Polymerase chain reaction amplification of pseudorabies virus DNA from acutely and latently infected cells

A characteristic of alphaherpesviruses, including pseudorabies virus (PRV), is that the acute phase of the disease is followed by lifelong latency. Latently infected animals are asymptomatic but can transmit reactivated virus. Corticosteroid administration, tissue explanation, blot- and in situ hybridizations have been used to demonstrate the presence of latent PRV infections. The use of blot hybridization as a convenient method for defining the incidence of PRV infections in swine herds has been hampered by the detection limit of this method. The objective of this study was to increase this sensitivity of blot hybridization by polymerase chain reaction (PCR) amplification of target sequences. Two sets of 20-mer primers were synthesized and used to amplify gX and gII glycoprotein gene sequences in two different strains of PRV. The specificity of the amplification was verified by Southern blot hybridization and restriction endonuclease analysis of the amplified fragments. Amplification of target sequences by PRC increased their detection limit by a factor of at least 10(5). Porcine ganglion samples, in which latency had been demonstrated by in vitro explanation, were analyzed by PCR together with positive and negative controls. Duplicate slot blot analyses of a portion of the amplified products were used to demonstrate latency in seven of eight samples. It was concluded that blot hybridization of PCR amplified DNA appears to be both a sensitive and convenient method for the detection of PRV induced latency.     

Characteristics of apparent derivatives of the 2512 strain of infectious bursal disease virus when used as vaccines

The 2512 infectious bursal disease virus (IBDV) strain maintained in the authors' laboratory was compared with apparent 2512 IBDV isolates designated I-2512, P-2512, and H-2512. The latter three viruses were obtained from different sources and, ostensibly, had their origin in the Purdue laboratory. Their effects on immunogenicity, transmissibility, pathogenicity, and cell cultures varied. One of these isolates was said to be only 2 embryo passages higher than the original seed virus in our laboratory. Included in the study, also for comparison, was a cell-cultured-adapted 2512-cloned attenuated virus. The findings emphasize changes that may occur in the identity of a virus from manipulation, mutation, storage, errors in labeling, or other factors. These characteristics should be identified if the virus is to be used as a vaccine, in research, or for other purposes. The need for well-characterized reference strains in repositories is discussed relative to their importance in potential vaccine research and development.     

Quantification of PCV2 capsid transcript in peripheral blood mononuclear cells (PBMCs) in vitro

The presence of PCV2 DNA or spliced capsid mRNA (Cap mRNA) for viral replication was assessed following addition of PCV2 to resting or concanavalin A (ConA) stimulated peripheral blood mononuclear cells (PBMCs). Real-time PCR or real-time RT-PCR assays were used to measure viral DNA or Cap mRNA, respectively. The study demonstrated that PCV2 replication increased in infected PBMCs over time. Replication within infected PBMCs was significantly (P<0.05) increased when PBMCs were stimulated with ConA, compared to unstimulated PBMCs. The data showed a strong correlation between the level of PCV2 Cap mRNA and the level of viral DNA in the ConA stimulated PBMCs. Replication of PCV2 was also assessed in T lymphocyte- and monocyte/macrophage-enriched or monocyte/macrophage-depleted PBMC populations which had been stimulated with ConA for 3 days. It was demonstrated that the enriched T lymphocytes and the monocyte/macrophage-depleted PBMCs had significantly higher Cap mRNA and viral DNA levels (P<0.05) compared to the monocyte/macrophage-enriched population, indicating that in addition to monocytes/macrophages, PCV2 replicates in lymphocytes, particularly T lymphocytes following stimulation. These results suggest that the presence of activated T lymphocytes may play an important role in PCV2 replication and potentially the development of clinical disease.     

Porcine circovirus type 2 (PCV2) distribution and replication in tissues and immune cells in early infected pigs

Replication of porcine circovirus type 2 (PCV2) in pigs, as measured by spliced capsid mRNA (Cap mRNA) and viral DNA, was investigated following experimental infection. Peripheral blood mononuclear cells (PBMCs), and tissue from bronchial lymph nodes (BLN), inguinal lymph nodes (ILN), tonsils, lungs, liver, kidneys, spleen and thymus from infected pigs on different days post-infection (DPI) were assessed. PCV2 replication differed dramatically between tissues from the same infected pig. The virus actively replicated in most tested tissues at 14DPI in association with increased PCV2 associated lesions and PCV2 antigen levels, although no clinical signs correlated with PCV2 associated disease were observed in infected pigs during the course of the study. The PCV2 Cap mRNA was detected only at 13DPI in PBMCs from infected pigs, suggesting replication of the virus in circulating blood is transient and not a major site for PCV2 replication in vivo. Evaluation of the Cap mRNA and viral DNA synthesis in T and B lymphocyte and monocyte populations from PBMCs and BLN at various intervals post-inoculation revealed replication of PCV2 in all cell subpopulations; however, viral replication in B lymphocytes was greater than observed in mononuclear cells isolated from BLN at 14DPI indicating that B lymphocytes may be an important cell population for PCV2 replication. These findings further our understanding of the cell types permissive for PCV2 replication and the pathogenesis of PCV2 infection in vivo.     

