Abstract
Several novel criteria have been tested to assist in the differential diagnosis of tumours induced by Marek's disease virus (MDV) from those induced by avian leukosis virus (ALV) and reticuloendotheliosis virus (REV). A collection of tumours induced by inoculation of specific strains of MDV, ALV and REV, alone or in combination, were tested for quantification of MDV DNA by real-time polymerase chain reaction, expression of the MDV oncogene Meq, expression of several cell markers associated with transformation (CD30, Marek's disease-associated surface antigen, and p53), and level of DNA methylation in the tumour cells. In addition, tissues latently infected with MDV and non-infected tissues were tested as controls. Tumours induced by MDV had about 102-fold more copies of MDV DNA than either tissues latently infected by MDV or tumours induced by retrovirus in MDV-vaccinated chickens. Moreover, the MDV antigen Meq was consistently expressed in all MDV tumours but it could not be detected in tissues latently infected with MDV or in tumours induced by retrovirus in MDV-vaccinated chickens. Other markers studied were not specific for MDV and therefore had limited value for diagnosis. Nonetheless, some of these markers might have potential value in research as they will help to identify transformed cells.
Nouveau critère pour le diagnostic des lymphomes induits par le virus de la maladie de Marek
Plusieurs nouveaux critères ont été testés pour aider à réaliser le diagnostic différentiel des tumeurs induites par le virus de la maladie de Marek (MDV) de celles induites par le virus de la leucose aviaire (ALV) et celui de la réticulo-endothéliose (REV). A partir de tumeurs induites par inoculation de souches spécifiques de MDV, ALV et REV, seules ou associées, il a été testé la quantité d'ADN de MDV par PCR en temps réel, l'expression de l'oncogène Meq de MDV, l'expression de différents marqueurs associés à la transformation (le CD30, l'antigène de surface associé à la maladie de Marek ou MATSA, et le p53), et le niveau de méthylation de l'ADN dans les cellules des tumeurs. De plus, les tissus infectés latents par le MDV et les tissus non infectés ont été testés comme témoins. Les tumeurs induites par le MDV avaient environ 102 fois plus de copies d'ADN de MDV que les autres tissus infectés latents par le MDV, ou que les tumeurs induites par le rétrovirus chez les animaux vaccinés MDV. En outre, l'antigène Meq du MDV a été exprimé de façon attendue au niveau de toutes les tumeurs dues au MDV mais il n'a pas pu être détecté dans les tissus infectés latents par le MDV ou dans les tumeurs induites par le rétrovirus chez les animaux vaccinés MDV. Les autres marqueurs étudiés n'ont pas été spécifiques du MDV et par conséquent présentaient une valeur limitée pour le diagnostic. Néanmoins, certains de ces marqueurs pourraient avoir une valeur potentielle en recherche du fait qu'ils aident à identifier les cellules transformées.
Neue Kriterien für die Diagnose von durch das Virus der Marekschen Krankheit induzierten Lymphomen
Zur Verbesserung der Differentialdiagnose zwischen Tumoren, die durch Marekvirus (MKV) induziert wurden, von den durch aviäres Leukosevirus (ALV) oder Retikuloendotheliosevirus (REV) verursachten wurden mehrere neue Kriterien getestet. Eine Reihe von Tumoren, die durch alleinige oder kombinierte Inokulation von spezifischen MDV-, ALV- und REV-Stämmen induziert worden waren, wurden hinsichtlich der Quantifizierung von MKV-DNS mittels Real Time-PCR, der Expression des MKV-Onkogens Meq, der Expression verschiedener mit Transformation assoziierterter Zellmarker (CD30, Marek-assoziiertes Oberflächenantigen oder MATSA und p53) und des Grades der DNS-Methylierung in den Tumorzellen untersucht. Als Kontrollen dienten latent mit MKV infizierte und nicht infizierte Gewebeproben. Durch MKV induzierte Tumoren wiesen 102 mehr MKV-DNS-Kopien auf als latent infizierte Gewebe oder durch Retroviren induzierte Tumoren in mit MKV geimpften Hühnern. Darüber hinaus wurde das MKV-Antigen Meq einheitlich in allen MKV-Tumoren exprimiert, konnte aber nicht in latent mit MKV infizierten Geweben oder in durch Retroviren induzierte Tumoren in mit MKV geimpften Hühnern nachgewiesen werden. Die anderen untersuchten Marker erwiesen sich als nicht spezifisch für MKV und haben deshalb nur begrenzten Wert für die Diagnose. Nichtsdestoweniger haben einige dieser Marker potentielle Bedeutung für die Forschung, da sie bei der Erkennung tranformierter Zellen hilfreich sind.
Nuevo criterio para el diagnóstico de los linfomas inducidos por virus de la enfermedad de Marek
Se han evaluado varios nuevos criterios para ayudar en el diagnóstico diferencial de los tumores inducidos por el virus de la enfermedad de Marek (MDV) frente a los inducidos por virus de la leucosis aviar (ALV) y virus de la reticuloendoteliosis (REV). Se evaluó una colección de tumores inducidos por inoculación de cepas específicas de MDV, ALV y REV, solos o en combinación, para la cuantificación de ADN de MDV por PCR a tiempo real, expresión del encogen Meq del MDV, expresión de varios marcadores celulares asociados con transformación (CD30, Marek's disease-associated surface antigen o MATSA, y p53), y nivel de metilación del ADN en células tumorales. Además, se evaluaron como controles, tejidos infectados de forma latente con MDV y tejidos no infectados. Los tumores inducidos por MDV contenían sobre 102 veces más copias de ADN de MDV que los tejidos infectados de forma latente con MDV o tumores inducidos por retrovirus en pollos vacunados con MDV. Además, el antígeno Meq del MDV se expresó de forma consistente en todos los tumores inducidos por MDV pero no pudo ser detectado en tejidos infectados de forma latente con MDV o en tumores inducidos por retrovirus en pollos vacunados con MDV. Otros marcadores estudiados no fueron específicos para MDV y tuvieron, pues, un valor limitado para el diagnóstico. A pesar de ello, algunos de estos marcadores podrían potencialmente tener valor en investigación, para identificar células transformadas.
