The value of biomedical research training for veterinary anatomic and clinical pathologists.

Veterinary pathologists traditionally have been actively engaged in research as principal investigators and as collaborators. Pathologists frequently obtain advanced training in research; however, it appears that in the last 10 years there has been a reversal of a previous trend toward increasing numbers of pathologists obtaining PhD degrees. This has arisen despite an established shortage of veterinarians engaged in research. This article evaluates the benefits of research training for individual pathologists, including a wide spectrum of professional opportunities and additional skill development beyond that usually provided by diagnostic pathology training alone. Various training models are discussed, including combined and sequential diagnostic residency and research degree training as well as the nondegree research fellowship programs more commonly pursued in human medicine. Best-practice recommendations for program infrastructure, mentorship, time management, and a team approach to research and research training are advocated to facilitate the development of successful programs and to encourage a continued emphasis on integrated training for pathologists as both clinical diagnosticians and experimentalists. This article is intended to help prospective and active pathology trainees, their mentors, and educational administrators optimize opportunities to ensure the future vitality of veterinary pathologists, and their contributions, in basic and applied research.

Association of GP51 expression and persistent CD5+ B-lymphocyte expansion with lymphomagenesis in bovine leukemia virus infected sheep.

Alterations in the circulating CD5+ B-lymphocyte population, in vitro GP51 expression, and in vivo tax/rex expression that may precede lymphomagenesis were characterized prospectively in ten experimentally BLV-infected sheep. Infection with pathogenetic BLV resulted in a significant expansion of the circulating CD5+ B-lymphocyte population in six infected sheep. Of the remaining four infected sheep that did not have persistently elevated CD5+ B-lymphocyte counts, three developed lymphoid neoplasia within 14 months post-inoculation. Neoplastic cells from two of these three sheep were CD5- B-lymphocytes, while cells from the third were CD5+ B-lymphocytes. In vitro GP51 expression was a consistent feature of circulating lymphocytes from all three sheep developing tumors, but high level tax/rex gene transcription was not detected in circulating lymphocytes prior to lymphomagenesis. Neither in vitro GP51 expression nor high level tax/rex gene transcription was associated with expansion of the CD5+ B-lymphocyte population in sheep with significantly elevated CD5+ B-lymphocyte counts. These observations indicate that BLV infection in sheep results in expansion of the circulating CD5+ B-lymphocyte population, and that this expansion is not required for the subsequent development of BLV-associated lymphoid neoplasia.

Quantitative characterization of the CD5 bearing lymphocyte population in the peripheral blood of normal sheep.

Quantitative characterization of the circulating CD5+ lymphocyte population in sheep was performed using monoclonal antibodies against ovine CD5, ovine CD2, and ovine sIgM. A total of fifteen sheep were examined biweekly over a period of 2.5 months. The total CD5+ population was measured using single color flow cytometry, while CD5+ cells co-expressing sIgM or CD2 were determined by two color flow cytometric analysis. The total CD5+ cell population comprised 31.5-65.4% of gated lymphocytes with a mean value of 44.9%. Absolute CD5+ cell numbers varied from 1075 to 8007 cells mm-3, with a mean value of 3149 cells mm-3. The mean proportion of CD5+ B-cells was 4.0%, with a mean absolute value of 270 cells mm-3. The mean proportion of CD5+/CD2+ cells was 12.1% while that of CD5+/CD2- cells was 28.6%, corresponding to mean absolute values of 727 and 1794 cells mm-3 respectively. Significant variation in CD5+ lymphocyte numbers in all individual animals occurred over the time span of the study. The results of this study provide a baseline from which to accurately assess CD5+ cell populations in neoplastic and infectious diseases.

Videoendoscopic techniques for collection of multiple, serial intra-abdominal biopsy specimens in HIV-negative and HIV-positive pigtail macaques (Macaca nemestrina).