An allogeneic hybrid-cell fusion vaccine against canine mammary cancer

Mammary cancer is among the most prevalent of canine tumors frequently resulting in death due to metastatic disease. Most tumors fail to raise an effective immune reaction making improving immune recognition a priority. Hybrid-cell fusion strategies have been employed to load dendritic cell populations with tumor cell antigens to stimulate immune recognition; however, recovery, heterogeneity and quality of primary cells from patients present enormous challenges. We employed allogeneic cell lines to develop an improved hybrid-cell fusion strategy and evaluated immune reactions in normal laboratory beagles. Such a strategy relies on enhanced immune recognition of allogeneic tumor cell antigens by antigen presenting cells. Optimized PEG-promoted fusions between uniquely stained canine mammary tumor CMT12 or CMT28 cells and a dendritic cell-like DH82 cell fusion partner resulted in greater than 40% hybrid-cell fusion populations by flow cytometry and fluorescence microscopy. Hybrid-cell fusions were delivered by direct ultrasound guided injection into popliteal lymph nodes of laboratory beagles. Only hybrid-cell fusions provided statistically significant enhancement of cell-mediated immunity ((51)Cr-release assay) compared to innate reactions in na?ve vehicle injected dogs while dogs vaccinated with either single cell component alone did not. Vaccination with hybrid-cell fusions enhanced IFN-gamma expression in sorted CD8+ and CD4+ cells but not in CD4-/CD8- cells consistent with a CTL response. Cell-mediated immune assays revealed strong reactions against matched (vaccine component) CMT cells and unmatched CMT cells indicative of an immune response to mammary cancer antigens common to both cell lines. These results provide proof of principle for development of an allogeneic vaccination strategy against canine mammary cancer.     

Agammaglobulinemia in a horse.

Immunologic deficiency was suspected in an 18-month-old Standardbred horse with persistent fever, multifocal bacterial infection, and neutropenia with a large number of immature neutrophils. Serum protein electrophoresis revealed marked depression of the gamma-globulin fraction (0.2 g/100 ml). Immunologic testing and histologic examination of lymphoid tissues identified the immune deficit as agammaglobulinemia. Serum concentrations of immunoglobulin (Ig)G and IgG(T) were initially low and declined with time; IgM and IgA were not detectable. The horse failed to produce antibodies when inoculated with foreign antigens but had a positive cell-mediated skin reaction to intradermal phytolectin injection, and lymphocytes responded normally to in vitro stimulation by mitogens. Histologic examination of lymphoid tissues revealed absence of germinal centers and plasma cells.     

Evaluation of local and systemic immune responses induced by intramuscular injection of a Mycoplasma hyopneumoniae bacterin to pigs

OBJECTIVE: To evaluate immune responses induced by administration of Mycoplasma hyopneumoniae bacterin to pigs. Animals-60 healthy 7- to 10-day-old cross-bred boars. PROCEDURE: Pigs were assigned to 1 of 4 pig groups (15 pigs/group): vaccinated, challenged; vaccinated, nonchallenged; nonvaccinated, challenged; nonvaccinated, nonchallenged. Vaccinated pigs received IM injections of a mycoplasma bacterin on days 0 and 14, whereas nonvaccinated pigs received saline (0.9% NaCl) solution. Pigs in the challenged groups were inoculated intratracheally with M hyopneumoniae on day 42. Pigs were euthanatized and necropsied 41, 44, 48, and 70 days after the first vaccination, and proportion of lung surface with pneumonic lesions was determined. Percentage of lymphocyte subpopulations and number of interferon-gamma (IFN-gamma) secreting lymphocytes in blood and tissues, cytokine and antibody concentrations in bronchoalveolar lavage (BAL) fluid, and serum antibody concentrations were determined. RESULTS: Vaccination against and infection with M hyopneumoniae induced a local mucosal immune response in the respiratory tract of pigs. Proportion of lung surface with pneumonic lesions in vaccinated challenged pigs was reduced on day 70, compared with nonvaccinated challenged pigs. Vaccination stimulated the production of M hyopneumoniae-specific IFN-gamma secreting blood lymphocytes. Tumor necrosis factor-alpha concentration in BAL fluid on day 70 was increased in nonvaccinated challenged pigs, compared with vaccinated challenged pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination against M hyopneumoniae induced local, mucosal, humoral, and cellular immune responses. Moreover, vaccination reduced the severity of lung lesions in challenged pigs, suggesting that mucosal antibodies, mediation of the inflammatory response, and cell-mediated immune responses are important for control of mycoplasmal pneumonia in pigs.