Introduction
Lymphomas in poultry are common and have economic relevance due to associated mortality and condemnations. Most of the lymphomas in poultry have a viral aetiology and result from infection with Marek's disease virus (MDV), avian leukosis virus (ALV) or reticuloendotheliosis virus (REV). An accurate diagnosis is very important as control measures differ depending on the aetiology of the lymphomas. In some cases, a specific diagnosis is needed to determine liability for losses.
Differential diagnosis of poultry lymphomas is still an unresolved problem. Critical points and currently available techniques have been recently reviewed (Witter et al., 2004). There are three major difficulties in the diagnosis of poultry lymphomas. First, lesions induced by the three viruses are very similar and, although some differences in lymphoid tropism are recognized, there are not truly pathognomonic lesions for any of the three diseases. Second, MDV, ALV and REV are ubiquitous and infection is not synonymous with disease. Most infected chickens will never develop tumours and, in the case of ALV-induced or REV-induced tumours, the infectious form of the causative virus might disappear. Finally, Marek's disease (MD) tumours are composed of a mixture of transformed and inflammatory cells, and in some instances chronic inflammatory lesions could be confused with MD lymphomas.
A variety of criteria are commonly used in the diagnosis of poultry lymphomas. Epidemiological criteria, mainly the age of affected chickens, can aid in the diagnosis since MD lymphomas tend to appear earlier than lymphomas induced by retroviruses. Although true in some cases, the age criterion is not absolute because MD lymphomas can appear also in older chickens, and non-bursal lymphomas induced by REV can appear as early as 6 weeks of age (Witter et al., 1986). In most cases, the diagnosis relies on pathological criteria, especially distribution of the lesions and histological characteristics of the tumour cells (Siccardi & Burmester, 1970). However, pathological criteria are not always adequate. Tumours in the bursa with exactly the same characteristics can be induced by both ALV and REV. Nerve lesions can be induced not only by MDV, but also by REV. In addition, there are a variety of other pathological phenomena that have emerged in the past decades and complicate the diagnosis—that is, peripheral neuropathy (Bacon et al., 2001), multicentric histiocytosis (Hafner & Goodwin, 2003), myeloid tumours (Fadly & Payne, 2003).
Tumour cell type and molecular characterization of integrated retrovirus are useful criteria for diagnosis, although not widely used (Neumann & Witter, 1979). Characterization of the cell type (B cells or T cells) will help to differentiate MD lymphomas from ALV-induced lymphomas (lymphoid leukosis [LL]) and bursal lymphomas induced by REV (RE bursal lymphomas) (Witter et al., 2004). However, this technique does not allow one to differentiate between MDV-induced tumours and non-bursal lymphomas induced by REV (RE non-bursal lymphomas) or between LL and RE bursal lymphomas. Also, characterization of the cell type in the lesions will not differentiate between peripheral neuropathy and MDV-induced type B lesions in the nerves (Payne & Biggs, 1967).
Detection of clonal insertion and c-myc alterations by molecular techniques is the best way to discriminate between LL and RE bursal lymphomas (Ewert, 1997; Fadly & Payne, 2003). Both tumours are initiated by insertion of the viral long terminal repeat (LTR) sequences into the c-myc gene, resulting in dysregulation of cellular replication. Single transformed cells clonally expand to form the respective tumour. Southern blotting techniques can also aid to discriminate between retroviral tumours and MD tumours.
The aim of this study was to identify new criteria that may assist in the diagnosis of MDV-induced lymphomas. A collection of tumours induced by inoculation of specific strains of MDV, ALV and REV, alone or in combination, were tested for quantification of MDV viral DNA by real-time polymerase chain reaction (PCR), expression of the viral oncogene Meq, expression of several cell markers associated with transformation (CD30, Marek's disease-associated surface antigen [MATSA], and p53), and level of DNA methylation in the tumour cells.
Materials and Methods
Chickens
All chickens were from the Avian Disease and Oncology Laboratory breeding flock, which is maintained in isolation and is free of common poultry pathogens including REV, ALV and MDV on the basis of periodic serologic tests. Chickens used were from unvaccinated breeder hens, free of antibodies to MDV and turkey herpesvirus. Line 0 (Crittenden et al., 1984) and a cross consisting of progeny of line 15I5 males and line 71 females (15×7) were used. Line 0 was used to produce RE non-bursal lymphomas subsequent to inoculation of the spleen necrosis virus (SNV) strain of REV (Witter et al., 1986). Line 0 is moderately sensitive to the development of MD lymphomas and LL, but when inoculated with strain SNV develop a high frequency of RE non-bursal lymphomas (Witter et al., 1986). Cross 15×7 is very susceptible to develop MD lymphomas and LL, and when inoculated with strain SNV to develop a high frequency of RE bursal lymphomas (Witter et al., 1986). Cross 15×7 was used to produce MD lymphomas, LL, RE bursal lymphomas, MDV latently infected tissues and uninfected tissues.
Viruses
Oncogenic MDV strains GA (virulent MDV) at passage 18 (Eidson & Schmittle, 1968), RB1B (very virulent MDV) at passage 23 (Schat et al., 1982), Md5 (very virulent MDV) at passage 7 (Witter et al., 1980) and 648A (very virulent plus MDV) at passage 8 (Witter, 1997) were used to induce MD lymphomas or to obtain MDV latently infected tissues. Attenuated serotype 1 MDV strains R2/23 at passage 106 (Witter, 1991) and CVI/988 at passage 42 (Rispens et al., 1972), non-oncogenic serotype 2 strain SB-1 at passage 12 (Schat & Calnek, 1978b) and non-oncogenic serotype 3 strain FC126 at passage 10 (Witter et al., 1970) were used to obtain MDV latently infected tissues. SNV strain (Trager, 1959) was used to induce either bursal lymphomas (cross 15×7 chickens) or non-bursal lymphomas (line 0 chickens). Strain RPL-40, an ALV of the subgroup A, was used to induce LL (Okazaki et al., 1982). Oncogenic MDVs were used at a dose of 500 plaque-forming units (PFU), with the exception of RB1B that was used at a dose of 2000 PFU. Non-oncogenic MDV vaccines (R2/23, SB-1 and FC126) were used at a dose of 2000 PFU. SNV and RPL-40 were used at a dose of 104 tissue culture infectious dose 50, median. Inoculation was generally conducted at hatch with the exception of chickens that were MDV vaccinated at hatch and challenged with an oncogenic MDV at 6 days of age. Inoculations were done via a subcutaneous route.
