[Rotavirus enteritis in buffaloes]. / Vurkhu rotavirusnite enteriti po bivolite.

Immunodiffusion was used to investigate a total of 492 serum samples of buffaloes--buffalo calves up to the age of 6 months and buffalo heifers--on both private and public farms. Positive serological reagents for rotaviruses were established in 73.4 per cent of the cases. The study of the same samples via the blocking ELISA test revealed the presence of 86.2 per cent positive cases. It was demonstrated that this type of the ELISA technique was a more precise method for the demonstration of group specific rotavirus antibodies in buffaloes. The results obtained spoke of the widely occurring rotavirus infections among the buffalo populations in this country. Particularly intense was the circulation of rotaviruses in buffalo calves aged up to six months. The study of 46 fecal samples from buffalo calves with diarrhea by means of the immunodiffusion and ELISA tests revealed that in as many as seven of them there were rotaviruses. This unquestionably demonstrated the participation of these agents in the etiology of enteritis in buffaloes.

[Experiments to culture the bovine pestivirus in heterologous cell cultures]. / Opiti za kultivirane na govezhdiia pestivirus v kheterolozhni kletuchni kulturi.

Attempts were made to adapt a strain of the bovine pestivirus to cell cultures of swine kidney in a series of 15 passages. The adaptation of the virus was followed up through the production of a cytopathic effect, through comparative investigations, and by means of cytologic preparations. It was found that the cytopathic effect induced and the titer of the virus grew with increasing the number of the serial passages. Immunofluorescence and cytologic data showed the progressive development of the virus in cell cultures of swine kidney. It was also established that the time of incubation of the infected cultures to produce an overall cytopathic effect was shorter.

[Attenuated vaccine against rota- and coronavirus enteritis in calves]. / Atenuirana vaksina sreshtu rota- i koronavirusnite enteriti po teletata.

Tested was the effect of the Bulgarian attenuated vaccine of rota- and corona-viruses 'PoKo-81', via treatment of dry and freshly calved cows (injected into the udder) and newborn calves (peritoneal injection). It was found that calves fed three times daily on milk obtained from cows that had had intramammary injections did not develop clinical signs of a disease following experimental infection with rota- and corona-viruses. When the cows on infected farms were treated in the same way the disease in calves, offered immune milk, dropped to 36 per cent as against calves of unvaccinated cows, with which morbidity ran as high as 85 per cent. Calves vaccinated via the peritoneum did not fall ill after infection with rota- and corona-viruses on the 6th day following vaccination. In experiments on 18 infected farms morbidity among control, unvaccinated calves was 76 per cent, and mortality--6.8 per cent. With vaccinated calves on the same farms morbidity dropped to 44 per cent, and mortality--to 2.5 per cent. The duration of diarrhea in the vaccinated calves was radically lowered. Discussed is the possibility to enhance the protective effect of the vaccine.

[Immunofluorescence demonstration of bovine pestivirus]. / Imunofluorestsentno dokazvane na govezhdiia pestivirus.

A high-titer hiperimmune calf serum was obtained via the manifold hyperimmunization with local strains of the bovine pestivirus, cultivated in homologous cell cultures. Its globulin fractions were treated with fluorescein isothiocyanate to produce a specific conjugate. The specificity of the immunofluorescent serum was demonstrated through the comparative study of cell cultures infected with various viruses. The method worked out was employed to investigate tissue cross sections from diseased calves, whereupon the opportunity for the rapid and specific diagnosis of mucous disease was demonstrated. The replication dynamic of a bovine pestivirus strain in cell cultures was followed up.

[Participation of coronaviruses in the etiology of respiratory diseases in calves]. / Uchastie na koronavirusi v etiologiiata na respiratornite zaboliavaniia po teletata.

