Aujeszky's disease virus strains with peculiar features.

Aujeszky's disease (AD) virus strains isolated from weak colostrum-deprived piglets were investigated for virulence in rabbits. Most of them (5 out of 9 investigated) proved avirulent. As clinical symptoms of AD were not observed during this period on the farms of origin, one can assume that the strains had a particular affinity for the pig fetus. It is proposed that the strains may be a result of recombination between the attenuated strains MK-35 (gI-) now in use as a live vaccine and MK-35 (gI+), used as a live vaccine until 3 years ago, as well as between the vaccine strain and a wild-type strain of AD virus, followed by the acquisition of tropism for the reproductive system. Virulent as well as avirulent strains were isolated in the same herd. The evaluation of a highly immunogenic killed AD vaccine in this complicated situation is recommended.

[2 strains of swine parvoviruses isolated from aborted fetuses]. / Prouchvane na dva shtama parvovirus po svinete, izolirani ot abortirani fetusi.

Two hemagglutinating virus strains were isolated (in primary cell cultures of pig kidneys) from viscera of aborted swine fetuses. A number of serologic, cytologic, physico-chemical, and laboratory investigations with the strains revealed that they belonged to the group of porcine parvovirus (PPV). The isolation of SPV from aborted fetuses pointed to the fact that the disease had been widespread among the swine population and plays a part in reproduction disturbances that have come to be known recently. The isolated strains did not produce a clear and distinguishable cytopathic effect in inoculated cell cultures. They, however, could be demonstrated in the cultures indirectly through cytologic investigation (the demonstration of intranuclear inclusion bodies, type B after Cowdry, through hemagglutination tests, and via immunofluorescent microscopy.

[Isolation of conjugated sera for the immunofluorescence demonstration of swine parvovirus]. / Poluchavane na koniugirani serumi za imunofluorestsentno dokazavane na parvovirusa po svinete.

High-titer, specific serum against porcine parvovirus was obtained via hyperimmunization of rabbits, with the use of a Bulgarian isolate that had been partially purified after a known, modified technique. A specific, high-titer conjugate was produced for the immunofluorescence diagnosis of porcine parvoviruses. The microscopic observation of the lamellae of cell cultures, treated with the conjugate, revealed the presence of a specific, typically granulated perinuclear (mostly unilaterally) and, in some cases nuclear fluorescence--at negative reaction in the control preparations. Immunofluorescent light was also established in cell cultures infected with the virus at highest dilution. It showed that immunofluorescence microscopy could be employed to demonstrate even the lowest amounts of the virus.

[Replication of a local strain of swine parvovirus in swine kidney cell cultures]. / Replikatsiia na mesten shtam parvovirus po svinite v kletuchni kulturi ot svinski bubrek.

The kinetics of replication of strain of parvovirus in swine was investigated in the primary cell cultures of swine kidney. The morphologic changes were traced in inoculated cultures by microscopic observation and the replication of the virus in the cells by immune-fluorescent examinations. The quality of the virus in the cell monolayer and in the nutritive medium, in the different periods after the infection, was determined by hemagglutination test. By the immune-fluorescent examinations, the virus was proved still in the first hours, while the first morphologic changes of the monolayer were determined 72 hours after the infection. The nuclear fluorescent appears at the 12-th hour and increases its intensity 24 hours after the infection. At the 72-nd hour and later predominates the cytoplasmatic fluorescent. The cell monolayer produces virus up to the 96-th hour, and the virus titer reaches its maximum 120 hours after the inoculation, which is the most appropriate moment for the yield of virus.

[Use of a microvirus-neutralizing reaction in diagnosing transmissible gastroenteritis (TGE)]. / Izpolzuvane na mikro-virusneutralizatsionnata reaktsiia v diagnostikata na transmisivniia gastroenterit (TGE).

A micro virus-neutralization reaction was designed and tested to detect antibodies to the virus of transmissive gastroenteritis. Use was made of a stable cell line, SPEV, and a laboratory strain of the virus that had been adapted to it. The optimal concentration values of the cell suspension and the normal calf serum contained in it were determined. A total of 90 blood serum samples from pigs were comparatively investigated for the presence of virus-neutralising TGE antibodies, the tube test being performed trough the inoculation of the cells in suspension. On the other hand, the micro virus-neutralization test was carried out in two variants: the sera were diluted via Mikrotiter micropipettes and micropipettes of the Takachi apparatus. It was found that the micro virus-neutralization test in its two variants was not inferior in terms of sensitivity to the tube test, was more readily applicable, and was less material consuming.

[Course of transmissible gastroenteritis on a swine-breeding farm]. / Protichane na transmisivniia gastroenterit v svinevudno predpriiatie.

