Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor in canine cutaneous fibrosarcomas.

The expression of five markers associated with tumour angiogenesis, proliferation and apoptosis was studied in 24 canine cutaneous fibrosarcomas. Tumours were assigned histological grades and were immunohistochemically evaluated for the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2). Additionally, intra-tumour microvessel density (iMVD) was assessed by immunohistochemical labelling for expression of von Willebrand factor (vWf) and tumour proliferation index (PI) was measured following labelling of Ki-67 antigen. Finally, tumour apoptotic index (AI) was determined by application of the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP end-labelling method (TUNEL). VEGF and VEGFR-2 expression were detected in 22/24 (92%) and 24/24 (100%) of fibrosarcomas, respectively. There was correlation between VEGF and VEGFR-2 expression (r = 0.51) and between histological grade and PI (r = 0.82). A significant difference in PI between tumours of different histological grade was found (P < 0.05). The median PI in grade 2 and 3 tumours (30.6 and 54.7, respectively) was significantly higher than in grade 1 tumours (6.4). Therefore, only PI correlates significantly with the histological grade of canine cutaneous fibrosarcomas. The potential for autocrine activity for VEGF exists in canine cutaneous fibrosarcomas, as VEGF and VEGFR-2 expression was found in most tumours.

Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor associated with tumor cell proliferation in canine cutaneous squamous cell carcinomas and trichoepitheliomas.

The expression of 5 markers associated with angiogenesis was studied in canine squamous cell carcinomas (SCCs) (n = 19) and canine trichoepitheliomas (TCPs) (n = 24). SCCs were assigned histologic grades, and tissue sections from both tumor types were immunohistochemially stained for the expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR-2), as well as intratumoral microvessel density (iMVD), tumor proliferation index (PI), and tumor apoptotic index (AI), using antibodies against VEGF, VEGFR-2, von Willebrand's factor, Ki-67 antigen, and the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate end-labeling method (TUNEL), respectively. VEGF and VEGFR-2 were detected in 17/19 (89.4%) and 19/19 (100%) SCCs and in 17/24 (70.8%) and 20/24 (83.3%) TCPs, respectively. In SCCs, there was substantial correlation between histologic grade and PI (r = 0.51); and moderate correlation between VEGF and histologic grade (r = 0.43), VEGFR-2 and histologic grade (r = 0.47), VEGF and PI (r = 0.47), and VEGFR-2 and PI (r = 0.47) (Spearman rank correlation coefficient). In TCPs, there was substantial correlation between VEGF and PI (r = 0.51) and a moderate correlation between VEGFR-2 and iMVD (r = 0.36). The median iMVD of SCCs (15.5) was significantly higher than the median iMVD of TCPs (9.05) (P value < .05). It was concluded that VEGF and VEGFR-2 may promote tumor cell proliferation in TCPs and SCCs. An autocrine pathway for VEGF probably operates in canine SCCs and TCPs, as VEGF and VEGFR-2 expression was found in most tumors and was associated with evidence for tumor cell proliferation.

Evaluation of formalin-fixed paraffin-embedded tissues from vaccine site-associated sarcomas of cats for polyomavirus DNA and antigen.

OBJECTIVE: To determine whether vaccine site-associated sarcomas (VSS) from cats contain polyomavirus antigen or DNA. SAMPLE POPULATION: 50 formalin-fixed paraffin-embedded tissue blocks of VSS from cats. PROCEDURE: Sections from each tissue block were evaluated for polyomavirus antigen by use of an avidin-biotin-complex immunohistochemical staining method, using rabbit anti-murine polyomavirus polyclonal antiserum as the primary antibody. The DNA was extracted from sections of each tissue block, and a polymerase chain reaction assay was performed, using primers designed to amplify regions of the bovine polyomavirus genome and consensus polyomavirus primers designed to detect unknown polyomaviruses. RESULTS: Polyomavirus antigen and DNA were not detected in any of the VSS. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that polyomaviruses likely do not have any direct involvement in the pathogenesis of VSS in cats.

