Investigations on the diagnosis and retroviral aetiology of renal neoplasia in budgerigars (Melopsittacus undulatus)

Abstract

The high susceptibility of budgerigars (Melopsittacus undulatus) to neoplasia, and specifically renal neoplasia, has often been reported. Further investigations led to a suspicion of a retrovirus as the causative agent for renal neoplasia in budgerigars, but definitive proof has yet to be found. In the present study, 32 budgerigars suspected of having renal neoplasia (based on the clinical presentation) were examined. The objectives were to investigate the use of different diagnostic methods for the ante-mortem diagnosis of this condition and to find more supporting evidence of a retroviral aetiology. The predominant clinical signs observed in budgerigars with renal neoplasia were lameness and absence of deep pain sensation of one leg. Alterations in haematology, plasma chemistry, and urine analyses could not pinpoint the cases of renal neoplasia. Contrast radiography of the intestinal tract proved to be diagnostically more useful compared with plain radiographic studies. Histology confirmed the renal neoplasia as adenocarcinoma. Investigations for virus identification included product-enhanced reverse transcriptase assay and enzyme-linked immunosorbent assay for the detection of avian leucosis virus group-specific antigen. Cell cultures and electron microscopy were performed on a limited number of patients. These investigations could find no presence of an exogenous, replicating retrovirus, neither could viral particles be detected by electron microscopy. Based on the current findings, it can be concluded that there is no evidence of retroviral involvement in the occurrence of renal neoplasia in budgerigars.

Introduction

Budgerigars (Melopsittacus undulatus) are popular pet birds. One of the most common single causes of death in these birds is neoplasia, and in particular renal neoplasia (Beach, 1962; Arnall, 1966; Blackmore, 1966; Petrak & Gillmore, 1969; Baker, 1980; Kummerfeld et al., 1983; Filip & Scope, 2002; Langenecker, 2006). Authors report an incidence of renal neoplasia in budgerigars in the range of 16 to 24%.

The clinical manifestation of this disease typically involves unilateral leg lameness, abdominal enlargement, central nervous system signs, and polyuria (Kummerfeld et al., 1983). These clinical signs, however, can be observed in other avian diseases; therefore additional diagnostic procedures are required (Woerpel & Rosskopf, 1984). Contrast and plain radiographic studies can be used, where one could determine the possible organ involvement by the location, filling, and displacement of the gastrointestinal apparatus (Krautwald et al., 1992). Further diagnostic improvement could be achieved by the use of ultrasonography. Ultrasonography as a diagnostic tool has already been used in birds, but the small size of the budgerigar is a challenge (Krautwald-Junghanns et al., 1994). Regarding the aetiology of renal neoplasia in budgerigars, Kummerfeld et al. (1983) were the first to suspect the involvement of a retrovirus (avian leucosis virus [ALV] ).

Subsequently, these authors used an enzyme-linked immunosorbent assay (ELISA) for group-specific (gs) antigen using the serum of 74 budgerigars suspected of having renal neoplasia (Neumann & Kummerfield, 1983). Although direct evidence for the presence of ALV gs antigen was obtained in 47% of the birds, the presence of this antigen was not specific for kidney neoplasia, as it was also found in budgerigars that did not suffer from this disease. An alternative approach was used by Gould et al. (1993). These authors tested for the hypothesized presence of ALV by applying an ELISA for viral antigen and by dot-blot hybridization for the presence of retroviral RNA sequences with a probe derived from Rous-associated virus-2 (RAV-2). All ELISA tests to detect the presence of p27 of ALV and attempts to isolate the virus were negative, which in itself is in contrast with the previous results of Neumann & Kummerfield (1983). It was speculated that the difference between the results of these two studies was due to the different ELISA tests used. Dot-blot hybridization of DNA extracted from renal neoplasms reacted positive in six of eight renal neoplasia samples (Gould et al., 1993). All eight specimens were further analysed by Southern blot hybridization. The most important finding was the presence of DNA in the tumours that hybridized to the RAV-2 probes. Since these studies were performed, the suspicion of a retrovirus as causative agent in renal neoplasia in budgerigars has persisted but was never proven.

