Comparison of serological and virological findings from subgroup J avian leukosis virus-infected neoplastic and non-neoplastic flocks in Israel

Abstract

Blood samples from nine broiler breeder flocks comprising five flocks clinically affected with myeloid leukosis tumours (ML+) and four tumour-free flocks from the same commercial background (ML−) were compared for avian leukosis virus subgroup J (ALV-J) serum antibodies by enzyme-linked immunosorbent assay (ELISA), for antigenemia (group-specific antigen) by antigen-trapping ELISA and for viremia. Group-specific antigen was detected in the sera of 58.1% of ML+ birds and 46.4% of the ML− birds (P=not significant), while 45.5% of ML+ birds and 24.1% of the ML− birds had ALV-J antibodies (P=0.065). In inoculated cell culture, 64.1% of the ML+ sera were viremic compared with 16.7% of the ML− sera (P=0.001). Similar significant differences were found between the two groups of flocks when ALV-J viremia was detected by immunofluorescence using a monoclonal env antibody (P=0.004), and for proviral DNA by polymerase chain reaction using two different sets of env-gene primers, H5−H7 (P=0.001) and R5–F5 (P=0.001). Using the primer pair R5–F5 the product size was approximately 1 kbp, while some heterogeneity in size among isolates was discernable. Our results indicate that a combination of diagnostic tests should be adopted in routine examination of tumour material in order to rule out false-negative findings.

Introduction

The recent emergence of myeloid leukosis (ML) as a major clinical entity affecting heavy breeds of poultry can be traced back to a novel retrovirus isolated by Payne in 1989 from a broiler breeder affected with a myeloid tumour. This was assigned to a new subgroup, J, of the avian leukosis virus complex (ALV-J) (Payne, 1998). In the following decade ML appeared in several breeding companies in the US and was causing serious economic losses to the broiler breeding industry (Fadly & Smith, 1997).

Eradication programs originally established for other exogenous ALV subgroups (Spencer et al., 1977; Payne & Howes, 1991) have proved effective in eradicating ALV-J from infected flocks with minor changes in emphasis (Venugopal, 1999; Witter & Fadly, 2001). Because of the presence of tolerant-shedders in ALV-J-infected flocks, these programs need to determine the ALV-J status of the breeding flock with the greatest accuracy, and multiple diagnostic tests repeated on several occasions during the reproductive life of the birds are considered necessary (Witter et al., 2000). Such tests may include a direct enzyme-linked immunosorbent assay (ELISA) that measures p27 antigen levels in tissues and secretions (Smith et al., 1979), an ELISA for antibodies to the envelope glycoprotein (gp85) of ALV-J (Hunt et al., 2000), a polymerase chain reaction (PCR) utilizing specific primers for the env gene (Smith et al., 1998a,b, Silva et al., 2000), isolation of the virus in C/E cells and detecting its replication by direct ELISA, PCR and immunofluorescence (IF) using a monoclonal antibody to gp85 (Venugopal et al., 1997).

In an attempt to evaluate the diagnostic value of each test when applied to clinically affected commercial birds, we have compared replacement and broiler breeding flocks affected with myeloid tumours with other commercial flocks from a similar genetic background undergoing non-specific losses. Our results show that PCR combined with virus isolation gave the clearest difference between the two groups of flocks.

Materials and Methods

Birds

All nine meat-type flocks included in this study had an anamnesis of high morbidity according to the attending veterinarian who examined them on the farm. The ages of the flocks are presented later in Table 4. Five flocks, referred to as ML+, had many sick and dead birds that on initial postmortem examination had characteristic pale-white lesions on the inner face of the sternum, a pelvic girdle throughout the liver and an enlarged spleen. Occasionally tumours were seen on the head. In these birds, the bursa of Fabricius was not visibly enlarged. Flocks were visited within days of being notified by the attending veterinarian. Sick birds were bled with ethylenediamine tetraacetic acid-washed syringes, scarified and their tissues together with the blood were transported on ice to our laboratory; only birds with tumours were processed for laboratory investigation. Tissues were either fixed in formalin for histopathological examination or frozen at −70°C for virological examinations. Blood was lightly centrifuged and the leucocyte-enriched plasma was either frozen at −70°C or processed for antigen or antibody ELISA.

The other four flocks (ML−) were suffering unusually high losses principally from egg peritonitis and other reproductive problems but no tumours were seen on routine postmortem examinations of sick or dead birds on several occasions over a period of 2 to 3 weeks from the initial report. Because these flocks were the offspring of contaminated grandparent or parent flocks, blood and tissues samples were processed as already described.