Epizootiologic studies were carried out from the introduction of the transmissive gastroenteritis on the Enterprise up to the end of 1984. Clinical observations and morphologic survey were conducted on the infected animal farms. The diagnosis was based on both virologic and serologic investigations with routinely employed methods. The basic reason for the occurrence and spread of transmissive gastroenteritis on the Enterprise were shown to be omissions in the system of prevention measures with regard to infectious diseases, i. e., the functioning of farms as open units and the intense connections between them. The basic way of spreading the infection was shown to be the removal of animals from healthy to affected farms. The part played by other ways of infection transmission proved negligible, and the strict adherence to veterinary and sanitary requirements of protection guaranteed to a high extent the prevention of transmissive gastroenteritis on swine-breeding complexes and farms. It was also found that the disease assumed a stationary character on the infected farms, with periodic enzootic outbreaks in some of them associated with the nonobservance of feeding and raising technologies.

[Associated vaccination of pigs against swine fever, erysipelas and Aujeszky's disease]. / Asotsiirano vaksinirane na praseta sreshtu chuma, cervenka i bolestta na Aueski.

Tested was the simultaneous vaccination of pigs against swine fever, swine erysipelas, and Aujeszky's disease. Parallel to this experiment a second group of test pigs were treated with each of the three vaccines at twenty-day intervals. The effect of vaccination was assessed through clinical observations with the test groups and by challenging part of the animals with virulent strains of the swine fever and erysipelas agents. It was found that the successive application of the three vaccines at 20-day intervals (at the age of 50, 70, and 90 days) could induce immunity that lasted for a longer period than the immunity resulting from the simultaneous treatment at the age of 90 days. In order to protect the pigs for sure up to the end of the fattening period a revaccination against swine fever with the simultaneous use of the vaccine against swine erysipelas in both cases was necessary.

[Sensitivity of various cell cultures to the swine parvovirus]. / Chuvstvitelnost na razlichni kletuchni kulturi kum parvovirusa po svinete.

Attempts were made to culture the swine parvovirus under laboratory conditions. A reference strain and a field isolate were used along with steady cell lines of pig kidney PK-15, IBAS-2, and SPEV as well as primary and secondary cell cultures of pig kidney. It was found that the steady cell lines were slightly sensitive or totally unsusceptible to the swine parvovirus. The could serve for its isolation from pathologic material and culturing in laboratory conditions. Both the primary and the secondary cell cultures proved strongly susceptible to the virus, and they could be used for the isolation of filed strains as well as for the laboratory maintenance of the virus. The strains used produced no clearly distinguishable cytopathic effects in the inoculated cell cultures. The morphologic changes that set in following inoculation with higher amounts of the virus could be seen under the light microscope and could be evaluated through cytologic investigations (the presence of intranuclear inclusion bodies). The virus could be most readily demonstrated in the infected cell cultures via the hemagglutination test.

[Trials of peroral vaccination of newborn pigs against transmissible gastroenteritis (TGE)]. / Opiti za peroralna vaktsinatsiia na novorodeni praseta sreshtu transmisiven gastroenterit (TGE).

Experiments were carried out to apply oral vaccination to newborn pigs against transmissive gastroenteritis on a stationary swine breeding complex at the time when the disease assumed an acute course and on newly infected farms. Used was an attenuated strain an hour prior to allowing the pig to suck. It was found that such vaccination approach was innocuous. It proved effective when applied at the moment of birth both on the infected (stationary) farms and in the new foci of infection--morbidity and mortality were lowered and the body development of pigs was improved. Such vaccination was shown to produce also an antiepizootic effect if systematically used on stationary farms--clinically, there were no new epizootic outbreaks of the diseases.

[Isolation and use of a farm-specific hyperimmune serum against transmissible gastroenteritis]. / Poluchavane i prilagane na obornospetsifichen khiperimunen serum sreshtu transmisiven gastroenterit.

A specific stationary hyperimmune serum was obtained in a pigbreeding complex with a transmissive gastroenteritis infection via trifold (at a fourteen-day interval) i/m injection of sows with 5 cm3 each of undiluted virus (10(6) TCCPE50 per cm3). Tested were hyperimmunization programmes with pigs in the final fattening period. It was found that the use of undiluted virus led to the equation of sows in terms of their immunologic state and to the essential rise of the titer of humoral antibodies. In order to obtain high titer immune serum against transmissive gastroenteritis it is sufficient to proceed with the i/m injection of pigs or adult swine in the final stage of fattening at rising amounts (from 3 to 10 cm3) of attenuated virus of sufficiently high titer (10(6) TCCPE50 per cu. cm). The hyperimmune serum produced a very good prophylactic effect with newborn pigs on the same farm - the twofold oral administration of 5 to 10 cm3 on the day of birth and a couple of days later led to a drop of both morbidity and mortality rate as well as to the improvement of body development.