Activation of the immune system
Sheep Blood Alsevers Solution was obtained from Baltimore Biological Laboratories (Cockeysville, Baltimore, Maryland, USA). Before use, the erythrocytes were washed three times with about 15 volumes of phosphate-buffered saline. After the final wash, the sheep red blood cells (SRBC) were suspended in a solution of phosphate-buffered saline mixed with Freund's complete adjuvant (FCA) (Becton Dickinson-Baltimore Biological Laboratories, Sparks, Maryland, USA) in a dilution 1:1 as described elsewhere (Bekierkunst et al., 1971). Each chicken was inoculated with 0.5 ml solution via the intramuscular route that contained 2×109 red blood cells.
Samples
All samples used in this study were generated by conducting a series of animal experiments in which the infection status of the birds was controlled. Several experiments were designed to generate four groups of samples: MD lymphomas, tissues latently infected by MDV, retroviral lymphomas, and uninfected tissues (Table 1). Chickens were kept in Horsfall Bauer units. The strain of chickens, age at inoculation, dose and strain of inoculum, and time of sample collection were carefully designed to obtain a particular group of samples.
Table 1. Classification of the samples used in the studya
MD lymphomas were generated by inoculating an oncogenic MDV strain in 15×7 chickens. Samples in this group include lymphomas in viscera or in nerves detected by gross inspection and confirmed by microscopic evaluation. There were two subgroups of samples within MD lymphomas. Tumours induced by an oncogenic MDV (GA, RB1B, Md5 or 648A) in chickens that had not been exposed to any other virus (MD single infection [MDS]) and tumours induced by the oncogenic MDV strain RB1B in chickens that had been infected at hatch with the ALV strain RPL40 and with the REV strain SNV (MD multiple infections [MDM]). MDS tumours were collected between 5 and 10 weeks post inoculation (w.p.i.). MDM tumours were collected at 5 w.p.i., an age when LL or REV bursal lymphomas should not be present (because they develop at older ages) and non-bursal lymphomas induced by SNV should be rare (because 15×7 chickens are relatively resistant) (Witter et al., 1986).
Tissues latently infected by MDV consisted of the spleen, bursa of Fabricius and thymus of MDV-vaccinated 15×7 chickens (MDV vaccinated [MDVVAC]) or the spleen, bursa of Fabricius and thymus of MDV-vaccinated 15×7 chickens that were also challenged with an oncogenic MDV (MDV vaccinated and challenged [MDVVAC + CHAL]). MDVVAC + CHAL tissues were obtained using a careful combination of vaccines and oncogenic MDV that provide at least 95% protection against development of tumours. Only tissues of chickens that did not develop any evident lesion by gross inspection and histopathology were included in the study. MDVVAC tissues were obtained by inoculating R2/23, SB-1 or FC-126. MDVVAC + CHAL tissues were obtained by any of the following treatments: vaccination at hatch with FC-126 and challenge 5 days later with GA; vaccination at hatch with a combination of FC-126 and SB-1 and challenge 5 days later with Md5; vaccination at hatch with CVI/988 and challenged 5 days later with 648A. Tissues of MDVVAC and MDVVAC + C were collected at 5 to 6 w.p.i.
Retroviral lymphomas include three subgroups of samples: lymphomas induced by the ALV strain RPL40 in 15×7 chickens (LL) that were collected 14 to 24 w.p.i.; tumours induced by SNV in 15×7 chickens (RE bursal lymphomas [REB]) that were collected at 19 to 26 w.p.i.; and tumours induced by SNV in line 0 chickens (non-bursal lymphomas [RENB]) that were collected at 7 to 17 w.p.i. All chickens inoculated with either RPL40 or SNV were also vaccinated at hatch with R2/23.
For uninfected tissues consisted of the spleen, bursa of Fabricius and thymus of uninfected chickens, there were two subgroups of non-infected tissues: tissues from chickens that had not received any treatment (control non-activated [CAN]); and tissues from chickens that had been treated with FCA and SRBC at 2 weeks of age (control activated [CA]). Tissues from the CNA and CA groups were collected at 6 weeks of age.
Fresh tissues and tumours were collected and snap frozen in liquid nitrogen. Samples were stored at −70°C until they were processed.
Real-time PCR
DNA was extracted from spleen and tumour homogenates using a Puregene™ DNA Isolation Kit (Gentra System, Minneapolis, Minnesota, USA), and each sample was amplified with primers specific for the glycoprotein B (gB) gene of MDV as previously described (Gimeno et al., 2004) as well as with primers specific for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of the chicken. Briefly, the respective forward and reverse primers were TM.5 (5′-CGGTGGCTTTTCTAGGTTCG-3′) and TM.3 (5′-CCAGTGGGTTCAACCGTGA-3′) for amplification of the gB gene, and GAPDH-TM.5 (5′-GGAGTCAACGGATTTGGCC-3′) and GAPDH-TM.3 (5′-TTTGCCAGAGAGGACGGC-3′) for amplification of the GAPDH gene. Amplifications were carried out using the GeneAmp®5700 (Applied Biosystems, Foster City, California, USA) in a 25 µl PCR reaction containing 50 ng DNA, 0.2 µM each primer and SYBR® green PCR master mix that contains the appropriate buffers, nucleotides and Taq-polymerase (Applied Biosystem, Warrington, UK). The reaction was cycled 40 times with 95°C denaturation for 15 sec and a 60°C combined annealing/extension for 60 sec. Fluorescence was acquired at the end of the annealing/extension phase. The melting curves were obtained at the end of amplification by cooling the sample at 20°C/sec to 60°C and then increasing the temperature to 95°C at 0.1°C/sec.