Serology

ALV-J antibody

Blood was collected from birds at the farm or at the laboratory. Serum was examined by ELISA for ALV-J antibody (IDEXX kit) (Venugopal et al., 1997).

ALV group antigen

ALV group-specific antigen (GSA) in serum was measured by ELISA according to the protocol previously described by Smith et al. (1979).

Virus isolation

Commercial specific pathogen free and line O embryos (ADOL, East Lansing, MI, USA) were trypsinized to prepare chick embryo fibroblasts (CEF). Secondary CEF were inoculated in suspension and grown for 7 to 9 days (Witter et al., 2000) in Leibovitz–McCoy medium (Biological Industries Ltd, Israel) with 0.5% fetal calf serum (Biological Industries Ltd). For each serum, two 60 mm plates of cells, with and without a glass coverslip, were inoculated. The coverslips were used for immunofluorescent staining while the remaining cells on the plate were assayed for GSA by ELISA. DNA was extracted from the cells in the other plate for PCR. The ELISA was performed on CEF extracts as already described. To prepare the antigen, the supernatant was removed and the cells were lysed with phosphate-buffered saline (PBS)–Tween 20 (2%), then frozen and thawed three times. Fibroblasts were also infected with 10-fold serial dilutions (10⁻¹ to 10⁻⁷) of three reference ALV-J isolates (ADOL 5701, ADOL 6803 and Hc1) (Fadly & Smith, 1999).

Immunofluorescence

After removal from the plate, the coverslips were washed briefly in PBS and fixed with cold acetone, then air-dried for a few minutes. Virus was identified with a monoclonal anti-gp85 antibody kindly supplied by Dr Venugopal (IAH, Compton UK). It was diluted 1/100 in PBS and incubated on the coverslips for 1 h at room temperature, followed by three washes with PBS of 5 min each. The anti-mouse FITC conjugate (Sigma #F0257) was diluted 1/30 in PBS and incubated for 1 h at room temperature. The first wash included 1% Evans blue for counter-staining followed by two more washes with PBS. The slide was air-dried for 1 min. Mounting buffer (90% glycerol, 10% of 0.05 M carbonate-bicarbonate buffer, pH 9.6) and coverslips were attached, and the slide was examined by ultraviolet microscopy.

Polymerase chain reaction

The medium was removed from the second plate and the cells were scraped off and pelleted by centrifugation. The pellet was treated with lysis buffer (0.5% SDS, 0.1 M NaCl, 10 mM Tris, pH 8.0, 1 mN ethylenediamine tetraacetic acid, 200 ng/ml proteinase K) for 1 h at 56°C. DNA was extracted with phenol–chloroform (Maniatis et al., 1982). The ALV-J env gene was amplified by three separate PCR systems (Table 1).

Table 1. The primer pairs used in the present study.

Subgroup	Product size (base pairs)	Primer	Sequence (5′ to 3′)
ALV-A to ALV-E	295 to 326	H5 (dir.)	GGA TGA GGT GAC TAA GAA AG
		AD1 (rev.)	GGG AGG TGG CTG ACT GTG T
ALV-J	545	H5 (dir.)	GGA TGA GGT GAC TAA GAA AG
		H7 (rev.)	CGA ACC AAA GGT AAC ACA CG
ALV-J	∼1000	F5 (dir.)	GGT ATT TTC TTG ATT TGT GGG G
		R5 (rev.)	GAT TCG CAA GTT TCA TAC CCC T

Primers H5–H7 and H5–AD1

The DNA was amplified according to Smith et al. (1998b), with two modifications for the H5-H7 primers; 2% formamide and 1.2 mM each primer.

Primers R5–F5

The PCR was performed according to Lupiani et al. (2000).

Reverse transcriptase-PCR

RNA was extracted directly from the serum using a Qiagen kit (QIAamp Viral RNA kit #52904). RNA (5 μl) was heated with 0.5 μl direct primer (H5 for PCR with primers H5–H7 and H5–AD1, F5 for PCR with primers F5–R5) at 60°C for 2 min. The reaction mix buffer (0.5 μl dNTP [each at 200 mM], 0.5 μl RNasin [#N2111; Promega, Madison, WI, USA], 0.12 μl super AMV reverse transcriptase [RT] [#1372-01; CHIMERX, WI, USA] complete to 10 μl with water) was then added. The reaction mixture was incubated at 39°C for 90 min. The PCR was performed as described earlier.