A recent development in the identification of retroviruses involves a new class of ultrasensitive tests for the detection of the retroviral enzyme reverse transcriptase, such as the product-enhanced reverse transcriptase (PERT) assay. These ultra sensitive tests are 10⁶ to 10⁷ times more sensitive than conventional reverse transcriptase assays and detect all replication competent retroviruses. The PERT assay was successfully used to detect reverse transcriptase activity in human melanoma cell lines contaminated with murine leukaemia virus, as well as the detection of an unclassifiable endogenous retrovirus in pythons (Huder et al., 2002; Deichmann et al., 2005).

The aims of the present study were to investigate the usefulness of different clinical diagnostic procedures—such as blood and urine investigation, ultrasonography and radiography—in the ante-mortem diagnosis of renal neoplasia in budgerigars. Furthermore, the hypothesis of a retroviral aetiology of renal neoplasia was tested by searching for the presence of retroviral reverse transcriptase in these birds.

Materials and Methods

Birds

Different groups of captive budgerigars were investigated. One group comprised 32 birds presented at the Clinic for Zoo Animals, Exotic Pets and Wildlife, Vetsuisse Faculty, University of Zurich, Switzerland, with a suspicion of renal neoplasia based on the clinical signs (A1 to A32). In addition, 12 clinically healthy budgerigars were included in a pilot study to establish the assay conditions of the PERT assay (B1 to B12), and 39 clinically healthy budgerigars were used as a control group (C1 to C39).

Diagnostic procedures performed in budgerigar Group A

Based on the history obtained from the owner, information on the birds' diet, their environment, age, sex, and the duration of clinical signs suggestive of renal neoplasia were noted. The budgerigars then underwent a thorough clinical examination. If lameness was present, both legs were compared for reflexes and sensitivity. Whenever possible, 0.3 to 0.4 ml blood was collected from the right jugular vein for haematology and plasma chemistry. The quick urine Combur® Test (Roche Diagnostics GmbH, Mannheim, Germany) was performed with the emphasis on the presence of haematuria. Plain and contrast radiographic examination in right lateral and ventrodorsal projection was performed in non-anaesthetized birds. Additionally, ultrasonography was attempted using a ventromedian approach (McMillan, 1988; Hofbauer and Krautwald-Junghanns, 1999) with a 5 to 8 MHz micro convex transducer (ATL HDI 5000; Philips AG Medical Systems, Zurich, Switzerland) under isoflurane (Attane™; Provet AG, Lyssach, Switzerland) anaesthesia.

Birds with a poor prognosis were euthanized with the owners' consent. A necropsy was performed as soon as possible after euthanasia, and organs suspicious for neoplasia were collected (fresh specimens, frozen specimens, and specimens in 10% neutral buffered formalin) for further investigation. Tissue specimens obtained from three budgerigars (collected in 10% neutral buffered formalin) were sent to the Institute of Veterinary Pathology, Vetsuisse Faculty, University of Bern, Switzerland for histological examination and transmission electron microscopy of thin sections (Zeiss EM 109; Oberkochen, Germany).

Virus detection

PERT assay

Reverse transcriptase (RT) activity was measured by a TaqMan real-time polymerase chain reaction (PCR) modification of the originally described PERT assay (Pyra et al., 1994; Buergisser et al., 2000). Ethylenediamine tetraacetic acid blood samples from budgerigars were separated by centrifugation (5 min at 1400×g) (Hettich, Bäch, Switzerland). The plasma fraction was collected in separate tubes and frozen at −20°C until further examination.

In order to define suitable assay conditions of the PERT assay for budgerigar plasma and to exclude the presence of a high RT activity due to endogenous retroviruses that might interfere with the search for an exogenous retrovirus, blood from six clinically healthy budgerigars (B1 to B6) was collected and their plasma tested without further pretreatment at a dilution of 1/100, 1/1000 and 1/10,000 in phosphate-buffered saline (PBS). Plasma at a dilution of 1/1000 was deemed suitable for direct testing in this study, because assay inhibition was not detectable and the levels of naturally occurring, measurable RT activity was only slightly above the limit of detection.

