A light and electron microscopic study was performed on pox-like epidermal lesions in an experimentally infected pig. Light microscopical investigation of semithin sections revealed the presence of nuclear vacuoles and of different types of cytoplasmic inclusions. In electron microscopical studies large numbers of both immature and mature virus particles and the cytological changes indicative of pox virus infection were observed. Various types of intra-cytoplasmic inclusions -- i.e. fibrillar inclusions, crystalloid-containing dense inclusions, complex membraneous inclusions and dense homogeneous inclusions -- were encountered in addition to viroplasms and nuclear vacuoles. Because of the presence of vacuoles in nuclei of stratum spinosum cells the diagnosis swine pox by swinepox virus was most probable. These nuclear vacuoles have not been described in swine pox caused by vaccinia virus, the only other known cause of pox in swine.

Purpose: The presence of replicating type C retrovirus in MBT-2 mouse bladder carcinoma cells is reported. This MBT-2 tumor cell line is nowadays globally distributed. The cells have been and are still used to study various aspects of bladder cancer. While studying the phagocytic capacity of MBT-2 cells for BCG organisms by electron microscopic methods, the presence of this retrovirus was noticed.Materials and Methods: MBT-2 cells that were cultured in vitro as well as cells from intravesically and intradermally grown MBT-2 tumors from syngeneic mice were investigated using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques.Results: All samples including the earliest generation MBT-2 cells that could be traced from stocks of other research groups contained the C type retrovirus, suggesting a contamination in all available generations of the MBT-2 cell line.Conclusions: As this tumor cell line is widely used in immunologic studies of the response to bladder cancer, it is important to consider the possible presence of type C viruses and associated antigens, since they could contribute to or interfere with the responses being measured. Studies should be initiated to determine whether viral antigen expression is involved in the immune rejection of MBT-2 bladder cancer. As a consequence, clinical implementation of immunological treatment strategies should not be based on results obtained with the MBT-2 model alone, but preferably should be confirmed with other (bladder) carcinoma models.

A virus with rhabdovirus morphology which proved to be antigenically distinct from rabies virus and vesicular stomatitis virus was isolated from a dolphin that had beached on the Dutch coast. Neutralizing antibodies to this virus were found in several European marine mammal species.

Pasteurellaceae notably P. pneumotropica, have been associated with severe outbreaks of respiratory disease in several species of rodents. Host-specific parasitism of Pasteurellaceae in rodents has hardly been studied. Since host tropism in many bacteria involves adhesive mechanisms, we examined the hemagglutinating (HA) properties of 44 isolates from different rodent species (mouse (15) rat (8), hamster (9), gerbil (10) and Mastomys (2)). Only 13 mouse isolates and the 2 Mastomys isolates hemagglutinated human (type O Rh+) and canine red blood cells (RBCs). No HA was found using RBCs from 10 other animal species. HA was not inhibited by simple sugars and glycoconjugates, but was completely inhibited by heating of bacterial cells for 10 min at 80 or 100 degrees C, partially inhibited by glutaraldehyde and inhibited in a dose-dependent mode by NaIO4, suggesting the involvement of bacterial polysaccharide structures in the HA process. Enrichment procedures did not reveal the presence of HA- subpopulations in HA+ isolates or the presence of HA+ subpopulations in HA- isolates. Electron microscopy revealed the presence of fimbriae both in HA+ and HA- isolates. A regularly structured (RS) layer was detected on cells of part of the HA+ isolates only. Our results suggest that Pasteurellaceae of mice and Mastomys may be related and differ from isolates isolated from other rodent species.

