Differential diagnosis of avian oncogenic viruses

Irit Davidson

Division of Avian and Fish Diseases, Kimron Veterinary Institute, Bet Dagan P.O.Box 12, Israel 50250

The avian oncogenic viruses

Tumors in commercial poultry are caused mainly by infection with avian herpes and retroviruses. Oncogenic Marek’s disease virus (MDV), serotype 1 alphaherpesvirus, has been widely disseminated, in spite of the extensive vaccination procedures employing avirulent strains of MDV of the three serotypes (1, 2 and 3) (Calnek and Witter, 1997). MDV infects chickens and causes a malignant transformation of T cells, but lately, using molecular differential diagnosis (see below), it was described also as a causative virus of turkey tumors also, where the target cells for transformation is still not completely clear.

The chicken oncogenic retroviruses include reticuloendotheliosis (REV) (Witter, 1997)), lymphoid leukosis, subgroups A-I (ALV) (Payne and Fadly, 1997), and the recently described avian lymphoid leukosis virus, subgroup J, (Payne, 1998). The avian oncogenic retroviruses are exogenic, and transmitted as infectious virus, in contrast to the avian endogenic viruses that are transmitted genetically and have no clinical effects.

REV is an avian type-C retrovirus which transforms pre-B and pre-T lymphocytes, causing bursal and T-cell lymphomas in chickens and turkeys.

ALV is an ubiquitous chickens that transforms B lymphocytes and causes B-cell neoplasia.

ALV-J transforms myeloid cells of meat-type chickens and causes predominantly late-onset myelocytomatosis. However, additional knowledge on this relatively new virus is being added continuously, and new types of ALV-J tumors, such as erythroblastosis were reported (Venugopal et al., 2000).

Turkeys are susceptible to MDV, REV and lymphoprolipherative disease virus (LPDV) (Biggs, 1997).

LPDV is a C-type retrovirus of turkeys only whose target cell for transformation has not been determined yet.

MDV and the retroviruses, both transform lymphocytes, and ALV-J, according to presently available data, transforms myelocytes, but their tissue affinity is believed to be considerably broader. Except for the ALVs of similar subgroups, there is no interference for infection of the same cell by more than one virus and it is feasible that one flock, or even an individual bird, may commonly be infected with multiple oncogenic viruses. These viruses are also immunosuppresive, cause growth retardation and are a source of major financial damage to the poultry industry. Infection with one virus might biologically alter the clinical outcome of birds that are already infected by another oncogenic virus and consequently, that they might even have a more profound molecular interaction that could influence the final outcome of the disease. Further observations revealed the joint ability of MDV-1 and ALV to cause tumors and to increase mortality in experimentally dually-infected chickens.

Diagnosis

The clinical signs caused by infection with the five avian oncogenic viruses will not be detailed now, as they have been detailed extensively in conjunction with each virus. In general, the symptoms of various viral infections overlap and are of a low degree of pathognomy, such as the diagnosis based on clinical signs is often indefinitive, therefore specific laboratory diagnosis is needed.

The clinical diagnosis of REV- and MDV-induced lesions is problematic, since the location and appearance of the tumors can be similar; REV induced T-cell lymphomas are similar both macroscopically and microscopically to those caused by MDV, while REV induced B-cell lymphomas resemble those caused by ALV. In general, tumors and /or immunosuppresion and paralysis are common, but few characteristics to each viral-infection can be mentioned.

Very generally, the enlargement of the peripheral nerves and proventriculus denotes the MDV involvement; the presence of bursal tumors is characteristic to lymphoid leukosis; the presence of tumors in the pancreas and intestines suggest infection with REV, and the appearance of tumors in the bones is characteristic to infection with ALV-J.

Classical assays

The classical differential diagnosis is based on virus isolation, demonstration of specific antibodies and the histopatological examination of the tumor tissues.

1. Virus isolation

The virus isolation is carried out by co-cultivation of peripheral blood leukocytes or tumor cells in chick or duck embryo fibroblasts or chicken kidney cells. MDV has to be cultivated for at least one passage of cells that last about 7 days, but sometimes additional passages of the infected cells is required, since MDV is a strictly cell-associated virus that replicates relatively at a slow rate. REV and ALV are not cell-associated, therefore are secreted to the medium, and the passages are made with media that contain secreted virions, and/or cells disrupted by three consecutive freezing cycles. ALV has to be cultivated for 7 days, while REV requires 14 days. LPDV can not be cultivated at all in vitro.