Studied was the Bovine corona virus as taking part in the epizootiology of respiratory diseases in calves. On 4 out of the 7 investigated farms there was a rise of the antibodies against the virus. Two strains were isolated from 2 diseased calves, using tissue culture of trachea. They were the III 213/81 and III 214/81 strains and were identified as bovine corona viruses. Test calves experimentally infected via the trachea with the III 214/81 strain responded with a slight and transient rise of body temperature and a slight cough. The virus was reisolated from the nasal discharge of 3 diseased calves as well as from the feces of one affected calf. The direct immunofluorescence method was employed to establish cytoplasmic fluorescence between the 4th and the 8 h day of infection. The immune response observed with the infected animals confirmed the development of the corona-virus infection. The clinical observations with calves on both the farms with spontaneous infections and those with experimentally induced infections showed that the bovine corona virus could not cause a strongly manifested respiratory disease.

[Immunofluorescent demonstration of respiratory syncytial virus infection in calves]. / Imunofluorestsentno dokazvane na respiratorno sintsitialnata virusna infektsiia po teletata.

Specific high-titre bovine conjugated antisera were obtained against respiratory syncytial virus. Use was made of the reference strain Nomi and the local isolate Antonovo/448. The conjugates produced were shown to have equal qualities to those of the Belgian conjugate used for comparison. The anti-Antonovo/448 conjugated serum was used in the direct immunofluorescent method to demonstrate a respiratory syncytial virus antigen in organs of animals with acute respiratory diseases as well as to control the replication of the virus in cell cultures. An infection with a respiratory syncytial virus was demonstrated with the employment of the same test in 16 per cent of a total of 119 samples taken from diseased animals. In the inoculation of the diploid cell culture of lamb thyroid gland with strains of the virus a specific antigen was established at the forty-eighth to the seventy-second hour following infection.

[Demonstration of coronavirus infection in buffaloes]. / Dokazvane na koronavirusna infektsiia po bivolite.

The hemagglutination-inhibition test and a bovine corona virus antigen were used to investigate a total of 293 samples of buffalo sera, of which 152 were taken from buffalo calves at the age of up to 12 months and 141 were taken from buffalo cows at the age of between 18 and 30 months. Corona virus antibodies were proven in 52.6 per cent of the sera of buffaloes originating from 3 cooperative farms and a number of private farms. The antibodies in the sera of buffalo calves had an average geometric titer of 182, and those in the sera of buffalo cows--74. The parallel study of 40 of the sera revealed a correlation between the hemagglutination inhibiting and the neutralizing antibodies. Twenty-nine fecal samples from buffalo calves with diarrhea, aged 5 to 30 days, were also studied via hemagglutination-inhibition on the same public farms. Corona virus of high titers was demonstrated in 7 of the samples, the hemagglutination activity of which had been suppressed by a bovine corona virus antiserum. It was concluded that a hemagglutinating coronavirus was circulating among the buffaloes. It seemed to be analogous to the bovine coronavirus and to participate in the etiology of part of the enteritis cases in growing buffalo calves.

[Serotyping of cytopathic rotavirus strains isolated from calves]. / Serotipizirane na tsitopatichni rotavirusni shtamove, izolirani ot teleta.

Antistrain sera were obtained against cytopathic rotavirus strains isolated from calves as well as against reference rotavirus strains, belonging to I and II serotype of the bovine rotaviruses with which cross antigenic studies were carried out. It was found that the isolated DS 39/82 and TR 248/82 strains belonged to I serotype of the bovine rotaviruses. Data was obtained speaking of some minor antigenic differences between strain TR 248/82 and strain DS 39/82 as well as between strain TR 248/82 and the reference rotavirus strain Lincoln (I serotype), which showed that there existed serologic variants between the strains of this serotype.

[Cultivation of bovine pestivirus in organ culture]. / Kultivirane na govezhdiia pestivirus v organni kulturi.