Evaluation of formalin-fixed paraffin-embedded tissues from vaccine site-associated sarcomas of cats for papillomavirus DNA and antigen.

OBJECTIVE: To determine whether vaccine site-associated sarcomas (VSS) from cats contain papillomavirus antigen or DNA. SAMPLE POPULATION: 50 formalin-fixed paraffin-embedded tissue blocks of VSS from cats. PROCEDURE: Sections from each tissue block were evaluated for papillomavirus antigen by use of an avidin-biotin-complex immunohistochemical staining method, using rabbit anti-bovine papillomavirus type-1 antibody. The DNA was extracted from sections of each tissue block, and polymerase chain reaction assays were performed, using primers designed to amplify regions of the E5 gene of bovine papillomavirus and consensus primers designed to amplify a region of the L1 gene of animal papillomaviruses. Sections from 20 of the tissue blocks were evaluated by use of nonradioactive in situ hybridization for bovine papillomavirus DNA. RESULTS: Papillomavirus antigen and DNA were not detected in any of the VSS. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that papillomaviruses likely do not have any direct involvement in the pathogenesis of VSS in cats.

Feline leukemia virus detection in corneal tissues of cats by polymerase chain reaction and immunohistochemistry.

OBJECTIVES: To determine the presence of feline leukemia virus (FeLV) in the corneas of FeLV-infected cats. ANIMALS STUDIED: Thirty-four random source cats. PROCEDURES: Seventeen cats were found positive for FeLV serum p27 antigen by enzyme-linked immunosorbent assay (ELISA). Twelve ELISA positive cats were also positive on peripheral blood samples by immunofluorescent antibody (IFA) testing. Seventeen ELISA negative cats served as negative controls. Full thickness corneal specimens were collected from the left cornea of all cats and analyzed for FeLV proviral DNA and gp70 antigen by polymerase chain reaction (PCR) and immunohistochemical (IHC) testing, respectively. RESULTS: Eleven (64.7%) positive corneal PCR results were obtained from 17 ELISA positive cats. Of 12 cats that were both ELISA and IFA positive on peripheral blood, 10 (83.3%) had positive corneal PCR results. All corneal tissues from ELISA negative cats were PCR negative. IHC staining of corneal sections revealed the presence of FeLV gp70 in corneal tissues of nine (52.9%) ELISA positive cats. Of the 12 cats that were both ELISA and IFA positive on peripheral blood, eight (66.7%) had positive corneal IHC results. Positive IHC staining was localized to the corneal epithelium. Corneal tissues of all ELISA negative cats and all IFA negative cats were negative on IHC testing. CONCLUSIONS: FeLV antigens and proviral DNA are present in corneal tissues of some FeLV-infected cats. Screening corneal donors for FeLV infection is warranted.

Immunohistochemical detection of tumor suppressor gene p53 protein in feline injection site-associated sarcomas.

Sarcomas associated with injection sites are a rare but important problem in cats. Immunohistochemical detection of p53 protein may correlate to mutation of the p53 tumor suppressor gene, a gene known to be important in oncogenesis. The expression of nuclear p53 protein in 40 feline injection site-assocated sarcomas was examined by immunohistochemical staining. In 42.5% (17/40), tumor cell nuclei were stained darkly; in 20% (8/40), tumor cell nuclei were stained palely; and in 37.5% (15/40), tumor cell nuclei were unstained. Immunohistochemical detection of p53 protein in a proportion of injection site-associated sarcomas suggests that mutation of the p53 gene may play a role in the pathogenesis of these tumors.

Comparison of endogenous feline leukemia virus RNA content in feline vaccine and nonvaccine site-associated sarcomas.