MDV replication in tissue culture is associated with a change in the cell morphology, the long infected fibroblasts become round cells that gradually detach from the vessel. The formation of centered plaques composed of round cells is typical for MDV, but is more defined and intense for viruses that are adopted for cell culture replication, and of a less magnitude for viruses that are isolated from field cases. In those cases, and for a definite MDV identification, the infected tissue culture cells are fixed and reacted with specific antibodies (Immunofluorescence – IF) to detect the viral antigens in the infected culture. The IF detection of MDV antigens is more sensitive than the plaque formation in tissue cultures. ALV and REV do not cause changes in the infected cell morphology, therefore their identification is done by either IF of the infected cells, or by the detection of the viral antigens in the tissue culture media by ELISA.

The diagnosis based on virus isolation is laborious and time-consuming, and can even be complicated by the presence of serum antibodies that neutralize the virus. Moreover, for the detection of two or three different viruses in the same clinical material, two-stage cultivation and triple IF are required. Since MDV passages require intact cells whereas the retroviruses the virions are secreted, the identification of a multiple virus-infection might be missed during the serial passages, if the tissue cultured infected cells from one passage are not kept intact for additional passages.

2. Antibody demonstration

The diagnostic importance of MDV antibody demonstration is limited because common epitopes are shared by the glycoproteins of oncogenic MDVs (serotype 1) and the vaccine virus strains of the three serotypes (Malkinson et al., 1992). The MD vaccines are administered at day-old, thus antibodies are produced in commercial chicks, and the identification of MDV antibodies has no diagnostic value in vaccinated birds.

Unlike for MDV, the antibody demonstration is an effective mean to assign exposure to retroviruses, and ELISA assays are widely used to detect ALV and REV antibodies. However, because retroviruses disseminate both horizontally and vertically, and upon the vertical transmission the virus is transmitted to the hatched chick, therefore it is recognized as self, and viral-specific antibodies are not produced. That situation is recognized as a tolerant infection, during which, the diagnosis is not absolute, as the virus might persist without the presence of antibodies. Antibodies to the infecting virus are produced sometimes several months later, when the birds overcome the tolerant state.

Histopathology

Histopathological analysis of the tumors is a valuable supportive procedure, but because of the great variability in the infiltrative patterns it may not be pathognomic. However, a pleomorphic type of cellular infiltration composed of a mixed population of small to large lymphocytes are found in MD tumors. Two types of MD typical changes were observed in the nerves: a) demyelination and Schwan cell proliferation, and b) a diffuse, light to moderate infiltration of plasma cells and small lymphocytes. The two features appear in different nerves of the same bird and even in various areas of the same nerve. In cases of infection with REV and ALV the tumors are infiltrated with monomorphic cells, composed of homogenous lymphoblasts in the case of ALV-infection, T or B lymphocytes in the case of REV-infection and myeloid cells in the case of ALV-J-infection. While the cytological picture is typical in the case of MDV and ALV-J, there is no possibility to distinguish histologically between ALV and REV-infections.

In turkeys, visceral lesions composed of pleomorphic cells are caused by MDV or LPDV (Iancunescu et al., 1979), while the monomorphic type is predominant in REV infections (Witter, 1997).

The novel assay of immunohistochemistry has been introduced in recent years by Dr. R.L. Witter, by which fixed tissue sections are immunostained with viral-specific antibodies. That test require the use of monoclonal antibodies, that are not commercially distributed and a very high level of professional expertise. However, there are several limitations for the usefulness of that assay in routine diagnosis; MDV is present in the bird in a latent state and expresses a limited amount of viral antigens on the infected-cell membrane, so detection might be difficult. Also when the retroviral infection is accompanied by the exclusion of the retrovirus genome from the cellular genome, the expression of retroviral antigens on the infected-cell membranes is minimal or absent, and the immunohistochemistry assays are difficult.

Molecular differential diagnosis

In an attempt to provide a rapid and sensitive means of differential diagnosis we have applied the polymerase chain reaction (PCR) for MDV and REV (Davidson et al., 1995a), and added ALV to the diagnostic complex (Davidson et al., 1995b). Later on the LPDV PCR (Davidson et al., 1996) and ALV-J env PCR (Davidson et al., 1998a) were introduced to the routine differential diagnosis. The various PCR primers and amplicons were detailed in these reports.

The primers used to detect MDV sequences flank the 132 bp tandem repeat of the BamH1-H fragment, whose PCR product is specific for serotype 1 oncogenic MDV (Becker et al., 1993). The primers selected for the detection of REV (Aly et al., 1993), ALV (Chen and Barker, 1984; Habel et al., 1993) and LPDV (Davidson.I., unpublished) were based on the long terminal repeat (LTR) sequences of each virus. Since the retroviral LTR is the genomic fragment that is integrated in the cellular genome in the most stable fashion while other retroviral genomic fragments tend to be excluded, the amplification primers were directed to obtain LTR amplicons. Besides, the LTR fragment include regulatory genes which deregulate the infected-cell cycle of replication. However, to identify the ALV-J virus-infection, a env-directed amplification system is used (Smith et al., 1998), because the LTR of the ten ALV subgroups are close in their genomic sequences and co-amplified similarly, unlike the env genes that differ between ALV and AL:V-J viruses.

