Screening for Avian Leukosis Virus Subgroup-J

Summary

The objective of this document is to clarify how to use the FlockChek* Avian Leukosis Virus Subgroup J Antibody Test Kit (IDEXX ALV-J) and the interpretation of results obtained under monitoring programs for parent stocks or broiler breeders. The following are key points to consider when establishing a program to eliminate, eradicate or determine the presence of avian leukosis virus subgroup J (ALV-J) when using the IDEXX ALV-J Test Kit:

Interpretation of results:

ELISA should be used as a screening tool. As with any screening tool, the focus is on sensitivity, so some level of nonspecific reactors can be expected. Any positive result with this test must be followed by a more specific test, such as virus isolation, with samples from the same flock of birds that showed positive ELISA results.

• A positive result on the ALV-J antibody test kit does not necessarily mean that there is virus in the flock. For clinical ALV-J disease to be present in a flock, a replicating exogenous ALV-J virus must be present in the population.
• The ALV-J antibody test kit has been developed as a flock-screening tool. ALV-J status for individual birds cannot be assessed.
• The ALV-J status of a flock can only be determined by testing for replicating exogenous virus through virus isolation in C/E chick embryo fibroblasts with detection on the ALV antigen test. Virus isolation is considered the gold standard for detection of exogenous virus. If cloacal swabs, albumin and meconium are tested for ALV p27 antigen, a low level of endogenous viral protein may be detected.
  (Note: the IDEXX ALV antigen test kit only has a claim for cloacal swabs and albumin.)
• ALV-J seroconversion is variable across lines and may be influenced by endogenous leukosis virus expression. Testing of meat-type birds less than 10 weeks of age is not recommended.
• Vertical transmission of ALV-J may result in seronegative, immune tolerant progeny.

Important: ALV antigen test kits (p27) do not discriminate between endogenous viral proteins and exogenous virus (such as ALV-J). Customers should not use serum on ALV antigen test kits; serum samples can have significant amounts of endogenous ALV antigen.

Characteristics, performance and recommended use

Introduction

ALV-J was isolated in the late 1980s and designated as a unique subgroup partly based on the heterogeneity of the envelope glycoprotein as compared to the other ALV subgroup envelope proteins (Payne et al,1991; Bai et al,1995). Diagnostics used in the eradication of ALV-J have focused on virus isolation methods or direct measurement of the virus in cloacal swabs, meconium or albumin. All of these methods detect group-specific antigen (p27). The basis of the IDEXX ALV-J Antibody Test Kit is the detection of antibody against the gp85 envelope protein of ALV-J. The solid-phase antigen used in the test is recombinant gp85 antigen from ALV-J.

Materials and Methods

The ALV-J antibody ELISA is an indirect format test with gp85 antigen coated on the solid phase and a goat anti-chicken HRPO conjugate. Samples are diluted 1:500 and then incubated on the plate for 30 minutes. After a PBS/Tween wash, the conjugate is added to the plate for an additional 30-minute incubation. Conjugate is washed from the wells and substrate is added. Sample and control ODs are used to calculate the sample/positive (S/P) values. S/P values >0.6 are considered positive.

Samples used to characterize the ELISA were obtained from different laboratories in which active virus isolation programs for ALV-J were ongoing. This included multiple commercial and government facilities. Dr. Aly Fadly (USDA/Avian Disease and Oncology Laboratory, East Lansing, MI) provided antisera against ALV subgroups A, B, C, D, E and J. Additional ALV-A antisera were provided by Dr. Mike Opitz (University of Maine, Orono, ME). Monoclonal antibody FE.9 was provided by Dr. K. Venugopal (Institute for Animal Health, Compton UK).

Results

ALV-J Antibody ELISA: To assess potential cross-reactivity with antisera against other pathogens, the USDA/NVSL Avian Specificity Panel was tested on the IDEXX ELISA. This panel includes antisera against most common poultry pathogens, as well as some mock vaccine antisera. All samples tested negative on the ALV-J antibdy ELISA (Table 1 and 2). Specificity of the test on an SPF population (Figure 1) was 100% (mean S/P= 0.037+0.073), while a field population of samples from virus isolation-negative flocks (Figure 2) had a specificity of 99.7% (mean S/P=0.055+0.100).
Note: Some individual samples yielded S/P results significantly > 0.6.

