Pre-registration efficacy study of a novel marker vaccine against classical swine fever on maternally derived antibody positive (MDA+) target animals.

Maternally Derived Antibodies (MDA) can have a negative effect on the efficacy of live attenuated vaccines against classical swine fever (CSF). For this reason, a marker vaccine candidate CP7_E2alf was tested for its efficacy in the presence of MDA. Pregnant sows were vaccinated four weeks before farrowing with CSF virus (CSFV) strain "Thiverval". A total of 40 piglets with MDAs were included in this study. At six weeks of age the piglets were allocated into three treatment groups using generalized randomized block design (GRBD) blocking on serological status and pen location. Of the 40 piglets with MDAs, 30 piglets were vaccinated either orally (n = 15) or intramuscularly (n = 15) with a single dose of vaccine candidate produced under Good Laboratory Practice (GLP) conditions. The ten remaining piglets were allocated into the untreated control group. All 40 piglets were oronasally challenged with 2 ml of the highly virulent CSFV strain "Koslov" 14 days after vaccination. It was revealed that presence of MDAs negatively influences the efficacy of the live marker vaccine candidate, however, the extent of this negative impact depends on the route of vaccine administration. Based on our observations, intramuscular vaccination is recommended during CSF control programs in order to develop superior immune protection.

Pre-registration efficacy study of a novel marker vaccine against classical swine fever on Maternally Derived Antibody negative (MDA-) target animals.

Classical swine fever (CSF) marker vaccine candidate CP7_E2alf produced under Good Laboratory Practice (GLP) conditions by Pfizer was tested on 40 six-week-old MDA-piglets according to the European Pharmacopoeia (Ph.Eur.) requirements. Single doses of CP7_E2alf were given to 15 piglets orally, while 15 other piglets were intramuscularly vaccinated. Ten additional animals were included as unvaccinated controls. All piglets were oronasally challenged with the highly virulent CSF virus (CSFV) strain "Koslov" 14 days after vaccination. CP7_E2alf administered i.m. provided a complete protection, while p.o. administratrion triggered only partial protection. The level of protection was determined by the development of clinical signs, viraemia and rate of mortality. The vaccine candidate met the criteria of Ph. Eur Monograph 0065, "Swine-fever vaccine (live, prepared in cell cultures), classical" 7th Edition, which claims the efficacy test is invalid if fewer than 50 per cent of the control piglets display typical signs of serious infection of CSF or die, and if fewer than 100 per cent of the control piglets show clinical signs of disease within 21 days following challenge. Fulfilling these validity criteria is a key step in the registration procedure for a vaccine candidate to become openly available.

Cytokine and immunoglobulin isotype profiles during CP7_E2alf vaccination against a challenge with the highly virulent Koslov strain of classical swine fever virus.

CP7_E2alf is a promising marker vaccine candidate against classical swine fever (CSF). To better understand the mechanisms of protection, cytokine and isotype-specific antibody profiles were investigated in CP7_E2alf vaccinated pigs before and after challenge with the highly virulent CSFV strain "Koslov" at 14 days or 6 months post-vaccination. The interference of vaccination with CSFV pathogeny-related cytokine responses, previously described following a moderately virulent challenge, was confirmed. However, the levels of additional cytokines, TNF-α and IL-6, were significantly attenuated by vaccination following highly virulent challenge. This vaccine interference with cytokine response was not dependent on the immunization route or the consequence of competition between vaccine and challenge strain. Interestingly, IFN-γ enhancement and persistent high IgG2 levels suggested an important role of cell-mediated immunity in long-term protection against CSFV induced by CP7_E2alf vaccination. IgA production also revealed a stimulation of mucosal immunity, especially after oral administration of the vaccine.

Occurrence of atypical myxomatosis in Central Europe: clinical and virological examinations.