The parameter Ct (the threshold cycle) was calculated for each PCR reaction by establishing a fixed threshold. Ct is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. Relative quantification of the amount of target in unknown samples was accomplished by two methods: the use of a standard curve and the comparative Ct method (DDCT). Standards used in this study were serial dilutions (six 10-fold dilutions starting at 100 000 copies) of the cosmid SN16 (Reddy et al., 2002) that includes the complete sequence of the serotype 1 MDV gene gB. The use of standards allows determining the number of copies per 50 ng or total DNA and results are expressed as the log of the number of viral DNA copies in 50 ng total DNA. This technique detects 100 copies of MDV as calculated using the standard curve. The DDCT method of quantification eliminates the need for standard curves when looking at relative number of copies of a target (gB gene) relative to a reference control (GAPDH). The Ct ratio value was established for each sample (Ct ratio = Ct gB gene/Ct GAPDH gene).
Immunohistochemistry
An avidin–biotin–peroxidase complex (Vectastain® ABC kit; Vector Laboratories, Burlingame, California, USA) was used for immunohistochemistry as described elsewhere (Gimeno et al., 2001). In particular, the immunohistochemistry staining of the antigens Meq and CD30 were amplified with the tyramide signal reaction following the manufacturer's instruction for the TSA™ Biotin System Kit (PerkinElmer Life Science, Boston, Massachusetts, USA). The monoclonal antibody (mAb) 23B46 (Liu et al., 1996), specific to MDV Meq, was used at a working dilution of 1:1000. The mAb H19.47 (Cui et al., 1990), which is specific for MD phosphoprotein pp38, was used at a working dilution of 1:3200. The mAb AV37 (Burgess et al., 2001), which is specific to the chicken CD30 antigen, was used at a working dilution of 1:25. The mAb 14B367 (Lee, unpublished data) was used at a working dilution of 1:2000. The mAb 14B367 is specific to MATSA as demonstrated by immunoprecipitation with MSB-1 cells (Lee, personal communication). The mAb PAb 240, which is specific to mouse p53 but has cross-reaction with chicken p53 (Gannon et al., 1990), was used at a working dilution of 1:25 (BD Biosciences Pharmigen, San Diego, California, USA). The mAb 5-methylcytosine (Ab-1), which is specific to 5-methylcytosine, was used at a working dilution of 1:25 (WWR International Oncogene Research Product, San Diego, California, USA). Detection of 5-methylcytosine can be used as an indicator of the total level of methylation in the cells and is decreased in most of the human tumours (Esteller, 2002).
Statistics
Data were analysed with the statistical program Statistica (Stat Soft, Tulsa, Oklahoma, USA). Comparison among groups was conducted by an analysis of variance test. In all cases, Scheffe test was used as a post hoc analysis. The level of significance considered was P<0.05.
Results
Load of MDV DNA evaluated by real-time PCR
The number of copies of MDV DNA was evaluated in 219 samples obtained from 157 chickens representative of four groups (MD lymphomas, tissues latently infected by MDV, retroviral lymphomas in MDV-vaccinated chickens, and uninfected tissues) and results are presented in Table 2. All MD tumours (MDS and MDM) were consistently positive and had high number of MDV copies (at least 103.2). No statistically significant differences were found between MDS and MDM tumours in MDV DNA copy numbers or in Ct ratios. Also, differences observed between MD tumours within the same birds and MD tumours from different chickens were not statistically significant. Within the group of MDVVAC tissues, only tissues of chickens inoculated with serotype 1 MDV strain R2/23 were positive as the technique was specific for serotype 1 MDV. Positive MDVVAC tissues had very low number of copies (up to 101.1). All MDVVAC + CHAL were positive but had low number of copies (up to 101.2). None of the retroviral tumours was found to be positive with this technique and the uninfected tissues were in all cases negative. No statistically significant differences were found between MD lymphomas of the subgroups MDS and MDM. Differences, however, were statistically significant between MD lymphomas and the other groups. MD lymphomas (MDS and MDM) had about 102-fold more copies of MDV DNA (103.2 to 104.3) than either tissues latently infected by MDV (100.8 to 101.2). MDV DNA was not detected in retroviral lymphomas in chickens vaccinated with R2/23 or in uninfected tissues of the subgroups CA and CAN.
Table 2. Quantification of MDV DNA in lymphomas, MDV latently infected spleens and uninfected spleens measured by real-time PCR
Statistically significant differences were also found in the Ct ratios between MD lymphomas (0.5 to 0.7) and the other groups of samples (0.9 to 1.1). Although there were no statistically significant differences within the MDS and MDM subgroups, there was some variability based on the nature of the infiltrates. Infiltrative lymphomas that included abundant parenchymal cells and enlarged nerves showing B-type lesions (characterized by marked oedema and infiltration of plasma cells) had the lower MDV DNA load (103.2 to 103.4) and the higher Ct ratios (0.7) (data not shown).
Expression of the MDV viral antigen pp38 in the MDV-induced tumours was inconsistent and was not related to the number of MDV DNA copies detected in the tumour or to the Ct ratios (Table 3). Undetectable levels of pp38 expression were found in tumours that had high number of MDV DNA copies (103.4) and low Ct ratios (0.6). On the other hand, high level of pp38 expression (2 + ) was not accompanied with statistically significant increase in the number of MDV DNA copies or in the Ct ratios. Expression of the MDV viral antigen Meq in the MDV-induced tumours, however, was consistently high and was related to the number of MDV DNA copies detected in the tumour or to the Ct ratios.