Intravesical administration of Bacillus Calmette-Guérin (BCG) has been shown to be effective in the treatment of patients with superficial bladder cancer. For a better understanding of the mechanism of this antitumor activity, scanning and transmission electron microscope (SEM, TEM) studies were carried out to investigate morphological aspects of the interaction of BCG with the bladder wall in vivo and in vitro. Adherence of BCG to the bladder wall in vivo was studied 1 and 24 h after single or multiple (6×) BCG instillations in intact and in electrocauterized guinea pig bladders. Despite extensive search with SEM for its presence, virtually no BCG was found on the intact urothelium, and BCG was only occasionally observed in the coagulation lesions. SEM and TEM studies revealed adherence and phagocytosis of BCG by the T24 human bladder carcinoma cell line in vitro. Time sequence studies on the phagocytosis and fate of BCG showed that T24 cells are capable of progressively degrading the mycobacteria in phagolysosomes. However, BCG did not alter MHC class II antigen expression on T24 cells in vitro. In contrast, 54 urine sediments and bladder washings of 11 bladder cancer patients, taken prior to or after several intravesical BCG instillations, failed to demonstrate urothelial (tumor) cells showing evidence of BCG phagocytosis (682 cells screened by TEM), while BCG was phagocytized avidly by leukocytes. These data suggest that a direct interaction of BCG with urothelial bladder cells in vivo can be called in question.

Recently morbilliviruses were isolated from harbour seals (Phoca vitulina) in North West Europe (phocid distemper virus-1: PDV-1) and from Baikal seals (Phoca sibirica) in Siberia (phocid distemper virus-2:PDV-2) during outbreaks of severe disease which resembled distemper in dogs. PDV-1 and PDV-2 were passaged in SPF dogs, in which they caused distemper-like disease symptoms, and were subsequently passaged in Vero cells in which they caused cytopathic changes. PDV-1, PDV-2, and canine distemper virus (CDV) were compared with respect to their biological, morphological, physical, protein chemical, and antigenic properties. It was concluded that PDV-1 should be considered a newly recognized member of the genusMorbillivirus, whereas PDV-2 proved to be quite similar if not identical to CDV.

During a recent disease outbreak among harbour seals (Phoca vitulina) in the North and Baltic seas, more than 17 000 animals have died. The clinical symptoms and pathological findings were similar to those of distemper in dogs. Based on a seroepizootiological study, using a canine distemper virus (CDV) neutralization assay, it was shown that CDV or a closely related morbillivirus (phocid distemper virus-PDV) was primary cause of the disease. The virus was isolated in cell culture from the organs of dead seals and characterized as a morbillivirus by serology (immunofluorescence neutralization and enzyme-linked immunosorbent assays) and by negative contrast electron microscopy. Experimental infection of SPF dogs resulted in the development of mild clinical signs of distemper and CDV-neutralizing antibodies. The disease was reproduced in seals by experimental inoculation of organ material from animals that had died during the outbreak. However, seals that had been vaccinated with experimental inactivated CDV vaccines were protected against this challenge. This fulfilled the last of Koch's postulates, confirming that the morbillivirus isolated from the seal organs, was the primary cause of the disease outbreak. The recent demonstration of the presence of a similar virus in Lake Baikal seals (Phoca sibirica), which infected these Siberian seals 1 year before the north-western European seals were infected, raises new questions about the origin of this infectious disease in pinnipeds.

The parallel application of two electron microscopic immunogold labeling procedures was used to assess the surface exposure and accessibility of gonococcal and meningococcal surface antigens. Monoclonal antibodies were used as markers for the surface antigens, i.e., outer membrane proteins and lipooligosaccharides. To evaluate the labeling densities obtained after incubation of whole bacteria in suspension or ultrathin cryosections of bacteria, a method of electron microscopic quantitation was developed. Incubation of whole bacterial suspensions with monoclonal antibodies and protein A-gold resulted in specific labeling of the bacterial surfaces. However, the labeling densities varied largely in each cell. By contrast, cryosections showed uniform heavy labeling densities at the surface of the outer membranes of all cells. Apparently, by sectioning the cells the antigen-masking barrier could be evaded, and steric hindrance was no longer restrictive. Thus, a better estimate of both the presence and the surface exposure, i.e., the accessibility of antigens, could be made. Such information is essential for us to better understand host-bacterial interactions and to develop new vaccines.