OBJECTIVE: To determine whether feline vaccine site-associated sarcomas (VSS) contain a higher amount of endogenous FeLV (enFeLV) RNA, compared with feline nonvaccine site-associated sarcomas (non-VSS). SAMPLE POPULATION: Formalin-fixed paraffin-embedded (FFPE) tissues from 50 VSS and 50 cutaneous non-VSS. PROCEDURE: RNA was extracted from FFPE sections of each tumor, and regions of the long terminal repeat (LTR) and envelope (env) gene of enFeLV were amplified by use of reverse transcriptase-polymerase chain reaction (RT-PCR). The density of each RT-PCR product band for enFeLV was compared with that of a constitutively expressed gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). An integrated density value (IDV) was determined by use of densitometry, and the IDV ratio for enFeLV to GAPDH was calculated for each enFeLV primer set. RESULTS: The median (interquartile range) of the IDV ratio for the enFeLV LTR primer set was 0.52 (0.26 to 1.17) for the VSS group and 0.84 (0.21 to 1.53) for the non-VSS group. The median (interquartile range) of the IDV ratio for the enFeLV env primer set was 0.60 (0.37 to 0.91) for the VSS group and 0.59 (0.36 to 1.09) for the non-VSS group. CONCLUSIONS: Because the amount of enFeLV RNA within the LTR and env gene was not significantly different between the VSS and non-VSS groups, enFeLV replication or expression is unlikely to be involved in VSS development.

Diagnosis of persistent bovine viral diarrhea virus infection by immunohistochemical staining of formalin-fixed skin biopsy specimens.

The objective of this study was to evaluate the efficacy of immunohistochemical (IHC) staining for diagnosis of persistent bovine viral diarrhea virus (BVDV) infection using formalin-fixed, paraffin-embedded skin biopsy specimens. Skin from 41 of 42 calves shown to be persistently infected (PI) with BVDV by repeated virus isolation more than 3 weeks apart were immunohistochemically positive for BVDV antigen. Positive IHC staining was most pronounced in the keratinocytes and in hair follicle epithelium, hair matrix cells of the hair bulb, and the dermal papilla. All of the skin sections from 10 calves experimentally infected postnatally with BVDV (10(5) median tissue culture infective doses [TCID50]) and biopsied on days 0, 5, 7, and 9 postinfection were negative for viral antigen. Ten calves from a second group experimentally infected with a higher dose of BVDV (10(8) TCID50) were biopsied when viremic between 10 and 14 days postinfection and 4 calves exhibited positive IHC staining for BVDV; however, staining in these skin biopsies was confined to small foci in the nonfollicular epidermis and follicular ostia. This staining was distinct from that observed in skin obtained from PI cattle. Skin biopsy represents an effective method for identifying animals PI with BVDV.

Mutational analysis of tumor suppressor gene p53 in feline vaccine site-associated sarcomas.

OBJECTIVES: To investigate the role of tumor suppressor gene p53 mutation in feline vaccine site-associated sarcoma (VSS) development and to evaluate the relationship between p53 nucleotide sequence and protein expression. SAMPLE POPULATION: Formalin-fixed paraffin-embedded tissues of 8 feline VSS with dark p53 immunostaining (high p53 expression) and 13 feline VSS with faint or no staining (normal p53 expression). PROCEDURE: DNA was extracted from neoplastic and normal tissue from each paraffin block. The following 3 regions of the p53 gene were amplified by polymerase chain reaction: 379 base pair (bp) region of exon 5, intron 5, and exon 6, 108 bp region of exon 7, and 140 bp region of exon 8. Amplified p53 products were sequenced and compared with published feline p53. The p53 mutations identified were correlated with p53 mutations predicted by immunostaining. RESULTS: Neoplastic cells of 5 of 8 (62.5%) VSS that had high p53 expression harbored single missense mutations within the p53 gene regions examined. The p53 gene mutations were not detected in the 13 tumors with normal p53 immunostaining. Nonneoplastic tissues adjacent to all 21 VSS lacked mutations of these p53 gene regions. CONCLUSIONS: The p53 gene mutations were restricted to neoplastic tissue and, therefore, were unlikely to predispose to VSS. However, p53 mutations may have contributed to cancer progression in 5 of the 21 VSS. There was very good (kappa quotient = 0.67 with a confidence limit of 0.3 to 1.0), although not complete, agreement between prediction of mutation by p53 immunostaining and identification of mutations by sequencing of key p53 gene regions.