Table 3. Expression of pp38 and Meq, number of copies of MDV DNA, and Ct ratio in nine MD lymphomas (MDS and MDM subgroups)
Expression of Meq, MATSA, CD30, p53 and methyl 3-cytosine
The MDV antigen Meq was consistently expressed in MD tumours (MDS and MDM) (Table 4). Level of expression in all lymphomas was very high and about 60% to 80% of the tumour cells were positive (Figure 1a,b). Within the subgroups MDS and MDM, expression of Meq was very similar in visceral tumours (Figure 1a) and in enlarged nerves with type A lesions (typically neoplastic lesions) (Figure 1b) but it was significantly lower in enlarged nerves with type B lesions (lesions characterized by abundant oedema and infiltration of plasma cells) (Figure 1d) (Payne & Biggs, 1967). No expression of Meq was detected in MDV latently infected tissues (MDVVAC and MDVVAC + CHAL), in tumours induced by retrovirus in MDV-vaccinated chickens (LL, REB and RENB) (Figure 1c), or in uninfected tissues (CA and CAN).
Published online:
18 January 2007Figure 1. Expression of the oncogene Meq in a variety of tissues: (1a) MD tumour in a liver, (1b) MD tumour in a nerve with type A lesions, (1c) REV non-bursal tumour in a liver, and (1d) MD tumour in a nerve with type B lesions. Bar = 35 µm.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2005/cavp20.v034.i04/03079450500179715/production/images/medium/cavp_a_10322208_o_cavp0332fig001.gif"})
Figure 1. Expression of the oncogene Meq in a variety of tissues: (1a) MD tumour in a liver, (1b) MD tumour in a nerve with type A lesions, (1c) REV non-bursal tumour in a liver, and (1d) MD tumour in a nerve with type B lesions. Bar = 35 µm.
Table 4. Expression of meq, MATSA, CD30, p53 and methyl-3-cytosine in lymphomas, tissues latently infected by MDV and uninfected tissues evaluated by immunohistochemistry
Expression of the antigens MATSA and CD30 was similar in most of the samples (Table 4). Both antigens were expressed in samples of the subgroups MDS, MDM, MDVVAC, MDVVAC + CHAL, RENB, CA, and CAN. The highest expression of MATSA and CD30 was found in MDS and MDM subgroups (2 to 3 + ), but samples of other subgroups were also positive. Difference in the expression of MATSA and CD30 were found at two levels. MATSA expression was found in spleens and thymus but not in the bursas of Fabricius of subgroups MDVVAC, MDVVAC + CHAL, CA, and CAN. CD30 expression, however, was found in the spleens, thymus and bursas of these subgroups. In addition, samples of the subgroup RENB were consistently expressing high levels of MATSA, but only few of them expressed CD30, and with much lower intensity.
Expression of the antigen p53 was detected in all studied lymphomas (MDS, MDM, LL, REB and RENB) but was not evident in any of the other tissues (Table 4). Expression of p53 in MDS and MDM tumours had similar distribution to the expression of Meq. Total level of methylation in the cell tumours measured by detection of methyl 3-cytosine was similar in all tested samples and no statistically significant differences were found between groups.
Discussion
This work studied the value of several criteria in the diagnosis of MDV-induced lymphomas. The objective was to find criteria that permit differentiation not only between MD lymphomas and lymphomas induced by retroviruses (REB, RENB and LL), but also between actual MD lymphomas and inflammatory lesions supporting MDV latency. Our results showed that high loads of MDV DNA and expression of the MDV oncogene Meq were specific for MDV-induced lymphomas and valid criteria for MD diagnosis. On the other hand, expression of MATSA and CD30 in the tumours was not specific for MDV lymphomas (MDS, MDM) and did not differ among the several types of tumours (MDS, MDM, REB, RENB and LL), MDV latently infected tissues (MDVVAC and MDVVAC + CHAL) and uninfected tissues (CA). No significant differences were found in the total level of methylation of the tumour cells between MD lymphomas and samples of other groups, either. Expression of p53, however, although not specific for MDV-induced lymphomas, was detected in all studied lymphomas (MDS, MDM, REB, RENB and LL) but not in other tissues and it might have some potential as tumour marker.
The value of PCR in the diagnosis of poultry lymphomas has been controversial (Davidson et al., 1995 1996; Silva & Witter, 1996; Witter & Schat, 2003). Standard PCR techniques that do not permit quantification of the MDV genome are valid only for the diagnosis of the MDV infection but not for the diagnosis of MD tumours as they cannot discriminate between latent infections and transformation. In this work a quantitative PCR assay has been designed to differentiate between MD lymphomas and latently infected tissues. Our results shows that MD tumours have much higher amounts of MDV DNA than latently infected tissues, and therefore quantification PCR techniques (i.e. real-time PCR) can be used for diagnosis of MD tumours. Variability between MD lymphomas was minimal regardless of the variable number of cells expressing the antigen pp38. It is generally considered that cells expressing MDV early antigen pp38 in MD lymphomas are those cells in which MDV infection have reactivated from either latency or transformation. The percentage of cells expressing pp38 in MD lymphomas is very variable. However, even in tumours with high expression of pp38, there is minimal if any expression of late MDV antigens (i.e. glycoproteins) (Witter & Schat, 2003). This differs greatly from cytolytically infected cells in the lymphoid organs during the early cytolytic infection or in the feather follicle epithelium, where both early and late MDV antigens are widely expressed. A possible explanation might be that cells expressing pp38 within MD lymphomas support an abortive replication of the virus. This hypothesis might explain why in the present work MD lymphomas with higher numbers of cells expressing pp38 did not have much higher numbers of MDV DNA copies than MD lymphomas without cells expressing pp38.