The possibility of a non-specific elevation of RT activity due to blood storage was investigated by collecting ethylenediamine tetraacetic acid blood samples from three clinically healthy budgerigars (B7 to B9). The blood was aliquoted and the plasma separated and stored for investigation of RT activity immediately after collection then after 2 and 4 days of storage. The influence of the time elapsed between death and sampling was further investigated by collecting blood from the heart of three deceased budgerigars (B10 to B12). Two of these birds (B10 and B11) were euthanized for reasons other than renal neoplasia, and one bird (B12) had renal neoplasia detected on post-mortem investigation. Blood from the heart of budgerigar B10 was collected after the bird was kept for 12 days at 4°C, from bird B11 after 2 days at 4°C and from bird B12 after the bird was kept for 1 day at 4°C.

Cell cultures

Fresh tissue samples from kidneys or kidney neoplasia obtained from four birds were collected in PBS (pH 7.2) and sent to the veterinary laboratory, Vetsuisse Faculty, University of Zurich for primary cell cultures. Samples were prepared in a medium as described by Gould et al. (1993). The culture medium was exchanged on days 5, 11, and 19, and supernatants were subsequently submitted for PERT assay. Samples were diluted 1/100 for the PERT assay.

ELISA test for retroviral gag antigen p27

An ELISA for ALV gs antigen (p27) (“FlockChek Kit”; IDEXX GmbH, Wörrstadt, Germany) was performed using plasma of clinically healthy budgerigars (C1 to C39), and budgerigars with renal neoplasia or other disease/tumours (A1 to A32). Because only small quantities of plasma were available, these plasma samples were diluted 1/10 in PBS and tested following the manufacturer's protocol with the exception that the turnover of 3,3′,5,5′-tetramethylenebenzidine was stopped after 20 min with phosphoric acid and the absorbance was measured at 450 to 630 nm. Using phosphoric acid improves the precision of the optical density (OD) measurement. The provided positive control reached an OD of 1.36 for 10 ng p27/ml.

Statistical analysis

Data were analysed by Mann–Whitney U-test and t-test using Statistika™ 5.0 (Stat-Soft®; Tulsa, Oklahoma, USA). The significance level was set to P<0.05. Data are presented as mean±standard deviation or, in the case of non-normal distribution, as median±upper/lower quartile.

Results

Diagnostic procedures

A summary of all the diagnostic procedures performed on the birds from Group A and the results are presented in Table 1.

Table 1.  Clinical manifestation and diagnostic procedures performed in budgerigars (M. undulatus) suspected of having renal neoplasia: histologically confirmed cases of renal neoplasia (A1 to A8) and budgerigars with other neoplasia or disease (A9 to A32)

			Clinical signs
Bird	Age (years)	Sex	Lameness	Coelomic enlargement	Blood uric acid	Urine Combur® Test	X-ray	Ultrasound	Histology
A1	8	M	+	–	+	NP	NP	NP	+
A2	5	M	+	+	NP	NP	NP	NP	+
A3	6	F	+	+	NP	+	NP	NP	+
A4	5	M	+	–	–	+	–	–	+
A5	2	F	+	–	+	+	+	+	+
A6	0.8	M	+	–	NP	NP	NP	NP	+
A7	6	M	+	–	–	NP	+	ND	+
A8	10	M	S	–	NP	NP	NP	NP	+
A9	4	F	–	+	–	+	+	+	–
A10	5	F	–	+	–	NP	+	NP	–
A11	4	M	–	+	–	+	–	–	–
A12	4	F	–	+	–	+	–	–	NP
A13	4	M	+	–	NP	+	NP	NP	–
A14	3	M	–	–	–	–	–	–	NP
A15	4	F	+	–	+	+	–	–	–
A16	3	F	–	–	NP	–	–	NP	NP
A17	6	F	+	–	NP	+	–	NP	–
A18	3	M	–	–	–	–	–	–	NP
A19	6	F	+	+	–	+	–	NP	–
A20	4	M	–	+	–	+	–	–	–
A21	12	M	+	+	–	+	–	+	–
A22	3	M	–	–	–	NP	–	–	–
A23	2	M	S	–	NP	NP	NP	NP	–
A24	2	M	+	–	NP	NP	–	NP	–
A25	2	M	–	–	–	+	–	–	NP
A26	9	M	+	+	+	+	–	–	–
A27	6	M	+	–	–	+	–	–	NP
A28	3	F	+	–	–	–	–	–	–
A29	4	F	–	+	–	–	–	–	–
A30	5	M	–	–	+	+	+	+	–
A31	9	M	–	+	+	+	–	–	–
A32	9	M	–	–	+	+	–	+	–
NP, not performed; +, positive result/results indicative of kidney neoplasia (within the scope of the respective diagnostic procedure); –, negative result for this investigation; S, stupor, it was not possible to perform evaluation for lameness. Bold data indicate budgerigars with histological diagnosis of renal neoplasia.

