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Studied was the carrier status with the Newcastle disease virus in experiments with a total of 665 birds at the age of 10 days, one month, and two months following an aerosol vaccination with a La Sota strain vaccine as well as the possibility to isolate the velogenic strain Texas GB after control infection of 10-day-old and one-month-old birds. Established was a short-term carrier status with the La Sota strain in the organs of birds up to the 6th-9th day following the aerosol vaccination. In an experiment with month-old birds originating from parental flocks immunized against Newcastle disease the La Sota virus was isolated also from the brain up to the sixth day. The resistance of birds to the infection was demonstrated at the aerosol immunization of 10-day-old and month-old- birds followed by challenging the birds with high doses of the velogenic virus strain Texas GB.

Routine methods of investigation were employed in parallel experiments for the isolation (in chick embryos), identification (serologically via HI), and typing (MDT, ICPI, IVPI, and via cloacal tests) of local isolates and standard strains of the Newcastle disease virus (NDV). The results were the same as those obtained by the single-phase complex investigation after the plaque technique method in a monolayer cell culture of chick embryo fibroblasts under agar cover. The cultures were inoculated with tissue suspension centrifugate in multiple dilutions of trachea and lung, spleen and liver, and brain of experimentally infected birds, using local isolates (13 in all) and standard strains of NDV (6 of the various types) as well as of birds that died of Newcastle disease (3 cases). The complex cell culture technique of investigation included plaque-forming under agar (and under agarose as well) following the direct inoculation with the infectious tissue suspensions in the presence of a normal (known negative), resp., specific hyperimmune serum against Newcastle disease. It also included a plaque-neutralization (plaque reduction) test and tests for hemadsorption and elution of erythrocytes--all made use of in a single planned programme of investigation. This technique, which is a modification of the one suggested by Beard et al. (1970) and known as single-phase complex plaque technique, provides the simultaneous isolation and identification of NDV isolates, which may well be coupled with some of the routinely practiced methods for definitive isolate typing (e. g., with the cloacal test for velogenic, and with MDT for lento- and mesogenic isolates). Thus, time can be saved, and labour and means are reduced, i. e., the laboratory diagnostics of ND may be perfected.

The period of virus harbouring was followed up in a total of 50 experimentally infected (Newcastle disease) birds. Two methodes were used to isolate the challenging virus: the ordinary virologic procedures and the method of tissue cultures. Studied was also the effect of the humoral immunity on the persistence of the virus. Investigations by the tissue culture method revealed a persisting latent infection in the course of 70 day, which in certain cases could assume an acute course and become apparent. The Newcastle disease virus was isolated from birds having either low or very high levels of serum agglutinins. The currently employed virologic methods failed to demonstrate the virus after the 16th day of infection.

A strain of the Newcastle disease virus, denoted GF, was isolated from birds vaccinated twice with a live vaccine. In some flocks of the farm this strain has caused oligosymptomatic disease, and in others the infection has run a latent course. It was shown that the GF strain possessed unusual biologic properties--an extremely high virulence in vitro combined with a total lack of pathogenicity in vivo. With regard to the tests mean death time with 10-day-old chick embryos (44.6 h) and plaque morphology (a heterogeneous plaque population of clear plaques, sizing 0.5 to 3.5 mm) it was referred to the velogenic group. With regard to the intracerebral pathogenic index (1.21) it belonged to the mesogenic group. In terms of the intravenous pathogenic index (0) it had to be classified with the lentogenic group of of strains of the Newcastle disease virus. All these properties of the strain were shown to be stable. Besides the difficulties with its differentiation the behaviours of this strain pose a number of problems with regard to its origin and the variability of the Newcastle disease virus in nature, which are subject to discussion.

Three local strains of lentogenic type of the Newcastle disease were used to produce live liquid vaccines. These were given per os to broiler chickens in order to test their immunogenicity (first vaccination at the age of 25 days and vaccination when 70 days old). The immunity thus acquired was followed serologically by the titer of the antihemagglutinins and the resistance manifested by the birds at the control infection with a proven lethal dose of a highly virulent velogenic strain of the Newcastle disease virus. The results obtained were compared with those observed with the use of liquid vaccines of the strains Hitchner-B1 and La Soto. It was concluded that the vaccines of the local Newcastle disease strains are completely harmless and immunogenic. The vaccinal strains are excreted by the immunized birds and serve to immunize the contact control chickens.

