Necropsy records at the New England Primate Research Center (NEPRC) were searched from 1991–2009 for intestinal malignancies in common marmosets. Excluding neonates and research necropsies, 384 common marmosets were necropsied during this time period. All cases recorded were localized to the small intestine. All animals were necropsied within 24 hours of death and representative sections of all major organs were collected, fixed in 10% neutral buffered formalin (NBF), embedded in paraffin, sectioned at 5μm, and stained using hematoxylin and eosin (HE). Additional sections of affected intestine were stained via the Warthin-Starry method.
To characterize the neoplasms using immunohistochemical methods we used standard immunoperoxidase staining for CD3, CD20, E-cadherin, cyotokeratin AE1/AE3, and β-catenin. Formalin fixed, paraffin embedded sections were deparaffinized, rehydrated and subsequently blocked with hydrogen peroxide. Pre-treatment for all antibodies involved microwaving for 20 minutes in 0.01M citrate buffer followed by 20 minutes of cooling. All steps were followed by a tris-buffered saline (TBS) wash. Prior to application of primary antibodies, all slides were treated with Dako protein block for 10 minutes. Sections were incubated with anti-human CD3 (Dako [Carpinteria, CA, USA], polyclonal, 1:600, 30′ at room temperature), anti-human CD20 (Dako, monoclonal, 1:175, overnight in refrigerator), anti-human E-cadherin (Labvision [Fremont, CA, USA], monoclonal, 1:100, overnight in refrigerator), anti-human cytokeratin AE1/AE3 (Dako, monoclonal, 1:140, overnight in refrigerator), and anti-human β-catenin (Santa Cruz Biotechnology [Santa Cruz, CA, USA], monoclonal, 1:100, overnight in refrigerator). Slides were then incubated with secondary antibody biotinylated goat anti-rabbit (Vector laboratories, [Burlingame, CA, USA],1:200, 30′ at room temperature) for CD3 and E-cadherin and biotinylated horse anti-mouse (Vector laboratories, 1:200, 30′ at room temperature) for CD20, cytokeratin AE1/AE3, and β-catenin. This was followed by 30 minute incubation at room temperature with Vectastain ABC Elite [Vector laboratories] (CD3, E-cadherin, cytokeratin AE1/AE3, and β-catenin) or Vectastain ABC standard [Vector laboratories] (CD20). All slides were developed with DAB chromagen (Dako) and counterstained with Mayer’s hematoxylin. In all cases, step sections were incubated with isotype-specific irrelevant antibodies for negative controls. Positive controls consisted of sections of lymph node (CD3 and CD20) and small intestine (E-cadherin, cytokeratin AE1/AE3, and β-catenin) from age matched common marmosets.
Isolation of DNA from affected frozen small intestine was performed on case 5 and 8 and was carried out using the DNeasy Tissue Kit (Qiagen, Valencia, CA). In order to amplify DNA, degenerate herpesvirus primers were used to screen the sections of frozen small intestine. The degenerate primers used corresponded to DFASA (5′-GTGTTCGACTTYGCNAGYYTNTAYCC-3′) and GDTD1B (5′-CGGCATGCGACAAACACGGAGTCNGTRRCNCCRTA-3′).15
Amplifications were carried out in 25 ul of 1x GoTag Green Master Mix (Promega, San Diego, CA) with 20 pmoles of each primer and 50 ng of template DNA. The cycling condition consisted of 2 minutes denaturation at 94C followed by 35 cycles of 1min denaturation at 94C, 1 min of annealing at 60C, and 1 min of extension at 72C followed by a final 10 min extension step at 72C. An aliquot (2 to 5%) of these amplification products (DFASA-GDTD1B) was then used as a template in a subsequent nested PCR with the primer pool VYGA (5′- ACGTGCAACGCGGTGTAYGGNYTNACNGG-3′) and GDTD1B. The products of the secondary nested PCR amplification were electrophoresed in a 2% agarose gel and visualized by irradiation with UV in the presence of ethidium bromide. The nested PCR product (-236 bp) were cloned into pCR 2.1-TOPO vector (Invitrogen) and sequenced (Retrogen, Inc., San Diego, CA).
In order to compare the relative levels of viral replication in neoplastic and non-neoplastic gastrointestinal tract, a 10 fold serial dilution was made with the DNA samples and a 306 bp fragment was amplified with CHV3 specific primers (CHV3F (5′-TTGACTTCGCCAGCCTCTAT-3′) and CHV3R (5′-TTGCAGGTGCACTTGATAGC-3′) derived from DNA sequences from the GenBank (AF319782.2). The cycling condition consisted of 3 minutes denaturation at 94C followed by 5 cycles of 30 sec at 94C, 30 sec at 60C, and 30 sec at 72C followed with 35 cycles of 30 sec at 94C, 30 sec at 55C, and 30 sec at 72C followed by a final 10 min extension step at 72C.