Of the 32 birds with clinically suspected renal neoplasia, eight (A1 to A8) were confirmed histologically. Six of these birds were male. Their age was between 10 months and 10 years. There was no age predisposition, neither were any blood parameters indicative of kidney neoplasia. Based on the history, there were no predisposing factors regarding the birds' diet or the environment. The urine Combur® Test reacted positive to budgerigars with renal neoplasia but also in birds with neoplasia of other origin or disease. Radiographic examination was performed in 25 birds. It was not possible to reach a conclusive diagnosis of urogenital neoplasia using plain radiographs in four budgerigars. In three of these birds a diagnosis of urogenital neoplasia was made on contrast studies, but there was still one bird where the diagnosis could not be made using radiography. Radiography could not differentiate clearly between renal and gonadal neoplasia. Ultrasonography was performed on 20 birds. In two birds, ultrasonography did not allow a conclusive diagnosis (A7 and A15) due to the small size of the budgerigars presented. Eleven birds (55%) were correctly diagnosed with disease (liver disease, salpingitis, coelomitis) or neoplasia (gonadal or renal), which was further confirmed by histology.

A diagnosis of renal neoplasia was confirmed histologically in eight out of 13 macroscopically suspicious cases. All cases with renal neoplasia were described as renal adenocarcinoma (solid, tubular, or tubular-papillary form).

Analyses for the detection of the presence of retroviruses

A summary of all the procedures performed for the detection of retroviruses and the individual results are presented in Table 2.

Table 2.  Retrovirus investigations performed in budgerigars (M. undulatus) with histologically diagnosed renal neoplasia (A1 to A8), birds with different neoplasia or disease (A9 to A32) and a control group of clinically healthy budgerigars (C1 to C39) where blood was collected twice within 12 weeks (Collections 1 and 2)

	PERT clinic cases					PERT C1 to C39, plasma (µU/ml)		ELISA C1 to C39 (OD)
Clinical case bird	Plasma (µU/ml)	Cell cultures	ELISA clinic cases (OD)	EM clinic cases	Control group budgerigars (n)	Collection 1	Collection 2	Collection 1	Collection 2
A1	52	NP	0.009	–	C1	4	1	0.020	0.018
A2	1645	NP	0.086	–	C2	0	0	0.046	0.029
A3	NP	NP	NP	NP	C3	8	0	0.064	0.035
A4	12	NP	0.017	NP	C4	52	NP	0.040	NP
A5	0	–	0.012	NP	C5	13	2	0.043	0.053
A6	NP	–	NP	NP	C6	229	31	0.026	0.040
A7	NP	NP	NP	NP	C7	0	6	0.014	0.020
A8	332	NP	0.037	NP	C8	70	0	0.079	0.115
A9	7	NP	0.023	NP	C9	0	0	0.014	0.019
A10	10	NP	0.018	NP	C10	156	79	0.021	0.022
A11	0	NP	0.021	NP	C11	8	5	0.030	0.025
A12	17	NP	0.044	NP	C12	43	16	0.026	0.028
A13	2	NP	0.016	NP	C13	13	0	0.017	0.032
A14	39	NP	0.023	NP	C14	102	18	0.044	0.033
A15	34	NP	0.014	NP	C15	91	15	0.080	0.046
A16	45	NP	0.021	NP	C16	222	NP	0.026	NP
A17	NP	NP	NP	NP	C17	6	1	0.022	0.017
A18	0	NP	0.022	NP	C18	316	NP	0.071	NP
A19	0	NP	0.011	NP	C19	3	2	0.083	0.017
A20	NP	NP	NP	NP	C20	163	203	0.022	0.024
A21	0	NP	0.020	–	C21	7	1	0.025	0.025
A22	15	NP	0.050	NP	C22	5	2	0.060	0.055
A23	NP	NP	NP	NP	C23	181	36	0.028	0.021
A24	0	NP	0.080	NP	C24	4	4	0.023	0.024
A25	NP	NP	NP	NP	C25	4	0	0.030	0.052
A26	9	NP	0.010	NP	C26	9	1	0.018	0.012
A27	6	NP	0.022	NP	C27	0	1	0.042	0.033
A28	10	NP	0.024	NP	C28	6	2	0.036	0.031
A29	22	NP	0.021	NP	C29	26	18	0.032	0.038
A30	136	–	0.016	NP	C30	10	0	0.090	0.125
A31	7	–	0.037	NP	C31	23	2	0.063	0.053
A32	17	NP	0.020	NP	C32	296	60	0.060	0.055
					C33	2	1	0.031	0.033
					C34	70	10	0.070	0.025
					C35	11	NP	0.055	NP
					C36	11	17	0.035	0.052
					C37	3	0	0.060	0.057
					C38	11	7	0.036	0.067
					C39	32	17	0.025	0.022
NP, not performed; −, negative result for this investigation; OD, optical density; EM, electron microscopy. Bold data indicate budgerigars with histological diagnosis of renal neoplasia.