The real-time PCR technique used in this study was specific for serotype 1 MDV strains and of moderate sensitivity (equal to or more than 100 copies). The latter characteristic was useful so that detection of latent infection could be minimized. For example, the technique was unable to detect serotype 1 MDV infection in any of the samples of the retroviral lymphomas group, even though chickens had been vaccinated at day of hatch with serotype 1 MDV strain R2/23. In addition to the lower sensitivity of the PCR technique used, the older age of the chickens when samples of the retroviral lymphomas group were taken might have contributed to the failure in detecting R2/23 infection since vaccine strains tend to decrease levels of infection with time (Purchase et al., 1972; Witter et al., 1995). A simple method of quantification has been established in this study by comparing the amount of the MDV gB gene and the chicken gene GAPDH. This method permits standardization of the technique without the use of a standard curve and therefore is easy to reproduce in different laboratories.
Meq is an oncogene of MDV that is highly expressed in MDV-induced lymphomas and in MD lymphoblastic cell lines (Ross et al., 1997). It has been recently shown that the Meq gene is necessary for the development of tumours and therefore is a key gene in the pathogenesis of MD (Lupiani et al., 2004). Although Meq is expressed mainly in MD lymphomas, low levels of Meq can be detected during cytolytic infection in the lymphoid organs (mainly thymus) and in the feather follicle epithelium (Gimeno, unpublished data). Expression of Meq has been also associated with latency (Kung et al., 2001). In this work, Meq was consistently found in MDV-induced lymphomas, both in viscera and nerves. Expression of meq was strongly correlated with the viral load. Levels of Meq expression in MD tumours varied slightly between tumours but it was high in all cases (60% to 80% of the cells). No Meq expression was found in latently infected tissues (MDVAC and MDVAC + CHAL), even when the latently infecting virus (detected by real-time PCR) was an oncogenic MDV (MDVAC + CHAL). The sensitivity of the technique used might have contributed to the failure to detect Meq in latently infected tissues or in the scattered cells where reactivation might have occurred. The high percentage of cells in the tumour expressing meq (60% to 80%) indicated that MDV genome is present in most, if not all, transformed cells and justified the greater number of MDV genome copies detected by the quantitative PCR. Also, this finding suggests that number of transformed cells in MD tumour might be higher than initially thought (Witter et al., 1975).
MATSA has been a controversial antigen since it was first described (Witter et al., 1975). Initially it was thought to be an antigen specific to cells transformed by MDV. However, later it was shown to be an antigen expressed in activated T cells (Schat & Calnek, 1978a), although it is still sometimes used for the diagnosis of MD tumours. Our results do not support the use of MATSA, as detected using mAb 14B367, for the diagnosis of MD tumours since MATSA expression was found not only in MD lymphomas, but also in latently infected tissues and in uninfected tissues, mainly of the CA group. A major limitation in the study of MATSA is that it is not well characterized. MATSA may not be a single antigen, as there is a whole family of antigens under this name. It has been proposed that the antigen CD30 could belong to the MATSA family (Burgess et al., 2004). In this work, expression of MATSA and CD30 were very similar. However, there was a major difference regarding the staining of B cells. While no expression of MATSA was found in the bursa of Fabricius, CD30 antigen was found in lymphocytes within bursal follicles. Expression of CD30 in B cells has also been reported (Burgess et al., 2004). This finding suggests that if CD30 belongs to the MATSA family (Burgess et al., 2004), then MATSA might not be restricted to activated T cells.
Overexpression of p53 occurs in numerous tumours in humans and other animal species (Bocchetta & Carbone, 2004). Mutations of the p53 gene had been described in poultry lymphomas induced by MDV and ALV (Takagi et al., 1996). Protein p53 moreover can interact with the MDV oncogene Meq, suggesting a role of this protein in the pathogenesis of MD lymphomas (Brunovskis et al., 1996). In this work, p53 was expressed in all the lymphomas studied, regardless of the aetiology. The antibody used detects not only the mutant type of p53, but also the wild type when used in immunohistochemistry (Said et al., 1992). No expression was detected in MDV latently infected tissues or in uninfected tissues, probably due to insufficient sensitivity of the technique. However, p53 was detected in all tumours, perhaps due to the overexpression of the protein. In MD lymphomas, the frequency and distribution of p53 expression resembled the expression of the antigen Meq. Both p53 and Meq were expressed at a high percentage of cells. Since interactions between p53 and Meq have been detected in vitro (Kung et al., 2001), further studies are warranted to determine whether this interaction also occurs in vivo.
Supermethylation of particular genes occurs in certain tumours but in most of the human tumours the total level of methylation in the cell decreases (Esteller, 2002). In this work, no difference in the level of methylation was detected between tumour and non-tumour tissues or within tumours of differ ent aetiologies.
Diagnosis of lymphomas in poultry is a multistep process. First, it is necessary to confirm that there are tumours and that they seem to be lymphomas by histopathology. Sometimes, diagnosis will be achieved using traditional criteria such as the location of the lesions and the age of the animals. However, in some cases these criteria will not be sufficient and further studies are needed. In cases suspected to be MD, there are several options: characterization of the cell type (B cells or T cells), quantification of MDV DNA or expression of Meq. The two new criteria proposed in this work have the advantage of being specific for MDV and it could be used not only to differentiate between MD lymphomas and retroviral lymphomas but also between MD enlarged nerves and peripheral neuropathy (Gimeno, unpublished data). In addition, quantification of MDV DNA does not require having frozen tissues and DNA can be easily extracted from tissues preserved in many different ways. These criteria are only valid when applied to tumours since a high load of viral DNA and expression of Meq can be found during early cytolytic infection in lymphoid organs or in the feather follicle epithelium.
This work thus presents two novel criteria, load of MDV DNA and expression of Meq, which will assist in the diagnosis of MDV-induced lymphomas. In addition, two cell markers (CD30 and MATSA) have been compared and the differences described; neither marker appears to have high value for diagnosis. Finally, overexpression of p53 has been detected in all poultry lymphomas and this finding might be of interest not only in the identification of tumour markers but also in understanding mechanism of transformation in avian viral lymphomas.
