Immunopotentiation of bovine herpes virus subunit vaccination by interleukin-2.

Cattle were immunized with glycoprotein IV (gIV) from bovine herpes virus-1 (BHV-1). Groups of five animals were then given either 2, 3, 4, or 5 doses of interleukin-2 (IL-2) (0.5 microgram/kg) at 12-hr intervals. Animals that received no IL-2 exhibited specific immune responses that are typical for BHV-1 infection, i.e. enhanced specific cytotoxicity, lymphocyte proliferative responses to gIV, and increased gIV-specific (ELISA) and virus-neutralizing antibodies. Treatment of animals with five doses of IL-2 significantly augmented all of these responses except serum neutralization (P less than 0.05). Furthermore, the dose of IL-2 that was selected did not induce any non-specific responses, i.e. hypergamma-globulinaemia, changes in blood chemistry, increased lymphokine-activated killer (LAK) cell activity, changes in mitogen responsiveness or alterations in the phenotypic profile of circulating lymphocytes. Nor were there any clinical changes associated with IL-2 therapy (e.g. depression, pyrexia, diarrhea). Animals that were treated with less than five doses of IL-2 also exhibited elevated immune responses, but they were not significantly different from untreated immunized controls. Interestingly, animals given five doses of IL-2 responded to minor contaminants present in the gIV preparation. This allows speculation that this dose regimen of IL-2 is not only a potent adjuvant for conventional vaccine immunizing doses, but will also allow the use of minute quantities of antigen for immunization.     

Susceptibility of bovine macrophages to infectious bovine rhinotracheitis virus infection.

Infectious bovine rhinotracheitis virus replicated in cultured bovine alveolar macrophages (AM). However, yields of infectious virus were low, with maximum titers approximately 100 times that of the residual inoculum. Immunofluorescence and electron microscopic studies indicated that the majority of macrophages produced viral antigen, but after infection at a multiplicity of 0.1, only 4.1% of AM produced infectious centers. Virus-infected AM culture supernatants possessed interfering activity, probably due to interferon. Incubation of fresh AM with these fluids rendered them refractory to infection. Although AM from infectious bovine rhinotracheitis virus-immune and -susceptible donors were equally permissive and their susceptibility was unaltered by incubation with bacterial lipopolysaccharide, bovine mammary macrophages which were elicited with lipopolysaccharide became nonpermissive when further incubated for 48 h with 1 microgram of lipopolysaccharide per ml. Under these conditions, infected mammary macrophages failed to synthesize viral DNA, and there was reduced synthesis of "late" viral polypeptides.     

Passive protection to bovine rotavirus (BRV) infection induced by a BRV VP8* produced in plants using a TMV-based vector

Summary. We have previously reported on the use of a tobacco mosaic virus (TMV) vector TMV-30B to express foreign viral antigens for use as experimental immunogens. Here we describe the development of an improved TMV-30B vector that adds a sequence of 7 histidine residues to the C-terminus of recombinant proteins expressed in the vector. We used this TMV-30B-HISc vector to express the VP8* fragment of the VP4 protein from bovine rotavirus (BRV) strain C-486 in plants. Recombinant VP8* protein was purified from N. benthamiana leaves at 7 days post-inoculation by immobilized metal affinity chromatography. The plant-produced VP8* was initially detected using anti-His tag mAb and its antigenic nature was confirmed using both monoclonal and polyclonal specific antisera directed against BRV. Adult female mice, inoculated by the intraperinoteal route with an immunogen containing 4µg of recombinant VP8*, developed a specific and sustained response to the native VP8* from the homologous BRV. Eighty five percent of suckling mice from immunized dams that were challenged with the homologous virus at the fifth day of age were protected from virus as compared to 35% of the pups from mothers immunized with a control protein. These results demonstrate that the plant-produced VP8* was able to induce passive protection in the new born through the immunization of dams. This suggests that the technology presented here provides a simple method for using plants as an inexpensive alternative source for production of recombinant anti-rotavirus antigens.Authors contributed equally to the results presented in this report.     

Retinal dehydrogenase-2 is inhibited by compounds that induce congenital diaphragmatic hernias in rodents

Currently, the etiology of the serious developmental anomaly congenital diaphragmatic hernia (CDH) is unknown. We have used an animal model of CDH to address this issue. We characterized four separate teratogens that produced diaphragmatic defects in embryonic rats that are similar to those in infants with CDH. We then tested the hypothesis that all these agents share the common mechanism of perturbing the retinoid-signaling pathway. Specifically, inhibition of retinal dehydrogenase-2 (RALDH2), a key enzyme necessary for the production of retinoic acid and that is expressed in the developing diaphragm, was assayed by measuring retinoic acid production in cytosolic extracts from an oligodendrocyte cell line. The following compounds all induce posterolateral defects in the rat diaphragm; nitrofen, 4-biphenyl carboxylic acid, bisdiamine, and SB-210661. Importantly, we demonstrate that they all share the common mechanism of inhibiting RALDH2. These data provide an important component of mounting evidence suggesting that the retinoid system warrants consideration in future studies of the etiology of CDH.     

Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome

A competitive PCR (cPCR) assay was developed for monitoring porcine circovirus (PCV) DNA in serum samples from piglets. The cPCR was based on competitive coamplification of a 502- or 506-bp region of the PCV type 1 (PCV1) or PCV2 ORF2, respectively, with a known concentration of competitor DNA, which produced a 761- or 765-bp fragment, respectively. The cPCR was validated by quantification of a known amount of PCV wild-type plasmids. We also used this technique to determine PCV genome copy numbers in infected cells. Furthermore, we measured PCV DNA loads in clinical samples. More than 50% of clinically healthy piglets could harbor both types of PCV. While PCV1 was detected in only 3 of 16 pigs with postweaning multisystemic wasting syndrome (PMWS), all the sick piglets contained PCV2. A comparison of the PCV2 DNA loads of healthy and sick animals revealed a significant difference, indicating that the development of PMWS may require a certain amount of PCV2.     

Assessing drug exposure in rodent toxicity studies without satellite animals

Five major objectives for pharmacokinetic investigations in support of toxicity studies are identified as follows: Assess whether animals exhibited measurable blood concentrations in a dose-dependent manner; estimate average area under the concentration- time curve (AUC)and maximal concentration (C _max )for each treatment group; elucidate general patterns in the concentration-time (CxT)profile, and summarize relationships between CxTand treatment group; determine CxTdependence on day into study; and judge interanimal variability and identify any animals with unusual concentration response. Such objectives are generally addressed in rodent toxicity studies by including satellite animals in the study. Satellite animals are extra animals dosed as per protocol but not subjected to toxicological and pathological observations and tests. Instead, they are used exclusively for the evaluation of pharmacokinetic characteristics of the test compound. In this paper, methods are described for achieving the five listed pharmacokinetic objectives in rodent toxicity studies without the use of satellite animals. A rat toxicity study is presented as an example.     

Particle-mediated DNA immunization of cattle confers long-lasting immunity against bovine herpesvirus-1

Particle-mediated delivery was used as a method to vaccinate ruminants with a DNA vaccine. The optimal conditions for gene gun-based delivery of gold particles into the epidermal layer of the skin were determined. After delivery of the gold particles, an inflammatory response was observed. This response occurred regardless of the presence of plasmid and therefore was a result of the physical disturbance of the skin by the gold particles. To identify transfected cells, a plasmid expressing a green fluorescent protein was delivered into the skin. Fluorescent cells were located primarily in the outermost layers of the epidermis and outside the core of gold particles deposited by the gene gun. Cattle were immunized by gene gun with a plasmid expressing a truncated, secreted form of bovine herpesvirus-1 glycoprotein D. Serum antibody responses, antigen-specific proliferation, and interferon-gamma secretion by peripheral blood lymphocytes were demonstrated. These immune responses were found to be of long duration and sufficient magnitude to protect cattle against challenge with bovine herpesvirus-1, which demonstrates the efficacy of gene gun-based delivery of DNA vaccines to target species.     

Broadening the approaches to developing more effective vaccines

Although vaccination has had a dramatic impact on reducing economic losses due to infectious diseases, vaccination technology has not changed dramatically over the last 200 years. However, with the advent of biotechnology and our understanding of virulence factors of infectious agents combined with our knowledge of the host immune response, we are now witnessing a revolution in the number of new agents which may potentially be controlled by vaccination, as well as the approaches being used to develop vaccines. These approaches include subunit vaccines, genetically modified live vaccines and most recently, polynucleotide vaccines. Pathogens involved in bovine respiratory disease are used as models to describe recent advances in developing new vaccines that have the potential to be safer, more economical and more efficacious. Emphasis will be placed on identification of specific proteins involved in inducing protective immunity and producing these in a mammalian expression system as subunit vaccines formulated with adjuvants. To increase the duration of immunity, the genes encoding these antigens have been introduced directly into animals as polynucleotide vaccines. The benefits and short-comings, as well as the practical problems associated (both scientific and regulatory) with eventual acceptance of these vaccines, are discussed.     

Bovine gut-associated lymphoid tissue--morphologic and functional studies. I. Isolation and characterization of leukocytes from the epithelium and lamina propria of bovine small intestine

A successful technique for the isolation of highly pure suspensions of viable leukocytes from the small intestine of cattle is described. Procedures ranging from mechanical mincing to enzymatic digestion of tissues were compared. The most reliable and reproducible procedure was the sequential treatment of tissues with dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA) in calcium-magnesium-free salt solutions, and collagenase. Two populations of mucosal leukocytes were obtained from the small intestine. One population was derived from within the epithelium (intraepithelial leukocytes, IEL), the second from within the lamina propria (lamina propria leukocytes, LPL). At least 2 X 10(6) viable leukocytes were obtainable from each square centimeter of the intestinal mucosa from either the epithelium or lamina propria. Erythrocyte rosetting and immunofluorescence characterization with conventional antisera and monoclonal antibodies (MAB) demonstrated that IEL were predominantly T cells (60%), with relatively few B cells present (10%), while LPL contained relatively high numbers of B cells (28%) and a reasonable percentage of T cells (45%). Both cell populations proliferate in response to stimulation with T and B cell mitogens. Addition of the thiol compound, 2-mercaptoethanol (2-ME) strongly augmented the mitogenic response of both cell isolates. Human recombinant interleukin-2 (hr-IL-2) in the presence or absence of additional stimuli was found to be able to induce the proliferation of both cell types. These results demonstrate that functional leukocytes can be isolated from the small intestine of cattle, and that they can maintain their responsiveness to both T and B cell mitogens and to exogenous cloned IL-2.