Since tumours induced by viruses of the ALV group develop only in viraemic birds, plasma was chosen as the specimen for the detection of retroviral RT activity by the PERT assay. To cope with the minute sample volumes, plasma was tested directly at a dilution of 1/1000. At this dilution no assay inhibition by plasma components was present, and the detection limit of 1 µU/ml was still sensitive enough to detect an exogenous retrovirus.

The PERT assay was performed with 25 budgerigar patients' specimens with a clinical suspicion of renal neoplasia and with 39 clinically healthy, control birds (C1 to C39). In Bird A2, histologically diagnosed with renal adenocarcinoma, the RT activity of 1645 µU/ml was noticeably higher than that of the other specimens. However, it was noted that the blood of this bird was obtained approximately 48 h post mortem. Subsequent investigations on potential effects of the time elapsed between death and blood collection indicated that the measurable RT activity increased significantly, and that this RT activity was not associated with viral particles (data not shown). The statistical analyses for RT activity (Table 3) using the Mann–Whitney U-test showed that there was no significant difference between budgerigars with renal neoplasia (A1 to A8) and budgerigars with another type of neoplasia or disease (A9 to A32; U=27.5, P=0.13), or between budgerigars with renal neoplasia (A1 to A8) and clinically healthy budgerigars (C1 to C39; U=68.5, P=0.28).

Table 3.  Statistical results from RT activity (µU/ml) measured in plasma of budgerigars (M. undulatus) with renal neoplasia (A1 to A8), budgerigars with different neoplasia or disease (A9 to A32) and a control group of clinically healthy budgerigars (C1 to C39), blood collected twice within 12 weeks

						Control birds
	Group A	Group A without A2	Birds with renal neoplasia^a	Birds with renal neoplasia^a without A2	Birds without proven renal neoplasia^b	Collection 1	Collection 2
Median	10	10	43	27	19	11	2
Upper quartile	34	28	332	181	19	86	17
Lower quartile	2	1	12	6	1	5	1
Number of budgerigars examined	25	24	5	4	20	39	35
Bird A2 was considered an outlier with an RT activity of 1645 µU/ml because blood was sampled 48 h post mortem. ^aBudgerigars examined with histologically confirmed renal neoplasia. ^bBudgerigars examined with other neoplasia or disease.

Cell culture

Primary cell cultures of kidney tissue from two budgerigars with renal neoplasia (A5 and A6) and two birds with other neoplasia (A30 and A31) were tested for the release and replication of retroviral particles by PERT assay. Starting at very low RT activities slightly above the detection limit, all measured RT activities gradually dropped in all cultures and did not show any increase in RT activity that would have been indicative of a transient virus release or even active retrovirus replication.

Electron microscopy

An ultra-structural study was carried out in the three budgerigars (A1, A2, and A21), of which two had renal neoplasia, and no distinct viral particles could be demonstrated.