Evaluation of formalin-fixed paraffin-embedded tissues obtained from vaccine site-associated sarcomas of cats for DNA of feline immunodeficiency virus.

OBJECTIVE: To evaluate the use of a polymerase chain reaction (PCR) method for detection of feline immunodeficiency virus (FIV) DNA, using formalin-fixed paraffin-embedded (FFPE) tissues, and to use this method to evaluate tissues obtained from vaccine site-associated sarcomas (VSS) of cats for FIV DNA. SAMPLE POPULATION: 50 FFPE tissue blocks from VSS of cats and 50 FFPE tissue blocks from cutaneous non-vaccine site-associated fibrosarcomas (non-VSS) of cats. PROCEDURE: DNA was extracted from FFPE sections of each tumor and regions of the gag gene of FIV were amplified by a PCR, using 3 sets of primers. Sensitivity of the method was compared between frozen and FFPE tissues, using splenic tissue obtained from a cat that had been experimentally infected with FIV. RESULTS: We did not detect FIV DNA in VSS or non-VSS tissues. Sensitivity of the PCR method was identical for frozen or FFPE tissues. CONCLUSIONS AND CLINICAL RELEVANCE: It is possible to detect FIV DNA in FFPE tissues by use of a PCR. We did not find evidence to support direct FIV involvement in the pathogenesis of VSS in cats.

Eccrine adenocarcinoma of the footpads in 2 cats.

Adenocarcinoma of sweat glands of the footpads was diagnosed in 2 cats. Clinical signs included lameness and swelling of multiple digits. Pulmonary metastasis was detected in one case. Diagnosis was based on histopathological and immunohistochemical findings. Eccrine adenocarcinoma should be included in the differential diagnosis of footpads lesions in aged cats.

The effect of formalin-inactivated vaccine on respiratory disease associated with bovine respiratory syncytial virus infection in calves.

The effect of vaccination with a formalin-inactivated, alum-precipitated (FI), bovine respiratory syncytial virus (BRSV) vaccine on BRSV induced respiratory disease in calves was investigated. Six month old BRSV-naive calves were vaccinated with either a FI, a modified live virus (MLV), or virus antigen negative control vaccine (n = 4 per group). One month after the second vaccination, the calves were aerosol challenged with lung wash from a newborn calf infected with a field isolate of BRSV. Moderate to severe clinical disease occurred in all calves. Calves that received FI vaccine had a significantly earlier (day 2 vs. day 4-5) onset of pyrexia and dyspnea (P < 0.05). Pulmonary lesions, consisting of cranioventral atelectasis and dorsal emphysema, occurred in all groups. Two calves that received MLV, and three that received FI vaccine, had reduced pneumonic lung area relative to controls. Vaccination with the FI vaccine resulted in more rapid onset of clinical disease, but ultimately, reduced pulmonary pathology in most recipients.

Pathogenesis of porcine Actinobacillus pleuropneumonia, part II: roles of proinflammatory cytokines.

The in vitro production of proinflammatory cytokines after stimulation with Actinobacillus pleuropneumoniae and the relation of these cytokines in vivo with the disease caused by A. pleuropneumoniae were investigated. Within 24 h, in vitro stimulation by A. pleuropneumoniae (serotype 1) preparations, including killed bacteria, bacterial culture supernatant, lipopolysaccharide, and bacterial extracts, porcine pulmonary alveolar macrophages (PAM) produced significant (P < 0.05) amounts of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) as measured by bioassays. The supernatants containing interleukin-8 from PAM after stimulation by bacterial preparations showed significant neutrophil chemotaxis, while bacterial preparations alone did not. After in vivo infection with A. pleuropneumoniae, the mean levels of TNF-alpha and IL-1 in serum, as measured by bioassays, were elevated 37- to 27836-fold for TNF-alpha and 11- to 5941-fold higher for IL-1 within 4 d post-infection, depending on the treatments, and remained elevated up to Day 7. Both cytokines were also detected in porcine lungs by bioassays and immunocytochemistry. The results indicated that both secreted and surface components of A. pleuropneumoniae can stimulate PAM to produce proinflammatory mediators. Neutrophil chemoattractants rather than bacterial components are the major factor causing acute lung inflammation. The elevation of TNF-alpha and IL-1 in pigs occurred coincident with the onset of acute clinical disease.