Translations of the abstract in French, German and Spanish are available on the Avian Patholgy website.
| Inoculum | Sampling | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Group | Subgroupb | Kind of sample | Lot | Chicken line | Strain | Oncogenicityc | Age | Age | Number |
| MD lymphomas | MDS | MD lymphomas | 1 | 15×7 | GA/22 | MDV oncogenic (virulent) | 1 day | 5 to 10 w.p.i. | |
| Single infection | 2 | 15×7 | RB1B | MDV oncogenic (very virulent) | 1 day | 5 to 10 w.p.i. | 10 | ||
| 3 | 15×7 | Md5 | MDV oncogenic (very virulent) | 1 day | 5 to 10 w.p.i. | 10 | |||
| 4 | 15×7 | 648A | MDV oncogenic (very virulent plus) | 1 day | 5 to 10 w.p.i. | 5 | |||
| MDM | MD lymphomas | 5 | 15×7 | RB1B | MDV oncogenic (very virulent) | 1 day | 5 w.p.i. | 11 | |
| Multiple infection | SNV | REV oncogenic | 1 day | ||||||
| RPL-40 | ALV oncogenic | 1 day | |||||||
| Tissues latently infected by MDV (no tumours) | MDVVAC | Tissues latently infected by MDV Vaccine strain | 6 | 15×7 | R2/23 | Non-oncogenic serotype 1 MDV | 1 day | 5 to 6 w.p.i. | 10 |
| 7 | 15×7 | SB-1 | Non-oncogenic serotype 2 MDV | 1 day | 5 to 6 w.p.i. | 9 | |||
| 8 | 15×7 | FC-126 | Non-oncogenic serotype 3 MDV | 1 day | 5 to 6 w.p.i. | 10 | |||
| MDVVAC + CHAL | Tissues latently infected by MDV | 9 | 15×7 | FC-126 | Non-oncogenic serotype 3 MDV | 1 day | 5 to 6 w.p.i. | 10 | |
| GA/22 | MDV oncogenic (virulent) | 6 day | |||||||
| Vaccine and oncogenic strains | 10 | 15×7 | FC-126 | Non-oncogenic serotype 3 MDV | 1 day | 5 to 6 w.p.i. | 10 | ||
| SB1 | Non-oncogenic serotype 2 MDV | 1 day | |||||||
| Md5 | MDV oncogenic (very virulent) | 6 day | |||||||
| 11 | 15×7 | CVI988 | Non-oncogenic serotype 1 MDV | 1 day | 5 to 6 w.p.i. | 9 | |||
| 648A | MDV oncogenic (very virulent plus) | 6 day | |||||||
| Retroviral lymphomas | LL | Lymphoid leukosis | 12 | 15×7 | RPL-40 | ALV oncogenic | 1 day | 14 to 24 w.p.i. | 30 |
| R2/23 | Non-oncogenic serotype 1 MDV | 1 day | |||||||
| REB | RE bursal tumours | 13 | 15×7 | SNV | REV oncogenic | 1 day | 19 to 26 w.p.i. | 30 | |
| R2/23 | Non-oncogenic serotype 1 MDV | 1 day | |||||||
| RENB | RE non-bursal tumours | 14 | Line 0 | SNV | REV oncogenic | 1 day | 7 to 17 w.p.i. | 30 | |
| R2/23 | Non-oncogenic serotype 1 MDV | 1 day | |||||||
| Uninfected tissues (no tumours) | CNA | Non-activated immune system | 15 | 15×7 | None | NA | NA | 6 weeks | 10 |
| CA | Activated immune system | 16 | 15×7 | FCA/SRBC | NA | 2 weeks | 6 weeks | 10 | |
| aSamples were generated in a series of experiments where the infections status of the chickens was controlled. Four general groups of samples were generated (MD lymphomas, latently infected MDV tissues, retroviral lymphomas and uninfected tissues). Each group of samples consisted of two or more subgroups. Lots in each subgroup represent different treatments. For each lot, the chicken line, viral strain, age at inoculation, time of sampling and number of samples are shown. | |||||||||
| bMDS includes lymphomas induced by an oncogenic MDV strain (GA, RB1B, Md5, or 648A). MDM includes lymphomas induced by the oncogenic MDV strain RB1B in chickens infected with the REV strain SNV and the ALV strain RPL-40. MDVVAC includes tissues (spleen, bursa of Fabricius and thymus) of chickens vaccinated with the attenuated serotype 1 MDV strain R2/23, the non oncogenic serotype 2 MDV SB-1 or the non oncogenic serotype 3 MDV FC-126. MDVVAC + CHAL includes tissues of MDV-vaccinated chickens and challenged with an oncogenic MDV strain that did not develop tumours. LL includes lymphomas induced by RPL-40. REB includes bursal lymphomas induced by SNV strain in 15×7 chickens. RENB includes non-bursal lymphomas induced by SNV strain Line 0 chickens. CNA includes tissues (spleen bursa of Fabricius, and thymus) of chickens that had not been infected or treated. CA includes same tissues of uninfected chickens that had been treated with Freund's complete adjuvant (FCA) and SRBC. | |||||||||
| cBrief description of the viral strain used is shown. | |||||||||
| Groupa | Subgroup | Virus treatment | Number of samples | Log (copies of MDV DNA)b | Ct ratioc |
|---|---|---|---|---|---|
| MD lymphomas | MDS | GA, RB1B, Md5, 648A | 30 | 3.4±0.1A (3.2–3.9) | 0.6±0.06 A (0.5–0.7) |
| MDM | RB1B + SNV + RPL-40 | 11 | 3.6±0.5A (3.2–4.3) | 0.7±0.07 A (0.6–0.7) | |
| Tissues latently infected by MDV | MDVVAC MDVVAC + CHAL | R2/23 | 10 | 1.0±0.2B (0.8–1.1) | 1.0±0.01 BC (0.9–1.1) |
| FC-126/GA | 29 | 1.1±0.3B (0.9–1.2) | 0.9±0.02 B (0.9–1.0) | ||
| FC-126 + SB1/Md5 | |||||
| CVI988/648A | |||||