Detection of retroviral p27 gag antigen by ELISA

There was a significant difference in ELISA results between clinically healthy budgerigars (C1 to C39) and diseased budgerigars (A1 to A32), with diseased budgerigars yielding the lower median value (U=208.5, P<0.001).

There was no statistical significance between the ELISA reactivity of budgerigars with non-renal neoplasia (A9 to A32) and budgerigars with diagnosed renal neoplasia (A1 to A8; U=47.0, P=0.839), nor between budgerigars with renal neoplasia (A1 to A8) and clinically healthy budgerigars (C1 to C39; U=62.0, P=0.189) Results are summarized in Table 4.

Table 4.  Statistical results from the ELISA test for detection of specific antigen (p27) of ALV, performed in budgerigars (M. undulatus) with histologically confirmed renal neoplasia (A1 to A8), budgerigars with different neoplasia or disease (A9 to A32) and a control group of clinically healthy budgerigars in which blood was collected twice within 12 weeks (C1 to C39)

	A1 to A32	A1 to A8	A9 to A32	C1 to C39 (collection 1)	C1 to C39 (collection 2)
Number tested	25	5	20	39	35
Mean value	0.024	0.032	0.022	0.041	0.039
Standard deviation	0.017	0.032	0.011	0.021	0.025
Results are measured as OD.

Discussion

The results of this study underline the difficulty in the ante-mortem diagnosis of renal neoplasia in budgerigars. In the eight proven cases of renal neoplasia, no signs of retroviral involvement could be detected.

Throughout the clinical examinations performed on the 32 budgerigars, lameness of one leg with absent deep pain sensation was the predominant clinical sign that was associated with renal neoplasia, but lameness was also observed in cases with gonadal and liver disease. These results agree with the publication of Neumann & Kummerfield (1983) that, whenever trauma or metabolic disorders can be excluded diagnostically, coelomic neoplasia should be considered as the cause of lameness in budgerigars. Abdominal enlargement was not always present in cases of renal neoplasia, which agrees with the study of Petrak & Gillmore (1982, pp. 606–637), who state that “it is possible for a bird to be in the terminal state of kidney neoplasia and have no perceptible bulging of the abdomen”. There was no single blood value or combination of blood parameters that could be linked to the presence of renal neoplasia.

The urine Combur® Test performed with the emphasis on haematuria was positive not only in birds with histologically confirmed renal neoplasia, but also in those with different neoplasia and/or liver disease. Radiography was followed by contrast radiography in cases of suspected renal neoplasia. From the results, if renal or gonadal neoplasia is suspected, contrast radiography could be more useful as a single diagnostic method rather than plain radiographic studies. Radiography as a diagnostic tool on its own was not able to differentiate between neoplasia of renal or gonadal origin or whether there was salpingitis or dystocia. Ultrasonography proved to be challenging in budgerigars. Some of the problems faced when performing ultrasonography were the critical general condition of the budgerigars with neoplasia and the small size of the birds. Nevertheless, the results of this study indicate that ultrasonography may be useful as a follow-up diagnostic method when the radiographic differentiation between neoplasia from urogenital tract, liver disease, and egg peritonitis or egg binding is not possible.

Histology confirmed the suspicion of renal neoplasia, based on the macroscopic post-mortem examination, in eight out of 13 cases (61.5%). In the other five birds that showed macroscopic changes in the kidneys suspicious for renal neoplasia, three were diagnosed with neoplasia of the epididymis and two had carcinomas of unknown origin. All of the renal neoplasms were identified as adenocarcinoma. Therefore the diagnosis of renal neoplasia should not be pronounced solely on the basis of the macroscopic appearance of the tumour, as microscopic examination is required to make a definitive diagnosis.