An outbreak of the atypical form of myxomatosis struck a rabbit farm in Hungary. The animals had previously been vaccinated with a vaccine containing Shope rabbit fibroma virus strain. The disease appeared in winter when the presence of mosquitoes and fleas is not common. The virus was isolated from an eyelid specimen of a naturally infected rabbit. The surviving animals were observed for four weeks, blood samples were collected and, after euthanasia, organ specimens were also examined by morphological methods including pathology and electron microscopy. Serum samples were examined by virus neutralisation for antibodies. Genetic analysis of the isolated virus was carried out by polymerase chain reaction (PCR) and direct sequencing. The primers were designed on the basis of the major envelope gene (Env) of the Lausanne reference strain in the GenBank. The viral proteins were examined by SDS-PAGE. The isolated virus (ref. no.: BP04/2001) was able to infect the susceptible animals directly, by contact. The disease was characterised by respiratory symptoms of the upper tracheal tract, conjunctivitis and high mortality by the 11th-14th day. Aerogenic infection with strain BP04/2001 resulted in 100% morbidity among the susceptible animals. Sequencing of the amplified 400-bp-long DNA revealed 97% homology with the Env gene of the Lausanne strain, which proves that strain BP04/2001 is a variant of the Lausanne strain having been enzootic throughout Europe. The live vaccine strain used in Hungary against myxomatosis, which is also a Lausanne-derived strain, protected the animals. According to the protein analysis a protein of 200 kDa in size is not expressed in strain BP04/2001. This is the first report on atypical myxomatosis in Central Europe. The virus spreads by airborne transmission and may cause severe losses in the rabbit population.

Pathogenicity of a bovine viral diarrhoea virus strain in pregnant sows: short communication.

The biological properties of bovine viral diarrhoea virus (BVDV) strain Oregon C24V were studied after intranasal and subcutaneous infection of pregnant sows. This virus strain is widely used in Hungary for immunising cattle against bovine viral diarrhoea (BVD). Based upon the results of the clinical, gross pathological, histopathological and virological examinations it can be established that the given strain caused asymptomatic infection and serological conversion in sows that were in the second third of gestation. The virus caused clinically apparent disease in some of the piglets born at term, which indicates that it had crossed the placenta. More than half (57%) of the live-born piglets died within 60 days of birth. The sows and their progeny did not shed the virus. BVDV infection has great differential diagnostic importance in pigs, as classical swine fever (CSF) virus strains of reduced virulence cause similar clinical symptoms and gross and histopathological changes.

Pathogenesis of primary respiratory disease induced by isolates from a new genetic cluster of bovine viral diarrhea virus type I.

The pathogenesis of infection induced by cytopathogenic isolates from the newly identified genetic cluster Id of bovine viral diarrhea virus (BVDV) type I was studied in two experimental infections of previously seronegative, immunocompetent calves. Experiment 1 focused on the evaluation of clinical patterns, viremia, and serological responses. All infected calves in this experiment developed respiratory symptoms and seroconverted to BVDV positivity. Contact calves also contracted a respiratory tract infection following exposure to infected animals. Viremia was demonstrated between postinfection days 2 and 17, and the virus was detected in organ specimens of all but one each of the infected and contact calves. In experiment 2, the distribution of BVDV in various tissues of calves euthanized at defined days postinfection was studied. In two of these calves recurrent shedding of BVDV in nasal secretions was shown. BVDV was detected in various tissues of all infected calves throughout the experiment and also following seroconversion and the clearance of BVDV from the circulatory system. Despite the widespread distribution of the virus in various organs, significant tissue damage was found mainly in respiratory tract and lymphoid tissues. These experiments revealed that viruses from cluster Id of BVDV are able to induce primary respiratory disease in previously seronegative, immunocompetent calves. Contact transmission and virus recurrence, contrary to observations from acute experimental infections with noncytopathogenic BVDV, are likely to reflect differences in biological features of these cytopathogenic isolates. Virus shedding and its presence in tissues following peripheral clearance and in the presence of antibodies may have implications in the diagnosis, pathogenesis, and epidemiology of BVDV-induced syndromes in cattle.

Synergistic potentiating effect of D(+)-mannose, orotic, and hippuric acid sodium salt on selective toxicity of a mixture of 13 substances of the circulatory system in culture for various tumor cell lines.

Despite global immune system abnormalities in the autoimmune deficiency syndrome, the incidence of only a few tumor types increases, and the degree of immunosuppression does not seem to be critical in the development of these tumors, indicating that the immune system does not prevent tumor development. Consequently, because tumors do not develop in most individuals, other defense systems may exist. We demonstrated previously that 13 substances in the circulatory system acting synergistically induced apoptosis in vitro and in vivo in different tumor cell lines, but not in normal cells and animals. We investigated another 17 compounds and five ions in the circulatory system to determine their participation in the defense provided by the 13 substances. Three of the 17 substances but no ions had a potentiating effect on the mixture of substances used previously. The new 16-component mixture suppressed in vitro growth of six human and murine tumor cell lines, including multidrug-resistant tumor cells, without cytotoxic effects in two normal cell lines. The selectivity also was demonstrated by investigating the mixture's effect over time on tumor and normal cell growth.