Additional non-neoplastic syndromes, such as paralysis are often encountered. The diagnosis of these cases have been accomplished by the amplification of brain DNA of paralysed birds. In most broiler cases a MDV infection was described by Davidson et al. (1998b), but in pullets of similar ages the cause of paralytic symptoms have not been solved yet. In several cases a ALV-J infection was also detected by PCR in the brain tissues. Moreover, recent studies demonstrate that very virulent MDV isolates cause early paralytic symptoms, prior to the appearance of neoplastic lesions (Witter et al., 1999; Gimeno et al., 1999).

The molecular differential diagnosis in practice

Chicken and turkey tumor-bearing flocks were surveyed over a period of seven years during which the viral cause of tumors was investigated molecularly (Davidson and Borenshtain, 2001). A total of 306 chicken and 59 turkey flocks were analysed, and these were the field veterinarians submissions, based upon clinical signs associated with oncogenic viral infections. About 3-6 birds from each flock were analysed. To obtain an optimal diagnosis it is recommended to sample the sick birds, however, the demonstration of viral sequences in at least one bird indicates that the entire flock may be infected, since the viruses are spread horizontally among the birds.

The differential diagnosis was determined on a cumulative basis of three tests, depending on the sample availability, and the results led to the flock classification according to the infecting virus. The three tests included the histopathology of the tumor tissue, ELISA, to measure REV antibodies and PCR of blood and tumor DNA. Chicken DNA was amplified separately with primers for MDV, REV, ALV-LTR (detecting subgroups A, C and J) and the ALV-J env gene, and turkey DNA with primers for MDV, REV and LPDV. We showed that 230/306 chicken flocks were infected with various oncogenic viruses; About 73% of the had a single virus infection, while 27% of the flocks had a multiple virus infection. A similar rate was also observed in turkeys; single virus infection was detected in 75% of the flocks, while multiple virus infection was detected in 25% of the flocks.

Several trends could be traced among the single virus infected flocks:

a) Both in chickens and in turkeys most flocks were MDV-infected, although a much higher rate was seen in turkeys: 36% vs. 66%, respectively;

b) REV-infection of chickens was prevalent in 16% of the flocks mostly until 1995, and sporadic afterwards;

c) ALV (subgroups A+C)-infection was sporadic over the entire period and prevailed in 6% of the flocks;

d) ALV-J-infection, that was analysed only from 1996, emerged during the years 1997-1998 in 16% of the flocks.

Molecular identification of a multiple oncogenic virus-infection

We were particularly interested in evaluating the incidence and the prevalence rate of flocks and birds that carry a multiple infection. It was apparent that about a quarter of the tumor-bearing commercial flocks carried a mixed MDV and retrovirus-infection. Although both viruses were found in such a remarkable rate of flocks, their presence in a bird or in a tumor does not necessarily reflect on the cause of a particular tumor, and the disease severity can not be predicted.

The major MDV virus-partner in mixed infected chicken flocks was REV (52%), while ALV (subgroups A+C) and ALV-J were similarly prevalent; 30% and 23%, respectively. Dual MDV and retrovirus-infections were detected in 22 flocks, in the case of REV, and in 9 and 13 flocks with ALV (A+C) and ALV-J, respectively. MDV was often accompanied by two retroviruses in mixed virus-infected flocks; 9 flocks had MDV+REV+ALV (A+C) and one flock had MDV+REV+ALV-J. Both REV and ALV (A+C) or ALV-J were the infecting viruses of 3 and 4 flocks, respectively.

Most multiple infected turkey flocks - 7/9 had a MDV+REV infection, and other two flocks had MDV+LPDV and MDV+REV+LPDV, each.

Summary

The PCR and the immunohistochemistry are novel emerging diagnostic assays that are potent to evaluate viral infections on the background of multiple virus infections.

The PCR appears to be the method of choice for the diagnosis of avian oncogenic viruses as it overcomes various difficulties encountered by the use of the classical diagnostic assays, on one hand, and enables the detection of multiple viral infections, on the other.

Some drawbacks for the use of the classical diagnostic assays (virus isolation and antibody detection is the presence of antibodies that might interfere with the retrovirus isolation in tissue culture because of the formation of complexes.

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