Immunohistochemical detection of canine distemper virus in haired skin, nasal mucosa, and footpad epithelium: a method for antemortem diagnosis of infection.

A reliable antemortem diagnostic method is needed for determining infection with canine distemper virus (CDV). The utility of immunohistochemical detection of CDV antigen was examined was examined for samples of nasal and footpad epithelium and haired skin in dogs with and without detectable CDV antigen in the lung and/or brain. Tissues from 57 dogs at risk of CDV infection were tested. Viral antigen was found in the lung and/or brain of 28 dogs. Among these dogs, viral antigen was demonstrated in the epithelial cells of the nasal mucosa in 24 of 27 dogs, in the footpad epithelium in 24 of 26 dogs, and in the haired skin of the dorsal neck in 26 of 27 dogs. Among the 29 dogs without CDV antigen in either the lung or brain, 1 dog had positive staining for viral antigen in the skin and nasal mucosa. Biopsies of haired skin of the dorsal neck, which is relatively simple to sample, can be used for immunohistochemical testing for acute and subacute infection with CDV.

A comparison of diagnostic methods for the detection of bovine respiratory syncytial virus in experimental clinical specimens.

Virus shedding was monitored in nasal secretions of 12 calves experimentally infected with bovine respiratory syncytial virus (BRSV) using an antigen capture enzyme-linked immunosorbent assay (ELISA) detecting the nucleoprotein (NP) antigen of BRSV, by a polymerase chain reaction (PCR) amplifying the fusion protein of BRSV, and by a microisolation assay combined with immunoperoxidase staining for the F protein of BRSV. Under the conditions of this study, similar limits of detection and quantitative results were obtained from all three assays. BRSV was detected in nasal secretions of all calves for a minimum of 4 d. Virus shedding began on Day 2 after infection, peaked on Days 3-5, and was cleared in most calves by Day 8. The PCR, and to a lesser extent the ELISA, may detect virus shedding for a longer period after infection than virus isolation, possibly due to neutralization of the virus by rising mucosal antibody. Simulated environmental conditions likely to be experienced during transport of clinical field specimens markedly reduced the sensitivity of virus isolation but had a minimal effect on the results of the NP ELISA. Actual field transport conditions (overnight on ice) had minimal apparent effect on the results of the PCR assay. The less stringent specimen handling requirements, combined with low limits of detection, of both the nucleoprotein ELISA and PCR, indicate either of these assays are more suitable for diagnostic applications than virus isolation.

Lesions and distribution of viral antigen following an experimental infection of young seronegative calves with virulent bovine virus diarrhea virus-type II.

During the past several years, acute infections with bovine viral diarrhea virus (BVDV) have been causally linked to hemorrhagic and acute mucosal disease-like syndromes with high mortality. The majority of BVDVs isolated in such cases have been classified as type II on the basis of genetic and antigenic characteristics. It was our objective to examine clinical disease, lesions and potential sites of viral replication, following experimental BVDV type II infection in young calves. On approximately day 35 after birth, calves that had received BVDV-antibody-negative colostrum were infected by intranasal inoculation of 5 x 10(5) TCID50 of BVDV type II isolate 24,515 in 5 mL of tissue culture fluid (2.5 mL/nostril). Calves were monitored twice daily for signs of clinical disease. Approximately 48-72 h after infection, all calves developed transient pyrexia (39.4-40.5 degrees C) and leukopenia. Beginning on approximately day 7 after infection, all calves developed watery diarrhea, pyrexia (40.5-41.6 degrees C), marked leukopenia (> or = 75% drop from preinoculation values), variable thrombocytopenia, and moderate to severe depression. Calves were euthanized on days 10, 11, or 12 after infection due to severe disease. Gross and histological lesions consisted of multifocal bronchointerstitial pneumonia (involving 10%-25% of affected lungs), bone marrow hypoplasia and necrosis, and minimal erosive lesions in the alimentary tract. Immunohistochemical staining for BVDV revealed widespread viral antigen usually within epithelial cells, smooth muscle cells and mononuclear phagocytes in multiple organs, including lung, Peyer's patches, gastric mucosa, thymus, adrenal gland, spleen, lymph nodes, bone marrow, and skin. This BVDV type II isolate caused rapidly progressive, severe multisystemic disease in seronegative calves that was associated with widespread distribution of viral antigen and few gross or histological inflammatory lesions.