Intense picosecond X-Ray pulses from laser plasmas by use of nanostructured "Velvet" targets

We describe the optical, radiative, and laser-plasma physics of a new type of nanostructured surface especially promising as a very high absorption target for high-peak-power subpicosecond laser-matter interaction. This oriented-nanowire material, irradiated by 1 ps pulses at intensities up to 10(17) W cm(-2), produces picosecond soft x-ray pulses 50x more efficiently than do solid targets. We compare this to "smoke" or metallic clusters, and solid nanogroove-grating surfaces; the "metal-velvet" targets combine the high yield of smoke targets with the brief emission of grating surfaces.

Epidemiology of syphilis in Hungary between 1952 and 1996.

Trends in morbidity from syphilis in Hungary between 1952 and 1996 were analysed. The incidence of syphilis/100,000 inhabitants declined rapidly owing to the public health and therapeutic measures of the early 1950s (1952: total=73.6, early infections=60.2; 1962: total=13.7, early infections=8.7). After a temporary, slight increase until 1973 the number of reported syphilis cases declined continuously between 1978 and 1989 (1989: total=0.9, early infections=0.84). In 1994 a marked increase occurred when compared with 1993 (1993: total=early: 1.4. 1994: total=2.3, early infections=2.2). Incidence trends were statistically analysed using Chi-square test and linear regression. Chi-square analysis showed that the changes in the incidence of total and early syphilis are significant (P<0.00001) comparing the time intervals 1952-1962 with 1962-1966 and 1975-1979 with 1988-1992. The same trends were found using the linear regression test, except for the time interval of 1960-1973.

Studies on the pathogenicity of bovine herpesvirus type 5 in sheep.

Four Merino lambs were intranasally inoculated with bovine herpesvirus type 5 (BHV-5) reference strain N569. Two lambs were mock-inoculated as negative controls. The virus-inoculated animals developed apathy, inappetence, rhinitis, nasal, ocular and genital discharge, slight diarrhea and neurological disorders, like tremor and salivation. BHV-5 was isolated from the nasal discharge in two of the animals, while the polymerase chain reaction (PCR) detected the virus in all the infected lambs. Two lambs died on post infection day (PID) 13, while the other two infected animals were euthanized on PID 15 and 30. Gross pathological changes were not observed, however, histopathological examinations revealed diffuse nonsuppurative meningo-encephalitis in all infected animals. Viral antigen was detected by immunohistochemistry and viral nucleic acid was revealed by in situ hybridization in the brain of the two lambs, which died on PID 13. The virus was demonstrated by virus isolation and by PCR from different organs of all the infected animals. Slight rise of antibodies was observed in the infected animals from PID 15. The results show that BHV-5 is able to cross the species barrier and may establish infection in sheep.

Persistently infected cattle stabilise bovine viral diarrhea virus leading to herd specific strains.

Animals persistently infected with BVDV are important in the epizootiology of the Bovine Viral Diarrhea (BVD) because they are a permanent source of contamination within a herd. These animals produce large quantities of virus and have, therefore, been proposed as responsible for generating antigenic variability. However, limited studies have failed to detect antigenic or genetic changes in viruses isolated at different time from persistently infected animals. One hypothesis to account for this stability is that the immunotolerance is accompanied by a selection against antigenic change. The presence of an immunotolerant persistently infected (IPI) animal in a herd would in turn lead to herd specific strains. To verify this hypothesis, we compared 17 BVDV strains isolated from IPI animals from 3 herds of Eastern Belgium. The comparison was based on the sequence of a 389 bp fragment of E2--a gene encoding for a highly variable glycoprotein. Sequences were strongly conserved within herds but were quite different between herds, indicating that BVDV herd-specific strains do exist and are associated with the presence of IPI animals.

Studies on tumourous and other urogenital patients with respect to antigens of oncogenic adenovirus.

The possible connection of viruses with tumours was investigated by serologic examinations. Concerning the presence of antibodies against adenoviruses, especially those against the early non-virion antigens of oncogenic adenovirus type 12, approximately 4000 tests were made with sera of 446 urogenital patients with and without tumours and 70 ones with internal diseases. It was found by complement fixation tests that antibodies against nonvirion antigens of adenoviruses were present in 53% of urogenital patients suffering from malignant tumours and prostatic hypertrophy, in 18% of non-tumourous urological patients and in 4% of patients with internal diseases, respectively. The results suggest that adenoviruses may play a role in tumourous diseases of the urogenital organs.