| Retroviral lymphomas | LL REB | RPL-40 + R2/23 | 30 | 0.0±0.0C | 1.1±0.00 C (1.1–1.1) |
| SNV + R2/23 | 30 | 0.0±0.0C | 1.1±0.00 C (1.1–1.1) | ||
| RENB | SNV (line 0) + R2/23 | 30 | 0.0±0.0C | 1.1±0.00 C (1.1–1.1) | |
| Uninfected spleens | CNA | None | 10 | 0.0±0.0C | 1.1±0.00 C (1.1–1.1) |
| CA | FCA/SRBC | 10 | 0.0±0.0C | 1.1±0.00 C (1.1–1.1) | |
| aGroups and subgroups of samples are explained in detail in Materials and Methods as well as in Table 1. | |||||
| bNumber of MDV DNA copies in 50 ng DNA sample measured by real-time PCR. The gB gene of MDV was amplified and the number of copies was calculated by using a standard curve. Results are presented as the log of the number of MDV DNA copies. Numbers represent the mean±standard deviation. Numbers in brackets represent the range of results in the subgroup. Differences between subgroups were statistically studied (analysis of variance test, P<0.05)—the same uppercase superscript means that differences were not significant. | |||||
| cThe Ct ratio was calculated for each sample by dividing the Ct value of the gB gene amplification by the Ct value of the GAPDH gene amplification. The lower the value of the Ct ratio, the higher the load of MDV DNA in the sample. Numbers represent the mean±standard deviation. Numbers in parentheses represent the range of results in the subgroup. Differences between subgroups were statistically studied (analysis of variance test, P<0.05)—the same uppercase superscript means that differences were not significant. | |||||
| Lymphoma | Subgroupa | Treatment | pp38 expressionb | Meq expressionb | Log (copies of MDV DNA)c | Ct ratiod |
|---|---|---|---|---|---|---|
| 1 | MDS | RB1B | 1 + | 3 + | 3.7 | 0.6 |
| 2 | MDS | RB1B | 2 + | 3 + | 3.7 | 0.6 |
| 3 | MDS | RB1B | 0 | 3 + | 3.4 | 0.6 |
| 4 | MDS | Md5 | 0 | 3 + | 3.4 | 0.7 |
| 5 | MDS | Md5 | 0 | 3 + | 3.4 | 0.7 |
| 6 | MDS | Md5 | 2 + | 3 + | 3.7 | 0.7 |
| 7 | MDM | RB1B + RPL40 + SNV | 0 | 3 + | 3.5 | 0.6 |
| 8 | MDM | RB1B + RPL40 + SNV | 2 + | 3 + | 3.7 | 0.7 |
| 9 | MDM | RB1B + RPL40 + SNV | 0 | 3 + | 3.4 | 0.6 |
| aSubgroups of samples are explained in detail in Materials and Methods as well as in Table 1. | ||||||
| bExpression of pp38 and Meq was evaluated by immunohistochemistry and subjectively scored on a scale from 0 to 3 in gradient of intensity. | ||||||
| cNumber of MDV DNA copies in 50 ng DNA sample measured by real-time PCR. The gB gene of MDV was amplified and number of copies was calculated using a standard curve. Results are presented as the log of the number of MDV DNA copies. | ||||||
| dThe Ct ratio was calculated for each sample by dividing the Ct value of the gB gene amplification by the Ct value of the GAPDH gene amplification. The lower the value of the Ct ratio, the higher the MDV DNA load in the sample. | ||||||
| Groupa | Subgroup | Virus treatment | Number of samples | Number of Meq-positive | Number of MATSA-positive | Number of CD30-positive | Number of p53-positive | Methyl 3-cytosine-positive |
|---|---|---|---|---|---|---|---|---|
| MD lymphomas | MDS | GA, RB1B, Md5, 648A | 30 | 30 (2–3 + )b | 30 (2–3 + ) | 30 (2–3 + ) | 30 (2–3 + ) | 30 (1–2 + ) |
| MDM | RB1B + SNV + RPL-40 | 11 | 11 (2–3 + ) | 11 (2–3 + ) | 11 (2–3 + ) | 11 (2–3 + ) | 11 (1–2 + ) | |
| Tissues latently infected by MDV | MDVVAC | R2/23, SB-1, FC-126 | 29 | 0 | 29 (1–2 + ) | 29 (1–2 + ) | 0 | 29 (1–2 + ) |
| MDVVAC + CHAL | FC-126/GA | |||||||
| FC-126 + SB1/Md5 | 29 | 0 | 29 (1–2 + ) | 29 (1–2 + ) | 0 | 29 (1–2 + ) | ||
| CVI988/648A | ||||||||
| Retroviral lymphomas | LL | RPL-40 + R2/23 | 30 | 0 | 0 | 0 | 30 (2–3 + ) | 30 (2–3 + ) |
| REB | SNV + R2/23 | 30 | 0 | 0 | 0 | 30 (2–3 + ) | 30 (2–3 + ) | |
| RENB | SNV (line 0) + R2/23 | 30 | 0 | 30 (1–2 + ) | 3 (1 + ) | 30 (1–2 + ) | 30 (2–3 + ) | |
| Uninfected tissues | CNA | None | 10 | 0 | 10 (1 + ) | 10 (1 + ) | 0 | ND |
| CA | FCA/SRBC | 10 | 0 | 10 (1–2 + ) | 10 (1 + ) | 0 | 10 (1–2 + ) | |
| aGroups and subgroups of samples are explained in detail in Materials and Methods as well as in Table 1. | ||||||||
| bResults showed number of samples positive for the stain (intensity of the stain). Intensity was subjectively scored on a scale from 0 to 3 as a gradient of intensity. Numbers in parentheses indicate the spectrum of intensity found in the subgroup. | ||||||||
Acknowledgments
The authors thank Barbara Riegle, Melanie Flesberg and Lonnie Milam for excellent technical assistance. They also thank Dr Shane Burgess for providing the antibody AV37 and Dr Lucy F. Lee for providing the antibodies 14B367 and 23B46.
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