PsHV-1 serotypes, parrots would have to be immunized with a polyvalent vaccine. The current commercial vaccine^a is monovalent, whether it would protect against infection with all serotypes of PsHV-1 is not known.

Surveys of wild parrots for PsHV-1 infections have been limited and there have been no surveys of wild parrots where the genotypes of the virus infecting them have been determined. It has been postulated that PsHV-1 variants have co-evolved with some species of neotropical parrots, whereas PsHV-2 may represent a herpesvirus that co-evoluted with the African grey parrot. Determining the PsHVs and their genotypes in captive parrots may not reflect what occurs in nature, as these parrots are exposed to many other species of parrots during importation allowing for infection with a herpesvirus that they might not be exposed to in nature. Three trends, however, emerged in this study that may suggest host-virus co-evolution. All but one of the parrots infected with the PsHV-1 genotype 4 were Patagonian conures. All of these birds were wild caught and imported and they came from at least 2 different sources, suggesting that they may have been infected with PsHV-1 genotype 4 in the wild. A similar pattern was observed for the hyacinth macaws. Five of the 6 positive hyacinth macaws were infected with PsHV-1 genotype 3 and at least 4 of these birds were wild caught and the 5 birds originated from at least 4 different sources. Finally, PsHV-2 was found in 3 more African grey parrots so than 6 of the 7 birds proven to be infected with this virus are African grey parrots suggesting that this virus has co-evolved with this species. Additional studies sampling wild birds will need to be done to verify these hypotheses.

	Acknowledgments

 
The authors thank Dr. Ian Tizard, Director of the Schubot Exotic Bird Health Center for his financial support for this work.

	Sources and manufacturers

TOP Sources and manufacturers Abstract Introduction Materials and Methods Results Discussion References

 
From The Schubot Exotic Bird Health Center, Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843-4467 (Tomaszewski, Phalen) and the Texas Veterinary Diagnostic Laboratory, College Station, TX 77843- 4471 (Wigle).

^a. Biomune, Lenexa, KS.

^b. Gentra Systems, Minneapolis, MN.

^c. Qiagen, Valencia, CA.

^d. Applied Biosystems, Foster City, CA.

^e. Gene Codes Corp., Ann Arbor, MI.

^f. TZ cloning kit; Invitrogen, San Diego, CA.

	References

TOP Sources and manufacturers Abstract Introduction Materials and Methods Results Discussion References

 

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