Studies on tumourous and other urogenital patients with respect to antigens of oncogenic adenovirus.

The possible connection of viruses with tumours was investigated by serologic examinations. Concerning the presence of antibodies against adenoviruses, especially those against the early non-virion antigen of oncogenic adenovirus type 12, approximately 4000 tests were made with sera of 446 urogenital patients with and without tumours and 70 ones with internal diseases. It was found by complement fixation tests that antibodies against non-virion antigens of adenoviruses were present in 53% of urogenital patients suffering from malignant tumours and prostatic hypertrophy, in 18% of non-tumourous urological patients and in 4% of patients with internal diseases, respectively. The results suggest that adenoviruses may play a role in tumourous diseases of the urogenital organs.

Theoretical and literary evidence for the existence of the passive antitumor defence system.

It is well documented that despite global abnormalities of the immune system in AIDS and other immune deficiency diseases or in immunosuppressed patients the incidence of only a few kinds of tumour increases and even in the development of tumours in question the degree of immunosuppression seems not to be a critical factor. It results from this that the known immune system has no significant role in the mechanism that prevents the development of tumours. Consequently, the fact that tumours do not develop in the majority of the population during their lifetime, indicates the existence of other defence systems. We assumed that the defence is made by small substances of the circulatory system. Substantial evidence exists that the uptake of the majority of these substances is increased and unregulated by tumour cells and proportional to their availability. This feature of tumour cells may be fatal when the number of cells is still low and there are abundant substances in their environment because some substances may be toxic if their concentrations can reach high level in the cells. Thus, the arising cancer cells die in the majority of the population during their lifetime if the number of cells arisen is not too high, or the concentrations of the required substances are not too low. To our hypothesis, the above effects of the physiological mixture of the given molecules in the blood form the Passive Antitumor Defence System (PADS). This hypothesis is confirmed by our experiments and supported by epidemiological, clinical observations and other literary data.

Apoptosis of tumor cells induced by substances of the circulatory system.

Despite global abnormalities of the immune system, such as in AIDS, the incidence of only a few kinds of tumor increases, and even in the development of these tumors the degree of immunosuppression seems not to be a critical factor. This means that the known immune system has no significant role in the tumor preventing mechanism. Thus, the fact that tumors do not develop in the majority of the population during their lifetime, indicates the existence of an additional defense mechanism of the immune system. We demonstrated previously that this defense is produced by the synergistic action of certain substances of the circulatory system. Here we report that the substances taking part in the defense induced, but only when they were used together, the apoptosis of tumor cells, but not normal cells, as was detected by different methods. Other substances of the circulatory system did not show similar effects. These results further support the existence of the mentioned defense mechanism called by us the Passive Antitumor Defense System.

Suspected adverse drug reactions in the Hungarian veterinary practice between 1982 and 1992.

The most characteristic adverse drug reactions observed after the use of home-manufactured or imported veterinary medicinal products in Hungary between 1982 and 1992 included toxicosis, severe local reaction, lack of efficacy, and presence of residues in the edible tissues of food animals. The causes of adverse drug reactions comprised manufacturing defects, lack of chemical or microbiological stability, misuse or extra label use, and neglecting the warnings in the directions for use. Collection and analysis of data relating to adverse drug reactions are indispensable for the prevention of similar cases. The authorities can facilitate data collection by supplying veterinary practitioners with the necessary report forms.

Inhibition of the growth of a murine and various human tumor cell lines in culture and in mice by mixture of certain substances of the circulatory system.