Table 1.				Table 2.
Sample number	Antisera ID	S/P	Result		Sample number	Antisera ID	S/P	Result
1	REV	0.00	Negative		13	IBV-Ark	0.04	Negative
2	REO	0.00	Negative		14	IBV-Conn	0.01	Negative
3	LLA	0.00	Negative		15	IBV-Mass	0.00	Negative
4	MG	0.00	Negative		16	IBV-JMK	0.00	Negative
5	MS	0.01	Negative		17	MOCK VAC cef C	0.06	Negative
6	CAA	0.00	Negative		18	MOCK VAC cef B	0.05	Negative
7	ILT	0.00	Negative		19	MOCK VAC cef A	0.17	Negative
8	IBD	0.00	Negative		20	MOCK VAC CH. EMB	0.00	Negative
9	HVT	0.00	Negative		21	MOCK VAC AAF	0.00	Negative
10	AE	0.00	Negative		22	CELO	0.00	Negative
11	NDV-C	0.00	Negative		23	FOWL POX	0.00	Negative
12	SB-1	0.00	Negative		24	SPF NEG	0.00	Negative

In a naturally infected ALV-J flock, (virus isolation-positive at 12 weeks of age) significant seroconversion was not seen before 14–18 weeks of age (Figure 3). A review of sixteen virus isolation-positive flocks, with virus isolation percentages between 1–40% (VI at 10–16 weeks of age), showed seroconversion percentages of 0–72% (Figure 4). Variable seroconversion levels may arise from differences in viral shedding, age of the flock when exposed to the virus and expression of endogenous leukosis sequences.

Studies conducted at IDEXX Laboratories; data on file.

Discussion

In developing an antibody test for ALV-J, the gp85 envelope protein was the best candidate antigen because of the significant sequence difference between the ALV-J gp85 protein as compared to the other ALV subgroups. Unlike the p27 antigen, in which there is a high degree of sequence homology between subgroups, the ALV-J gp85 protein shares only ~40% sequence homology with the other gp85 antigens (Bai et al,1995). Antisera against the other ALV subgroups do not cross-react on the IDEXX ALV-J antibody ELISA. Conversely, ALV-J antisera is not detected by the IDEXX ALV subgroup A & B antibody ELISA (data not shown).

While the ALV-J antibody ELISA is based on an antigen of UK origin, we have previously demonstrated reactivity with Hc1 antisera and antisera against other U.S. origin isolates. (Myrick et al,1999). Additionally, the recombinant gp85 used in this test was detected on immunoblots by monoclonals derived from both U.S. and UK origin ALV-J isolates. The overall specificity of the ALV-J antibody test is theoretically greater than 99%. However, there have been some reports of virus isolation-negative flocks in which >20% of the flock has shown reactivity on the test. These reactors are being investigated and there is some evidence that they may be associated with expression of endogenous viral proteins.

Interpretation of Results: The ALV-J antibody ELISA was developed as a flock-screening tool; it should not be used to monitor individual birds. Seroconversion can be very slow in lines that have endogenous ALV (Smith et al, 1990); testing on the ALV-J antibody ELISA is not recommended before 12–14 weeks. Flock status must be confirmed with virus isolation. Vertical transmission can lead to immune tolerant, seronegative progeny.

References

Bai J, Howes K, Payne LN, Skinner MA. Sequence of host-range determinants in the env gene of a full-length, infectious proviral clone of exogenous avian leukosis virus HPRS-103 confirms that it represents a new subgroup (designated J). J. Gen Virol. 1995;76:181–187.

Myrick B, Jackson T, Leathers V, Fadly A. Evaluation of an antibody test specific for subgroup J avian leukosis virus. Presented at: American Association of Avian Pathologists/AVMA conference. 1999.

Payne LN, Brown SR, Brumstead N, Howes K, Frazier JA, Thouless ME. A novel subgroup of exogenous avian leukosis virus in chickens. J. Gen.Virol. 1991;72:801–807.

Smith EJ, Fadly AM, Crittendeb LB. Interactions between endogenous virus loci in ev6 and ev21. 1. Immune response to exogenous avian leukosis virus infection. Poultry Science. 1990;69:1244–1250.

Venugopal K, Howes K, Barron GS, Payne LN. Recombinant env-gp85 of HPRS-103 (subgroup J) avian leukosis virus: antigenic characteristics and usefulness as a diagnostic reagent. Avian Diseases. 1994;41(2):283–288.

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