Comparative studies were carried out with 4 local velogenic strains (2 viscerotropic and 2 neutropic) and 6 standard strains of the Newcastle disease virus of the various pathogenic groups with regard to their plaque-forming capacity in a monolayer of tissue cultures (TC) of chick embryo fibroblasts (CEF) under agar cover, after a routinely employed method. It was found that the velogenic strains produce a heterogenic (in terms of plaque size) population of bright, mat-white plaques regardless of their belonging to the viscero-, resp, neurotropic pathogenic type. The mesogenic produced only small, bright plaques, while the lentogenic strains produced no plaques. The plaque-forming unit titers per cm3 in TC of CEF of the strains as well as their ratio to the respective infectious titers for chick embryos (EID50) and for TC of CEF overlaid with liquid nutrient medium by microscopic record of the cytopathic effect (TCID50) varied within a wide range. It was in favour of EID50, and in most of the strains - in favour of TCID50 no matter to which type of strains belonged. The results obtained with the use of the plaque method in TC of CEF confirmed the concept of a number of authors abroad that they could be used successfully as an index in typing also for the differentiation of the Newcastle disease virus strains.

It has been demonstrated that Musca domestica L. could harbour the Newcastle disease virus. It has been isolated from the surface of the flies body 96 hours after these had been in contact with it, and from their digestive tract--after 240 hours. It has also been found that virus excretion in infected flies continues for 96 hours. It is believed that Musca domestica L. flies can participate in the spreading of the ND virus.

Mass prophylactic vaccinations have radically chaugned the clinical syndrome, pathogenesis, and morphological picture of the Newcastle disease: clinically it is manifested with respiratory troubles, and morphologically--with tracheitis. The method for isolating the virus on chick embryos from immune birds have proved undependable: the higher the immunity of birds, the lower the probability of virus isolation. Following the vaccination of birds after the adopted prophylactic programme and at the three-fold vaccination with a lentogenic La Sota strain and four-fold vaccination with a mesogenic Komarov strain precipitating antibodies have been detected in single serum samples only. In the presence of respiratory troubles in immune birds the rise of antiheamagglutination titers and the mass manifestation of precipitating antibodies in the blood serum is said to be an indication of the presence of a field of velogenic virus, while HI and AGPT are suitable methods for the detection of immune carriers of Newcastle disease. These can be used also for the differential diagnosis of the latent forms of infectious bronchitis, infectious laryngotracheitis, the diphtheroid form of pox, and CRD. In order to restrict and do away with the velogenic field strains of the Newcastle disease virus a suggestion is made to kill all birds that have shown respiratory troubles and positive results in virologic and serologic examinations through HI and AGPT.

A rapid agglutination method was worked out for the demonstration of the Newcastle disease virus (NDV) and the bovine leukosis virus (BLV) with the use of specific antibody-charged Staphylococcus aureus cells. It consists in the production of formalin-treated St. aureus cells, their sensibilization with specific antibodies, and their agglutination with the respective viral antigens. The latter were obtained from cell lines infected with NDV, resp., BLV as well as with purified NDV. It is concluded on the basis of the results obtained that the agglutination test suggested is rapid, specific, and sensitive and can be employed as an adjunct means in various immunochemical investigations and in the practice for the demonstration of the virus in infectious material.

Five-day-old chickens decendants of hens vaccinated several times under conditions existing in the practice were ophtalmo-nasally inoculated with 10(5),5ELD50 velogenic viscerothropic Newcastle Disease virus NDV. Thirty two per cent of them were stricken by the disease and died, but the remaining 68% resisted the provocation and showed no clinical symptoms 80 days post inoculation. The virulogical investigation of the chickens resistant to inoculation proved they are spreading the virus for 45 days and remained virus-carriers for 54 days post inoculation. NDV did not change its pathogenic properties even after persisting for long periods of time. Two virulogical methods for virus isolation were used -- tissue homogenates and cloaca washings inoculated on hen embryos and tracheal organ culture. The advantages and the great possibilities for discovering latent NDV infection of the organ culture are pointed out. Latent fowl pest infection in birds is discussed.