The following observations support the conclusion that an exogenous retrovirus is not involved in renal neoplasia in budgerigars. For an exogenous retrovirus, which is expected to replicate in cells and cause neoplasia, one would assume that the expression of the virus in neoplastic tissue and in the blood circulation would be high. RT activity determinations performed in healthy budgerigars and in budgerigars with a histological diagnosis of renal neoplasia were unable to show an elevated RT activity in cases with renal neoplasia. Overall the detected RT activity in plasma specimens and supernatants of budgerigars was low compared with RT activity tested by PERT in other samples. For comparison, culture supernatants of normal chicken embryonic fibroblasts or a chicken stem cell line show activities of up to 2×10⁵ µU/ml, and 3.6×10⁶ µU/ml, respectively (unpublished results). The strong signals in dot-blot hybridization observed in the analyses of Gould et al. (1993) with DNA from renal tumours hybridized with RAV-2-labelled DNA and the predicted retrovirus are in strong contrast to the low RT activity in birds with renal tumours in this study. A retrovirus detected by hybridization as the causative agent for renal neoplasia is expected to be easily demonstrated in its replicating form by PERT assay, because the direct hybridization technique is several orders of magnitudes less sensitive. A clear asymmetric distribution in RT activity, corresponding to the hybridization signals, between birds with and without neoplasia was also not found.

The measured activity of RT in cell cultures with confirmed renal neoplasia dropped within 20 days at approximately the same rate as negative control cultures did. The results showed no elevation of the RT activity in cell cultures in the budgerigars with renal neoplasia compared with the positive control (cell culture from a sheep infected with Maedi-Visna). This decrease was in contrast to a predicted replicating retrovirus in neoplastic tissues; therefore an active replicating retrovirus as the causative agent of renal neoplasia in the examined tissues was excluded. This conclusion is also supported by the absence of retroviral particles in renal neoplasms on electron microscopy and the absence of a significant difference in absorbance values of the ELISA test for ALV gag antigen p27 in healthy controls and birds with renal neoplasia. While there was a statistically significant difference between diseased budgerigars (with renal neoplasia, other neoplasia or disease) and clinically healthy birds, with lower values in the diseased birds, the opposite was expected; and this might be explained with a generally decreased synthesis of proteins in the diseased birds. These findings also argue against the presence of a cross-reacting viral antigen in the diseased birds. Consistent with the absence of an association of a retroviral involvement, there was no correlation found between the values of OD for the ELISAs and the measured RT activity. Kummerfeld et al. (1983) described in their study that no correlation was found between the ALV specific antigen and certain neoplasia manifestation. However, a control group of clinically healthy birds was not tested in their study.

The possibility that another type of virus (with no retrovirus nature) could be involved in budgerigars with renal neoplasia was not investigated in the present study. According to the study of Gould et al. (1993) such a possibility is still conceivable, because there was cross-reactivity observed in dot-blot and Southern-blot hybridizations with a probe containing RAV-2 polymerase gene sequences. To further investigate this possibility, tests should be made according to the same protocol used by Gould et al. (1993) but supplemented with additional negative controls. If the results of those hybridizations can be reproduced, further work at the molecular level should be performed. Isolation of DNA and/or RNA should be carried out and the nucleic acid sequences then used for identification.

Extensive studies performed in humans with renal cell carcinoma have examined the possible aetiological and predisposing factors. These studies suggest that obesity is related to an increased risk of renal cell cancer (RCC) (Boeing et al., 1997; McLauchlin & Lipworth, 2000; Bergstorm et al., 2001; D'Amico et al., 2001; Moyad, 2001; Pischon et al., 2006). Other factors such as aristocholic acid (Volker et al., 2002), smoking, hypertension, cardiovascular disease, reduced intake of fruit and vegetables, analgesics, and amphetamines have also been implicated. The possible role of a genetic involvement has also been discussed (Scanlan et al., 2004). In our opinion, further epidemiologic research should focus on the connection between renal neoplasia in budgerigars and obesity/lipoma formation. In one study in humans, the increased risk of RCC among women was related to obesity irrespective of fat distribution, whereas low hip circumference was related to increased RCC in men (Pischon et al., 2006). Furthermore, a genetic predisposition of certain breeds should also be investigated.

Acknowledgements

The authors would like to thank PD Dr Marcus Clauss for his help, advice and much appreciated suggestions with regard to the whole work, Dr Hanspeter Steinmetz for the statistical analysis, and all the owners of budgerigars that participated in this study. Without their cooperation it would not have been possible to collect these data.

Additional information

Notes on contributors

Jon B. Huder

Joint first author