Statistics

The incidence of positives to total number of birds of the ML+ and ML– groups in all the assays were compared by chi-square analysis using SAS software.

Results

Titration of ALV-J virus in chicken embryo fibroblast cells by ELISA, IF and PCR with primers H5–H7 and R5–F5

To establish the sensitivity of the three assays for detecting ALV-J, we initially compared them with each of the three ALV-J ADOL prototype strains: Hc1, ADOL 6803 and ADOL 5701. The sensitivity of each assay is presented in Table 2. End points of 10⁻⁴ TCID₅₀ for ADOL 6803 and Hc1, and 10⁻⁶ TCID₅₀ for ADOL 5701 were determined by GSA ELISA on medium harvested on day 7 post-infection. IF was observed in dilutions that were positive, giving end points of 10⁻³ TCID₅₀. PCR tests performed with the two primer pairs, H5−H7 and R5–F5, were equally sensitive in detecting proviral DNA after 7 days in culture. The agarose gel of the PCR is shown in Figure 1.

Figure 1. Comparison of PCR detection of ALV-J DNA prepared from C/E cells inoculated with 10-fold dilutions (10⁻¹ to 10⁻⁷ noted 1 to 7) of three reference viruses, ADOL 6803, ADOL 5701 and Hc1, on day 7 post-infection. M, molecular weight (1 kb DNA ladder [# 15615-016; Gibco BRL USA]); –, negative control; 1018 bp and 517 bp, molecular weight of the marker in base pairs; ‘high’ and ‘low’, PCR product.

Table 2. Comparison of three methods to detect the ALV-J strains ADOL 6803, ADOL 5701 and Hc1 replication after 7 days in C/E cells

	GSA ELISA		Immunofluorescence				PCR
			Hc1	6803	5701	Hc1	H5–H7			R5–F5
Dilution	6803	5701					6803	5701	Hc1	6803	5701	Hc1
10^−1	NT^a	NT	NT	+	+	+	+	+	+	+	+	+
10^−2	NT	NT	NT	+	+	+	+	+	+	+	+	+
10^−3	+	+	+	+	+	+	+	+	+	+	+	+
10^−4	+	+	+	−	+	+	+	+	+	+	+	+
10^−5	−	+	−	−	+	−	−	+	+	−	+	+
10^−6	−	+	−	−	+	−	−	−	−	−	−	−
10^−7	−	−	−	−	−	−	−	−	−	−	−	−
^a Not tested.

For strain ADOL 6803, the ELISA and PCR tests were 10-fold more sensitive than IF in yielding an end point of 10⁻⁴ TCID₅₀. With ADOL 5701 both PCR tests were 10-fold less sensitive than IF and ELISA, giving an end point of 10⁻⁶ TCID₅₀, while for Hc1 the PCR tests detected virus at a dilution of 10⁻⁵ TCID₅₀ that was 10-fold higher than that detected by ELISA and IF.

Comparison of two PCR systems for detecting field isolates of ALV-J

The nine flocks that were positive by ELISA or by IF were further examined by PCR. The sizes of the PCR product with the R5–F5 primers were of the order of 1 kbp (denoted ‘+ low’ and ‘+ high’ in Table 3). Differences in the size of the products are related to differences in the length of the 3′ untranslated region (Fadly, 2000; Silva et al., 2000). Amplification revealed that, within individual flocks, the size of the PCR product with primers R5–F5 was uniform, whereas some differences in product size were observed between flocks. These findings are evidence for the clonality of the isolates from an individual flock, and also indicate the degree of heterogeneity of ALV-J strains circulating in Israeli flocks (Figure 2 and Table 3).

Figure 2. Comparison of field isolates with primers R5-F5. For identity of the isolates, see Table 3. For definitions, see caption for Figure 1.