Circovirus-like viral associated disease in weaned pigs in Indiana.

Inclusion bodies with staining affinity and ultrastructural characteristics typical of circoviruses that stained positive for porcine circovirus (PCV)-like virus were demonstrated in association with granulomatous lesions in multiple tissues of three clinically ill 10- to 12-week-old pigs. A syndrome of poor growth and wasting in 5-15% of weaned pigs was an intermittent problem on a 450-sow one-site farrow-to-finish swine farm in Indiana. Routine diagnostic testing did not demonstrate a cause. Gross examination of three representative weaned pigs from two farrowing groups over a 1-month period revealed generalized lymphadenopathy and interstitial pneumonia. A unique microscopic finding for all three pigs was granulomatous inflammation of lymphoid tissues associated with large numbers of multinucleate giant cells and characteristic viral inclusions in the cytoplasm of macrophages. These inclusions were round, homogeneous, and magenta to basophilic, varied in size (5-25 microm), and either were single or formed botryoid clusters. Ultrastructurally, these inclusions were composed of electron-dense paracrystalline arrays of small nonenveloped icosahedral viral particles that were approximately 17 nm in diameter. The sizes and shapes of the virus particles, the unique microscopic appearance of the inclusions, and the positive staining of the intracytoplasmic viral inclusions by the Feulgen technique are consistent with circoviruses. Immunohistochemistry for PCV-like virus demonstrated viral antigen in the cytoplasm of macrophages that were within inflammatory infiltrates in a variety of organs. The described inclusion bodies stained positively.

Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe.

Samples of lung, liver, kidney, pancreas, spleen, and lymph node from pigs with postweaning multisystemic wasting syndrome from California (USA) and samples of mesenteric lymph nodes from similarly diseased pigs from Brittany (France) were examined by light microscopy, in situ hybridization (ISH), and/or virus isolation. Whole genomic probes for porcine circovirus (PCV) and chicken anemia virus (CAV) were used for ISH. Tissue homogenate supernatants were inoculated onto PK/15 cells for virus isolation, and the presence of viral antigen and viral particles was verified by indirect immunofluorescence, ISH, and electron microscopy. Histologic examination of lung from pigs from California revealed interstitial pneumonia, alveolar epithelial hyperplasia, and basophilic nuclear and cytoplasmic inclusions in mononuclear cell infiltrates and various pulmonary epithelial cells. Granulomatous lymphadenitis with syncytial cells typified the lesions seen in the pigs from France. PCV-like nucleic acid was detected by ISH in lung, pancreas, lymph node, kidney, and liver in pigs from California. Positive signal was also obtained in lymph node sections from pigs from France. Probes for CAV were consistently negative. PK/15 cell cultures inoculated with lung preparations from diseased California pigs and mesenteric lymph node preparations from pigs from France had positive fluorescence by indirect staining for PCV using pooled polyclonal pig sera and hyperimmune rabbit serum and had variable staining with a panel of 7 monoclonal antibodies specific for cell culture contaminant PCV. PCV-like nucleic acid was also detected by ISH in cell cultures. Cytopathic effect was not observed. Electron microscopic examination of inoculated cell cultures revealed 17-nm viral particles morphologically consistent with PCV. No other virus particles were observed. Although genomic analysis for the definitive identification of these viral isolates remains to be done, the evidence provided strongly suggests that these tissue isolates are closely related to, although antigenically distinct from, the original PCV cell culture contaminant.