[Pathology in hybrid pig breeding due to mixed infections]. / Patologiia na khibridnoto svinevudstvo, prichineno ot smeseni infektsii.

A total of 7,500 samples from 5 swine breeding complexes with 200,000 hybrid Camborough pigs were studied morphologically and bacteriologically. The raising technologies used on all complexes were of industrial type or semi-industrial and extensive. A description is given of the simultaneous participation of the following nosologic units: colibacteriosis, responsible for 13 to 14 per cent of the total mortality rate in newborn pigs; bronchopneumonia--causing 6 to 39 per cent losses in the other age groups; and dysentery with salmonellosis--inflicting 5 to 9 per cent losses. Underlined is the great advantage of the industrial technologies of animal raising as against the conventional ones in the prevention of these diseases in hybrid pig breeding. Tested were effective programmes for the prophylaxis associated with the individual nosologic units. More essential were the following: active immunoprophylaxis with live attenuated vaccines against colibacteriosis and salmonellosis; elimination of the alimentary stress factor in dysentery; disinfections of premises by gassing with a mixture of ammonium nitrate and chlorinated lime in a 1:2 ratio. However, all measures proved effective when the principle of "all in, all out' was observed which is essential part of the Camborough industrial raising technologies.

[Demonstration of a transmissible gastroenteritis virus by contrast immunofluorescence]. / Dokazvane na virusa na transmisivniia gastroenterit chrez kontrastniia imunofluorestsenten metod.

Tested were a number of procedures to employ the immunofluorescence method for diagnosing transmissive gastroenteritis in pigs--impression preparations, frozen cross-sectioned material, and infected cell cultures of the established cell line SPEV. It was found that immunofluorescence microscopy was a dependable method for the express diagnosis of transmissive gastroenteritis. It could be employed in its three variants. The use of impression preparations, however, did not prove as dependable as the remaining ways of applying the method, and this drawback had to be compensated for with the study of a greater number of impression preparations taken from more pigs that had contracted the disease. It was also established that most promising and effective was to apply the method with the use of cell cultures infected with suspensions of organs. Cell cultures of the established SPEV cell line infected with material that contained the virus could produce dependable positive results in immunofluorescence investigations at the 24th hour following inoculation. This method could be employed for the express diagnosis of transmissive gastroenteritis.

[Experiments to produce an attenuated strain of the transmissible gastroenteritis virus and its use as a live vaccine]. / Opiti za polchavane na atenuiran shtam na virusa na transmisivniia gastroenterit i izpolzuvaneto mu kato zhiva vaksina.

Serial passages have been performed of a field virus of the transmissive gastroenteritis to obtain an attenuated strain adapted to a permanent cell line of pig kidney, "SPEV". The strain is innocuous for test animals, for pigs of all ages, and for swine during the entire period of pregnancy. It has proved genetically stable. The strain is immunogenic for swine and pigs both under laboratory and under field conditions (in new foci and on stationary farms) and can be used as a live vaccine against transmissive gastroenteritis, either untreated or in a freeze-dried state.

[Isolation of the transmissible gastroenteritis virus in a continuous pig embryo kidney cell line]. / Izolirane na virusa na transmisivniia gastroenterit v postoiannata kletuchna liniia SPEV.

Attempts were made to isolate the virus of transmissive gastroenteritis in a permanent cell line SPEV in view of the diagnostics of the disease. Used were small intestines of pigs during the first 24 hours after the setting in of clinical symptoms. The successful isolation of the virus in a SPEV line depended on the methods employed to handle the material as well as on the ways of inoculating the cell cultures. In the conditions of the investigation most proper proved centrifugation of the organ suspensions and the suspension method of infection. The SPEV line, however, was shown to be insufficiently sensitive to isolate the virus of transmissive gastroenteritis. About 30 per cent only of the positive material could be used to demonstrate the virus following direct infection, while the study of the remaining (up to 62 per cent) material required additional passing. The SPEV line could replicate the virus after its infection with organ suspensions containing the virus, and this could be demonstrated through immunofluorescence investigations. The same method could likewise be employed in the express diagnosing of the disease. The passing of the virus strains in the SPEV line led to their attenuation. In order to retain the virulence of the strains passing should take place in nonimmune pigs or should alternate with the use of both pigs and cell cultures.

[Cultivation of the transmissible gastroenteritis virus in a continuous cell line]. / Kultivirane na virusa na transmismisivniia gastroenterit v postoianna kletuchna liniia.