Table 3. PCR of laboratory strains and proviral cDNA field isolates inoculated in C/E cells with the two sets of primers

		PCR				PCR
Sample number	Identity of the flock^a	H5–H7	R5–F5	Sample number	Identity of the flock^a	H5–H7	R5–F5
1	C/E cells	−	−	16	KY	−	−
2	Z	+	+high^b	17	G	−	−
3	Z	+	−	18	G	−	−
4	Z	−	−	19	G	−	−
5	Z	−	−	20	SB	+	+low
6	Z	+	+high	21	Ca	+	+high
7	Z	−	−	22	An	+	+high
8	Z	+	+high	23	An	+	+high
9	Z	−	−	24	An	+	+high
10	S	+	+low^b	25	An	+	+high
11	B	+	+high	26	An	+	+high
12	KM	+	−	27	ADOL Hc1	+	+high
13	KY	+	−	28	ADOL 6803	+	+low
14	KY	+	+high	29	ADOL 5701	+	+low
15	KY	+	−
^a Flocks Z, KY, G, Ya and SB are ML+, while flocks N, S, B and KM are ML−.
^b High and low refers to the size of the PCR product (greater or smaller than 1 kbp).

Comparison of detection of ALV-J antigen and antibody by ELISA, IF and PCR, respectively, in flocks with clinical ML and in flocks experiencing non-specific losses

The numbers of GSA-positive serum samples from the ML+ and non-ML flocks did not differ significantly (58.1% versus 46.4%), nor did the numbers of ALV-J antibody positive birds in each group (45.5% versus 24.1%, P=0.068) (Table 4).

Table 4. Comparison of antigenemia, and serum antibodies by direct GSA ELISA, IF and PCR in C/E cells, inoculated with sera from ML+ and ML− flocks

			Serum		Tissue culture
			ELISA				PCR
Flock	Age (weeks)	ML status^a	Ag	Ab	ELISA Ag	IF	H5–H7	R5–F5
Z	34	+	4/8	5/6	4/8	3/7	4/8	3/8
KY	30	+	4/6	3/6	3/4	0/2	3/4	1/4
G	18	+	3/13	10/13	4/8	NT^b	0/3	0/3
Ya	28	+	5/6	1/6	4/6	4/6	NT	NT
SB	14	+	9/10	1/13	10/13	9/12	10/13	10/13
Total (%)			25/43 (58.1)	20/44 (45.5)	25/39 (64.1)	16/27 (59.2)	17/28 (60.7)	14/28 (50)
N	50	−	4/5	3/6	2/7	2/7	NT	NT
S	38	−	4/8	1/8	1/8	1/8	1/8	1/8
B	6.5	−	4/8	0/8	1/8	NT	1/8	1/8
KM	36	−	1/7	3/7	1/7	1/7	1/7	0/7
Total (%)			13/28 (46.4)	7/29 (24.1)	5/30 (16.7)	4/22 (18.2)	3/23 (13)	2/23 (8.7)
^a Clinical and histopathological examination.
^b Not tested.

The presence of infectious virus in some of the surveyed flocks was established by IF and GSA production in cell culture, and in the others by PCR. A high level of viremia was detected in ML+ flocks with a 64.1% GSA-positive rate after 7 days in culture. This was significantly more than 16.7% of the ML− flocks (64.1% versus 16.7%; P<0.001). Of the isolates, approximately 60% were confirmed by PCR as ALV-J. The failure to detect all the isolates by PCR may be due either to the narrow specificity of the primers or the possibility that other ALV subgroups were circulating in these flocks. Also, both primer pairs yielded a statistically significant difference between the groups (P=0.001). Moreover, there was a close agreement between the ratios of positive ALV-J samples in the ML+ flocks as expressed by each of the three methods (PCR=50% to 60.7%, IF=59.2%, GSA=64.1%). In the ML− flocks, the absolute number of positive birds detected by each method was almost the same (PCR=two to three birds, IF=four birds, GSA=five birds).

Farm G

A flock of 18-week-old roosters was investigated further because it presented clinical signs of myeloid leukosis, but virus was not detected by PCR in cell culture (Table 3). The sera were tested directly by RT-PCR with primers H5–H7 and H5–AD1 (Figure 3 and Table 5). High antibody levels were recorded but only a relatively low antigenemia was detected. The RT-PCR yielded three positive ALV-J and seven positive ALV subgroup A to subgroup E samples. The ELISA detected GSA in CEF inoculated with four of the serum samples (all four were RT-PCR-positive with H5–AD1 and two were positive with H5–H7). None of the three tissue culture samples tested was ALV-J-positive by PCR.

Figure 3. RT-PCR of flock G serum amplified with primers H5–H7 and H5–AD1. Product of H5–H7, 545 base pairs; product of H5–AD1, 295 to 326 base pairs. For definitions, see caption for Figure 1.