The use of immunohistochemistry and the polymerase chain reaction for detection of feline leukemia virus and feline sarcoma virus in six cases of feline ocular sarcoma.

Ocular sarcoma was diagnosed by light microscopic examination in enucleated globes (n = 4), orbital tissue biopsy (n = 1) and ocular evisceration contents (n = 1) from six cats. To determine if feline leukemia virus (FeLV) or a replication-defective FeLV, feline sarcoma virus (FeSV), was present in these ocular sarcomas, immunohistochemistry (IHC) and polymerase chain reaction (PCR) for FeLV were utilized. Immunohistochemical staining for FeLV glycoprotein 70 (gp70) was performed on all six formalin-fixed, paraffin-embedded tumors using an avidin-biotin complex technique. DNA was extracted from each specimen and a 166 bp region of the FeLV long-terminal repeat (LTR) was amplified by PCR. All tumors were composed primarily of spindle cells; two neoplasms had PAS-positive basement membrane enveloping areas of spindle cells. All tumors involved the uvea and five of six tumors showed transcleral extension, one of which invaded the optic nerve. Immunohistochemical staining for FeLV gp 70 was negative. PCR to amplify a portion of the FeLV LTR was negative. Based on these findings of these limited number of cases, FeLV/FeSV may not play a role in the tumorigenesis of feline ocular sarcomas. However, additional tumors representing all morphological subtypes should be investigated for the presence of viral antigen and DNA. It is important to determine the etiology and pathogenesis of these malignant ocular sarcomas. If the cell of origin and pathogenesis involve ocular and lenticular injury, and FeLV/FeSV is not present, then the clinical management of cases of feline ocular trauma, uveitis and glaucoma may prevent the development of this tumor.

Cell-mediated cytolysis of equine herpesvirus-infected cells by leukocytes from young vaccinated horses.

The objective of this study was to determine whether the administration of modified-live equine herpesvirus (EHV-1) to young horses with residual maternal antibodies stimulated EHV-specific cytolytic responses, and whether these responses were crossreactive between EHV-1 and EHV-4. Eighteen clinically normal Belgian cross-foals were used in the study and were commingled in two adjacent pens. Skin biopsies were harvested from 16 foals within 24 h of birth and fibroblast cultures were established, expanded and cryopreserved. Beginning at approximately 10 weeks of age, 10 randomly chosen foals were inoculated on days 0, 21, and 43 of the study with a vaccine containing modified-live EHV-1. Blood mononuclear leukocytes were obtained on days 0, 32, and 50 for the assessment of EHV-specific cytolytic activity using 5 h and 18 h chromium release assays. EHV-1-specific antibodies were assessed by enzyme-linked immunosorbent assay using serum collected on days -21, 0, 32, and 50 of the study. Lymphocyte blastogenic tests and bioassays for interferon activity were conducted on day 50. After two vaccinations, mononuclear leukocytes from seven of ten vaccinated foals had cytolytic activity against autologous EHV-1 cells and leukocytes from six of ten lysed EHV-4-infected cells when tested in an 18 h assay. This activity was enhanced by exogenous interleukin 2 and was markedly reduced using target cells from unrelated horses. Cytotoxicity was not detected in a 5 h assay following in vitro stimulation of leukocytes. After three vaccinations, blood leukocytes from 6/6 vaccinated foals and 0/6 unvaccinated foals had proliferative responses EHV-1. There were no significant differences in interferon production by leukocytes from these foals. Twelve foals tested had low concentrations of (maternal) EHV-1-specific antibody prior to vaccination. Five of eight foals tested had increases in EHV-specific antibodies, while 4/4 commingled unvaccinated foals had a decrease or no change in EHV-specific antibodies. These results demonstrate cytotoxic cellular immune responses can be induced in young horses with maternal antibodies following administration of modified-live vaccine.