Cell cultures of the permanent cell line SPEV to which the transmissive gastroenteritis virus had already been adapted were used to culture the virus and carry out the virus-neutralization test. Use was made of a cell suspension of a variable density--300 and 500 thou cells per cm3. Both variants of the cell suspension were comparatively studied in terms of growth, the production of a monolayer, susceptibility to infection, and titer of the virus obtained, using 4 test tubes with the virus at various rates of dilution which were kept under observation daily, keeping a record of the infected and noninfected cell cultures. The amount of the virus was determined by titration. It was found that the monolayer was produced more rapidly in the suspension containing 500 thou cells/cm3. In that case infection could be performed at the 24th hour. The cytopathic effect was more pronounced, and the titer of the virus obtained was higher. Successful attempts were made with the virus-neutralization test with the infection of the cell cultures in suspension. Thus, the entire procedure was shown to be labour-saving as the time for investigation of the sera was shortened.

[Stability of Aujezky's disease virus strain MK25 in an aerosol]. / Stabilnost na virusa na bolestta na Aueski--shtam MK25 v aerozol.

Twenty-two tests were carried out to evaluate the stability of the virus of Aujeszky's disease in aerosol. Use was made of a aerosol chamber of a flow dynamic type, 450 l, of 60 l/min of passing air, 5 min. exposure, 80-100 per cent relative humidity, and temperature range of 14-21.2 degrees C. The size of aerosol particles surpassed 5 micron, and changed according to the composition of the dilution medium of the vaccine. The aerosol was sampled via impinger at the rate of air flow of 5.2 l/min. Tested were the stabilizing properties of 2% glycerin, 10% pepton, 0.8-2.0% saccharose, 2% gelatin, and 5% skimmed milk added to the extender consisting of a 0.2 M phosphate buffer saline, pH 7, specifically for the MK25 strain of the Aujeszky's disease virus in a dispersed status. Highest stability, reaching 11.2% survival rate of the virus in aerosol was recorded at the dispersion of the vaccine in a 0.2 M buffer saline with the addition of 10% pepton and 0.8% saccharose at pH 7.

[Protein fraction changes in swine raised under different conditions]. / Promeni v beltuchnite fraktsii u svine, otglezhdani pri razlichni usloviia.

The total protein and the protein fractions were studied in pigs raised both under industrial conditions and on the personal farms. It was found that the process of immunogenesis in pigs varied largely parallel to the various conditions of raising. The numerous immunoprophylactic treatments under industrial conditions were found to have an adverse effect on the forming of the immune response, even without apparent antigen competition. The investigations aimed at elucidating the expediency of the adopted and very often unsubstantiated, repeatedly effected treatments for the rational use of mixed vaccines.

[Control of enzootic Aujeszky's disease on pig farms]. / Opit za ozdraviavane na enzootichna za bolestta na Aueski svineferma.

Virologic and serologic investigations were carried out to demonstrate the persistence of the virulent virus of Aujeszky's disease. Threefold vaccinations were conducted with a live vaccine, embracing all animals on the farm and those that were born during the time when sanitation measures were applied. The first vaccination of the sucking pigs was performed with 10 doses of the vaccine in each case. During the time of applying sanation measures the farm was kept closed, the pigs after weaning being transferred to another farm. After sanitation was over some new virologic and serologic studies were carried out along with the serologic examination of nonimmune pigs that had stayed on the farm. These investigations did not prove the presence of a wild virus on the farm. It was found that the manifold vaccinations and use of higher amounts of the vaccine might well suit the base in working out sanitation programmes for farms infected with Aujeszky's disease.

[Control of latent enzootic Aujeszky's disease]. / Opit za prekusvane na skritiia epizootichen protses pri bolestta na Aueski.

An attempt was made on a swine-breeding complex, where the virus of Aujeski's disease was demonstrated to interrupt the inapparent epizootic process of the disease. The persistence of the virus on the complex was demonstrated via virologic investigations of organs of pigs with respiratory symptoms and studies of sera of various groups of animals. A batch of pigs was divided into three groups of which one was left untreated, one was vaccinated at the time of weaning, and one was vaccinated at the age of 14 days, using as many as 10 vaccinal doses with one dose of a live vaccine being used at the moment of weaning. During fattening the pigs were kept in isolation and under clinical observation with the study of organs and sera. Wild virus was demonstrated only in the group of twice vaccinated pigs. It was established that the manifold vaccinations of sucking pigs on farms that were enzootic in terms of Aujeszky's disease, using a live vaccines, with the isolation of the animals right after weaning could contribute to the interruption of the inapparent epizootic process.