Tissue biopsy sampling by laparotomy is considered major surgery, which precludes serial sampling. This increases variability and requires a larger n value for pathogenesis studies. To address this problem, a study was conducted to develop and validate the feasibility of performing multiple, serial biopsy sampling by laparoscopy in pigtail macaques. Tissues were obtained laparoscopically from 2 HIV-negative and 2 HIV-positive (late postinoculation) macaques on days 0, 3, and 7, followed by necropsy on day 21. Anesthesia was induced with ketamine and atropine and maintained with isoflurane. Carbon dioxide pneumoperitoneum was maintained at 6 mm Hg. A triangulated threeport technique was used for insertion of pediatric (3.5-5.0 mm) laparoscopic instrumentation. Biopsies of kidney and spleen were obtained with a core-sampling biopsy needle, of small intestine and mesenteric lymph node with a pretied loop, and of liver with 3.5-5.0 mm biopsy forceps. Analgesics were administered for 24 h post operation, and animals were evaluated for postoperative complications. All monkeys maintained a good appetite. Mild postoperative pain was observed in one animal after the second surgery. There was no excessive bleeding or intestinal stenosis at biopsy sites. Skin infection, observed in 1/36 (2.8%) port sites, resolved with systemic antibiotics. Significant adhesions formed at 23/114 (20.2%) sites. Out of 34 samples evaluated for histopathology, 29 (85.3%) were satisfactory (minimal to mild tissue crushing). In situ hybridization results revealed few (4 of 29 samples tested) positive cells, which is consistent with the low level of HIV-2 virus found in cells late in the postinoculation period in pigtail macaques. The results of this study suggest that laparoscopic serial abdominal biopsy collection in healthy and immunocompromised pigtail macaques may be considered a minor procedure, and can be used to expedite serial tissue collection in survival studies.

Uterine epithelioid leiomyosarcoma in a pig-tailed macaque.

During ultrasonographic examination following spontaneous abortion, an approximately 6-cm diameter mass of mixed echogenicity is detected in the uterine wall of a mature pig-tailed macaque (Macaca nemestrina). Grossly, the mass is associated with the uterine fundus, multilobulate and tan with red-black mottled foci on cut surface. Microscopically, the invasive mass is composed of poorly differentiated round cells arranged in sheets and bundles supported by a fine fibrous stroma. On immunohistochemical evaluation, neoplastic cells contain vimentin, desmin, muscle actin, and smooth muscle alpha-actin antigens and do not react with antibodies to low and high molecular-weight cytokeratins. Cell morphology and immunohistochemical results indicate a diagnosis of epithelioid leiomyosarcoma (clear cell variant).

Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.

Bovine leukemia virus (BLV) encodes at least two regulatory proteins, Rex and Tax. Tax, the transactivating protein, stimulates the long terminal repeat to promote viral transcription and may be involved in tumorigenesis. Rex is involved in the transition from early expression of regulatory proteins to later expression of viral structural proteins. We have targeted ribozymes against the mRNA encoding Rex and Tax. The ribozymes consist of the hammer-head catalytic motif flanked by antisense sequences that hybridize with the complementary rex/tax mRNA. To evaluate cleavage in a cell-free system, we transcribed portions of rex/tax mRNA and incubated them with synthetic RNA ribozymes. A ribozyme was identified that cleaves > 80% of the target RNA. Synthetic DNA encoding this ribozyme was cloned into the expression vector pRc/RSV and transfected into BLV-infected bat lung cells. Intracellular cleavage of rex/tax mRNA was confirmed by reverse transcriptase PCR. In cells expressing the ribozyme, viral expression was markedly inhibited. Expression of the BLV core protein p24 was inhibited by 61%, and reverse transcriptase activity in supernatant was inhibited by 92%. Ribozyme inhibition of BLV expression suggests that cattle expressing these sequences may be able to control BLV replication.

Genomic diversity and evolution of papillomaviruses in rhesus monkeys.

We are studying the diversity of and relationships among papillomaviruses (PVs) to understand the modes and timescales of PV evolution and in the hope of finding animal PVs that may serve as model systems for disease caused by human PVs (HPVs). Toward this goal, we have examined 326 genital samples from rhesus monkeys and long-tailed macaques with a PCR protocol optimized for detecting genital HPV types. In 28 of the rhesus monkey samples, we found amplicons derived from 12 different and novel PV genomes, RhPV-a to RhPV-m, with the likely taxonomic status of "type." The frequency with which novel RhPVs were detected suggests that rhesus monkeys may play host to PVs with a diversity similar to that of humans. In phylogenetic trees, all 12 of the different RhPVs and the previously described type RhPV-1 were members of the genital HPV supergroup and formed three minor branches distinct from the 11 branches formed by genital HPVs. We also identified a novel PV amplicon, MfPV-a, from a long-tailed macaque, a species belonging to the same genus as rhesus monkeys. MfPV-a turned out to be a close relative of five RhPVs. It appears that the evolution of primate lineages leading to the genus Macaca and to humans created transmission barriers for PVs, resulting in viral evolution closely linked to the host. Additional support for the linked-evolution hypothesis comes from considering the phylogenetic association of two other ape and monkey PVs with the genital HPVs, the supergroup formed by at least seven ungulate PVs, and the isolated phylogenetic position of the only known bird PV.