It is well documented that despite global abnormalities of the immune system in AIDS and other immune deficiency diseases or in immunosuppressed patients, the incidence of only a few kinds of tumor increases, and that the degree of immunosuppression seems not to be a critical factor in the development of even these tumors. The fact that tumors do not develop in the majority of population during their lifetime, despite the ineffectiveness of the known immune system against the majority of tumors, can only be explained by hypothesizing that the living system has an additional defense mechanism against tumors. On the bases of literary data, it can be assumed that the effective agents of this defense mechanism are certain substances of the circulatory system. We proved this hypothesis by being able to select thirteen substances of the circulatory system from 71 compounds tested, using the synergistic tumor cell-killing effect as criteria. The mixture containing the thirteen substances (L-tryptophan, L-tyrosine, L-methionine, L(-)-malate, L-ascorbate, L-arginine, L-phenylalanine, L-histidine, 2-deoxy-D-ribose, d-biotin, pyridoxine, adenine and riboflavin) had a cytotoxic effect against Sp2/0-Ag14 mouse and K562, HEp-2, HeLa and Caco-2 human tumor cell lines in well-controlled conditions, but it was not cytotoxic against Vero normal cell line. The mixture of the above substances increased significantly the survival time of mice (T/C% 148.1) injected i.p. with Sp2/0-Ag14 mouse myeloma cells by killing more than 2 logs (99%) of the cells. Approximately the same 2 logs cell kill was found counting the Sp2/0-Ag14 cells in the ascitic fluid of control and treated animals after finishing treatment. The above mixture slowed down the growth of HeLa solid tumor significantly (T/C%, the least value 35.7). The weight loss of control and treated group during treatment did not differ significantly.

Suppressed phagocytosis but promoted bacterial growth upon the effect of virus free supernatants of infected lymphocytes.

The pathomechanism of suppressed phagocytosis and bacterial superinfections which follow viral diseases is not completely understood. Both polymorphonuclear leukocytes (PMNL) and mononuclear phagocytes (MNPh) as well as bacterial growth are controlled by several cytokines produced by other immune cells. The effect of disturbed production of cytokines on phagocytes and microbes have not been studied yet. Peripheral T lymphocytes were infected with human adenoviruses (Ad) and herpes simplex virus type 1 (HSV-1) or were activated by phytohaemagglutinin (PHA), then culture supernatants containing no infectious virus were mixed to phagocytes swallowing viable Staphylococcus aureus. Influence of supernatants on eukaryotic and bacterial cell growth was compared to cytokine assays. Supernatant mediators, different from interferon (IFN) and tumour necrosis factor (TNF), induced by oncogenic Ad-12 or HSV-1 diminished phagocytosis of both PMNL and MNPh in a dose dependent manner, promoted bacterial growth free and inside MNPh, while those of latent Ad-5 and nononcogenic Ad-8 exerted moderate effects. Plating efficiency of HEp-2 cells was decreased by all of them. Supernatants of PHA treated lymphocytes containing IFN-gamma enhanced both phagocytosis and bacterial replication free and inside MNPh, while it suppressed HEp-2 plating. Neither viruses nor PHA affected phagocytic process directly. Staphylococci inside PMNL were not affected. Presumably, production of a single mediator by infected T lymphocytes is responsible for the multiple effects. Relationship with another soluble factors is discussed.

Follow-up of the effect of BCG vaccine treatment in bladder tumour patients.

The effect of intravesical BCG vaccine treatment in 38 patients with superficial bladder tumour after TUR (Transurethral Resection) was followed by the lymphocyte transformation test (LTT) and the staphylococcus phagocytosis test of in vitro washed leukocytes. The results have confirmed the immunostimulating and hence anti-tumour effect of intravesical BCG vaccine. Monitoring of the cellular immune response is suitable for the continuous follow-up of the BCG effect. Comparing the tolerable side-effects with their favourable therapeutic results, BCG vaccine is considered to be effective for the prevention of recurrences in treating superficial bladder tumours.

Carriage, transfer and interaction of oral viruses and bacteria.

Carriage of antigens and infectious herpes simplex virus type 1 (HSV-1) and human adenovirus type 1 (Ad-1) by salivary leukocytes was compared with the antibacterial activity of oral polymorphonuclear leukocytes (PMNL) and with the spectrum of oral bacterial and fungal flora. Risk of iatrogenic infections by microbes was assessed by detecting these viruses and microbes after disinfecting dental instruments. The results indicate carriage of antigens and infectious viruses in each age group between 6 and 60 years. Phagocytic activity by PMNL of virus carrier persons was found to be decreased as compared to virus-free subjects. The species number and survival after disinfection of oral bacteria and fungi were significantly higher in virus carrier persons. Infectious viruses were also obtained after disinfecting instruments used in their dental treatment. It is concluded that, virus infection of immune cells can contribute to the oral suppression of phagocytosis by PMNL. Intracellular viruses hidden from disinfectants can also result in infection of other subjects, especially if contemporary immunosuppression exists.