Studied was the opportunity to demonstrate the Newcastle Disease Virus in tissue cultures with the employment of an immunofluorescence method. The development was followed up of four strains--the vaccinal ones Hitchner B1, La Sota, and Komarov and the velogenic one, Texas GB, using chick fibroblasts. It was found that the velogenic strain cumulated earlier and more intensely than the vaccinal ones, where the virus was seen to explicitly locate perivascularly. The most dependable demonstration of the virus proved possible with the velogenic strain at the 4th hour, and with the vaccinal strains--from the 6 th to the 10 th hour following the infection of the cultures. The virus antigen cumulated progressively, and with the velogenic strain it was accompanied with the production of a cytopathic effect which destroyed the cells by the 20th hour; with the vaccinal strains the cells remained intact up to the 48th hour following infection.

The mean death time in a cloacal test (MDT/CT) was employed to define the pathogenic type of isolates of the Newcastle Disease virus (NDV) with 5-week-old birds. The results were compared with the use of standard methods for typing--the mean death time with 10-day-old chick embryos as defined by the minimal lethal dose (MDT/MLD), the intracerebral pathogenic index for day-old chicks (ICPI), the intravenous pathogenic index for 6-8-weak-old birds (IVPI). Sixteen local isolates and 6 reference strains of NDV were used in the experiments. Using the author's modified technique to determine MDT/CT the velogenic strains of NDV were strictly differentiated from the local meso- and lentogenic NDV isolates and reference strains. At the same time differentiation was made of the velogenic-viscerotropic and of the velogenic-neurotropic isolates (strains). The values of MDT/CT in the experiments with isolates of the velogenic pathotype (14 in number) varied from 4.0 to 7.8 (mean = 5.32 +/- 0.26), and by them the isolates were graded for virulence within the respective (viscero- and neurotropic) group. For the lentogenic (5) and the mesogenic (3) isolates and strains these values were approx. 10. With 6 local NDV isolates of the velogenic-viscerotropic pathotype the test birds showed edema of the head and neck, with swelling of the eyelids, and post mortem the accumulation of semi-liquid fibrinous mass under the skin in these parts of the body and conjunctivitis were found. MDT/CT in the modification employed proved simple and readily applicable, and superior to IVPI in reliability, and is, therefore, suggested for use in typing and differentiating NDV isolates.

Epizootiological, clinical, morphological, and virological investigations have indisputably demonstrated the persistence of the Newcastle disease virus (NDV) in vaccinated flocks of layers at the age of 5-6 months for a period of three months. The influence was followed up of NDV on mortality rate and the egg performance of birds that had built immunity after ocular/nasal instillation of the Komarov vaccine, birds that had been simultaneously vaccinated and infected, and birds that had been infected and then subjected to i/m treatment with Komarov vaccine. An attempt was made to elucidate the persistence of the virus in various organs and tissues of birds manifesting variations in their immune status as well as to demonstrate it at different intervals of time through isolation in chick embryos. It has been suggested to investigate virologically tracheal swabs or the trachea itself and the nasopharinx of birds that have recently contracted the disease when respiratory troubles are established in vaccinated flocks. The hemagglutination-inhibition test is a valuable adjunct also in the case of employing it as a diagnostic reaction. The difference between the agglutination titers of log2-2 and more in newly affected birds and survivals may dependably serve in establishing the presence of a virulent virus. The atypical course of the Newcastle disease at the rhythmical course of the technologic processes contributes to the creation of a rather dangerous, 'smouldering' focus of NDV.

Studies were carried out on four strains of the Newcastle disease virus, isolated in Bulgaria and referred, as typed by the routinely used methods, to the velogenic group. The strains proved to be hemagglutinin - thermostable. Two of the local virus strains (Rosa and Toutrakan) belonged to the velogenic-neurotropic pathogenic type, and two (Bregovo and Haskovo) - to the velogenic - viscerotropic pathogenic one. All four strains were slowly eluated from chicken erythrocytes. Full immunologic similarity was established between these strains by means of the live vaccines used in this country for the specific prevention of Newcastle disease, produced with the lentogenic strains Hitchner B1 and La Sota and the mesogenic Komarov.

Two NDV glycoproteins--hemagglutinin-neuraminidase and F-protein--were separated from the remaining structural virus proteins through treatment of the virions with a 2 per cent triton-x-100 and chromatography on a Koh A-sepharose 4B column. A subsidiary ion-exchange chromatography on DEAE--Sephadex technique was employed to separate the two products and obtain them in a pure state. Biologically active NDV glycoproteins were obtained through solubilization of the virions with 10 per cent triton-x-100 at high ion strength, and the two glycoproteins were separated on a DEAE-bio gel column with the use of 0.01 M phosphate buffer of pH 7.2, containing 0.5 M NaCl.