A scanning electron microscopic study was carried out to compare the in vivo pathogenicity of two strains of Vibrio cholerae in an adult rabbit ligated-gut test model. V. cholerae C5 (serotype Ogawa, biotype El Tor), a motile strain possessing hemagglutinating activity in vitro, and C21 (serotype Ogawa, classical biotype), a nonmotile strain possessing no hemagglutinating activity, were tested. Tissue samples from small intestinal loops were examined 3, 6, 9, and 12 h postinoculation. Contradictory to most published data, neither hemagglutinating activity nor motility appeared to be essential prerequisites for the pathogenesis of cholera in the experimental animal model used: nonmotile hemagglutinin-negative strain C21 adhered to and colonized the small intestine at least to the same extent as did motile hemagglutinin-positive strain C5. Maximum colonization was seen at 9 h postinoculation for both strains. C5 and C21 vibrios caused comparable damage to the villi of the small intestine. The villous epithelium showed only mild changes during the first 9 h postinoculation. However, after 12 h the epithelium was seriously damaged concomitant with a decrease in the number of vibrios. Many villi showed partial or total denudation, owing to repelled epithelium, leaving a bare basal lamina with only some to moderate numbers of vibrios attached. Since similar changes were induced by pure cholera enterotoxin, these changes were likely the result of excessive fluid accumulation. From this study it is concluded that, at least in the animal model used, factors other than hemagglutinating activity and motility may also play a role in the association of V. cholerae with the small intestinal surface.

The frequency of precursor cells capable of giving rise to cells with characteristics of mucosal mast cells in tissues from thymus-bearing and non-thymus-bearing (nude) mice orally infected with Trichinella spiralis was determined with an in vitro assay. Analysis of the frequency of mast cell precursors in bone marrow, blood, spleen and small intestinal tissue revealed similar frequencies of mast cell precursors in bone marrow from both thymus-bearing and athymic mice. These frequencies in bone marrow were not affected by infection. However, in blood and spleen from thymus-bearing mice at Day 7 post-infection (p.i.), and in the gut at Day 14 p.i., significant increases of mast cell precursor frequencies were detected. In contrast, no significant increase was observed in the tissues of infected nude mice. These data are in accordance with in vivo findings, indicating that a mucosal mast cell response in the gut is both thymus and antigen dependent. It was concluded that a mucosal mast cell response to infection with T. spiralis is probably due to local proliferation and maturation of residing mast cell precursors, that this response might be amplified by an influx of precursor cells from the blood into the gut, and that both phenomena are T-cell dependent.

Immuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture. The procedure was monitored by testing the respective fractions in an infectivity titration system, in an ELISA, in a haemagglutination assay and by negative contrast electron microscopy to quantify CPV or CPV antigen. The degree of purification was further estimated by testing the fractions for total protein content in a colorimetric method, for bovine scrum albumin content in an ELISA and by SDS-PAGE. Over 99% of the contaminating proteins proved to be removed, and 20% or 70–90% of infectious CPV or CPV antigen, respectively, was recovered.

This report describes the first isolation and partial characterization of a herpesvirus from the harbor seal(Phoca vitulina). The virus was isolated during a disease outbreak in a group of young seals nursed in an seal orphanage in The Netherlands. Almost half of the seals died with symptoms of acute pneumonia and focal hepatitis and the virus was isolated of organs of most of the dead animals. Seven out of ten seals of which paired serum samples were obtained showed seroconversion in a virus neutralization test during this outbreak. The virus was tentatively characterized as a herpesvirus (seal herpesvirus: SeHV or phocid herpesvirus 1) on the basis of its characteristic morphology in electron microscopy, buoyant density in sucrose, sensitivity to ether and heat treatment and its antigenic relationship with other probable members of the Alphaherpesvirinae subfamily. The virus caused cytopathic changes within 24 hours after inoculation in seal kidney cells, consisting of a focal rounding of cells and syncytium formation. No cytopathic changes were observed in the cells of nine other mammalian species tested.

The origin, kinetics and role of eosinophilic granulocytes in immune defense and inflammatory response in parasitism are reviewed. Particular attention was paid to the toxic action of eosinophilic granulocytes on parasites and the biochemical reactions which are of importance in these cases. Reference was also made to the regulatory role of eosinophilic granulocytes in inflammatory reactions, particularly in regard to mast cells. It was concluded that (1) several new insights into the role of eosinophilic granulocytes were gained in recent years, (2) that these cells can at any rate be said to have a dual function (viz. a directly parasitotoxic and regulatory role) and (3) a definite statement regarding the role of eosinophilic granulocytes in helminthic infections cannot so far be made.