Table 5. Broiler breeder rooster flock G

	Serum				Tissue culture
	ELISA		RT-PCR			PCR
Bird number	Ag	Ab	H5–H7	H5–AD1	ELISA Ag	H5–H7	R5–F5
1	−	+	−	+	−	NT	NT
2	−	+	+	+	−	NT	NT
3	−	+	−	+	−	NT	NT
4	−	−	−	−	−	NT	NT
5	+	−	−	+	+	−	−
6	+	+	+	+	+	−	−
7	−	+	−	+	+	NT	NT
8	−	+	NT^a	NT	NT	NT	NT
9	−	+	NT	NT	NT	NT	NT
10	−	−	NT	NT	NT	NT	NT
11	+	+	+	+	+	−	−
12	−	+	NT	NT	NT	NT	NT
13	−	+	NT	NT	NT	NT	NT
^a Not tested.

Discussion

In 1997, ML was recognized as an economic problem mainly due to high mortality in broiler breeder flocks in the Israeli poultry industry (Banet et al., 2000). Retrospective histopathological examination of archival tumour material, however, revealed that the disease was present as early as in 1995.

Having established the parameters of the ELISA, of IF and of PCR using three standard ALV-J isolates, and after demonstrating that each virus has its own replicative pattern, we proceeded to show that with minor exceptions these tests could be applied to detect ALV-J infection in commercial flocks. When considering the nature of the specificity of the immunological reagents employed in the ELISA kits for detecting gag antigenemia, however, no significant difference between the groups was detected reflecting the presence of endogenous (subgroup E) and possibly exogenous ALVs in these flocks. Hwang & Wang (2002) have recently compared differences in GSA levels in the serum of infected and non-infected ALV-J chickens by ELISA and concluded that, despite the existence of endogenous ALV in their flocks, they were able to demonstrate significant differences between the two groups at 1 and 6 weeks of age. Early sampling points may be useful in screening flocks using this assay provided that it is appreciated that undetectable GSA levels do not necessarily imply absence of ALVs.

The ALV group contains the most important naturally occurring oncogenic retroviruses of domesticated birds. Five subgroups are characterized as exogenous (subgroups A to D and subgroup J) while subgroup E is endogenous and non-oncogenic. Molecular characterization of HPRS-103, the prototype of subgroup J, shows that it has a characteristic ALV genomic structure with gag, pol and env genes. However, the env gene is distinct from the other ALVs by being closely related to that of novel endogenous retroviral elements designated EAV-HP. As the other regions of the genome are closely related to ALV, it is believed that subgroup J has evolved by recombination with env sequences of EAV-HP (Bai et al., 1995a,b). Recent data have shown that ALV-J isolates are evolving rapidly, as reflected by sequence changes within the variable regions of the env gene. Some of these changes are reflected as antigenic variants (Fadly & Smith, 1999) and provide a possible explanation for the prevalence of false-positive ELISA values in ML− flocks if EAV-HP is being expressed. Moreover, the failure of the commercial ALV-J antibody kit to reveal a marked difference (P=0.065) between the two groups of flocks may be due to factors associated with the isolates used to produce the coating antigen in the kit and to interpretation of the test readings (Hunt et al., 2000). In contrast, highly significant differences between the flocks could be demonstrated in the GSA assay of the C/E cells inoculated with viremic sera. These differences were paralleled by the higher incidence of immunofluorescent cytoplasmic staining of the duplicate-inoculated monolayers and also by PCR performed on proviral DNA extracted from these cells.

These laboratory findings emphasize the problems that may be encountered in confirming the diagnosis of ALV-J despite the presence of tumours on the inner face of the sternum of some birds, which are generally considered to be pathognomonic. Furthermore, our results indicate that a combination of diagnostic tests should be adopted in routine examination of tumour material in order to rule out false-negative findings. The strategy could be applied to advantage in all exogenous ALV eradication programs. In doing so, we have included the example of flock G where we were unable to unequivocally confirm the identification of ALV-J by PCR. We concluded that the serum antigen detected by ELISA may belong to another ALV subgroup that was detected also by RT-PCR or a possible double infection with ALV-J and another ALV subgroup. It is pertinent to recall that ML was recognized as a pathological entity long before ALV-J was characterized, although it was generally associated with individual birds rather than causing a flock problem (Campbell, 1969).

Acknowledgements

The authors wish to thank Dr A. Lublin for performing the statistical analysis. This project was supported by Research Grant Project No. IS-3006-98 from BARD, The United States–Israel Binational Research and Development Fund.