Attempts were made to culture strains of the bovine pestivirus in organ cultures of tracheal rings. Contrary to other scientists the authors used roller organ cultures of calf tracheal rings. The development of the virus in the cultures was judged by its presence in the maintaining medium through ELISA or by titration in cell cultures of fetal calf kidney. It was found that the two strains tested could be replicated in organ cultures of calf trachea, the intensity of multiplication being strongest between the third and the sixth day as demonstrated by ELISA. It was also established that the various batches of tracheal tissue had varying susceptibility.

[Experimental infection of lambs with strains of the bovine respiratory syncytial virus]. / Eksperimentalno zaraziavane na agneta s shtamove na govezhdi respiratorno-sintsitialen virus.

Lambs were successfully infected with bovine strains of a respiratory syncytial virus. When the Nomi reference strain and the local Antonovo strain of the RSV were used i/v and via the trachea the animals reacted on the 3rd to 5th day with dizziness, dyspnea, conjunctivitis, nasal discharges, etc., more strongly manifested with the use of the Antonovo strain. The temperature reaction started on the 3rd day and receded on the 7th day, with a second peak on the 8h day. Serologically, RSV-antibodies were released on the 14th day, and that correlated in dependence on the severity of the clinical manifestations. Such observations made it possible to use lambs as a sensitive animal species to differentiate the virulence of the tested strains. Testing along these lines rendered is feasible to obtain attenuated strains and use various immunoprophylactic agents against the respiratory syncytial virus.

[Immunofluorescent diagnosis of rota-, corona- and pestivirus infections in calves]. / Immunofluorestsentna diagnostika na rota-, korona- i pestivirusni infektsii po teletata.

Bovine hyperimmune fluorescent sera were obtained for indication of the rota- and corona-virus infections in calves, which were highly specific and of immunofluorescent titers within the 1:32-64 range. Their testing was carried out via the direct immunofluorescence method, using histologic cross-sectioned material and impression preparations of the mucous membrane of calves died of enteritis as well as cell cultures of fetal calf kidney and organic cultures of calf trachea all contaminated, resp., infected with feces. The study of the intestinal membrane of a total of 100 diseased calves, employing this method, has demonstrated a rotavirus antigen in 38.0 per cent of the cases, and a coronavirus one--in 37.0 per cent. The causative agent of mucous disease (a pestivirus) was demonstrated by means of the respective conjugate in 47.3 per cent of the cases, using abomasal membrane of a total of 55 calves. Rota- and corona-virus infections were also established in the abomasal membrane of diseased calves, and pesti-virus infections--in the intestinal mucosa as well. Established were also mixed infections with the three viruses taking part at various combinations. A celloidin method was worked out for making immunofluorescent preparations of infected monolayer cell cultures. In the cell cultures infected with feces the totavirus antigen was demonstrated in 19.4 per cent of the cases, and the coronavirus one--in 25.6 per cent.

[Isolation of the respiratory syncytial virus from calves with respiratory diseases]. / Izolatsiia na respiratorno-sintsitialniia virus ot teleta s respiratorni zaboliavaniia.

Described is a respiratory enzootic in calves aged 30 to 90 days. Two cytopathic strains of the bovine respiratory syncytial virus were isolated from diseased calves in a cell culture of lamb thyroid. Clinical observations and the results of virologic and serologic investigations have revealed that mixed infections of the respiratory syncytial virus and the mucosa disease virus take part in the etiology of the respiratory enzootic described.

[Experiments to isolate cytopathic strains of bovine rotaviruses]. / Opiti za izolirane na tsitopatichni shtamove govezhdi rotavirusi.

Experiments were carried out to isolate cytopathic strains of bovine rotaviruses. Material from a total of 129 positive fecal samples, taken from calves affected with rotavirus gastroenteritis, was used to infect cell cultures. In the roller culturing of 19 of the positive samples in suspension of the MA-104 cell line in the presence of trypsin two cytopathic strains were isolated. The cell line used was more sensitive than the cells of calf kidney.

[Demonstration of respiratory syncytial virus infection in calves by the complement fixation reaction]. / Dokazvane na respiratorno-sintsitialnata virusna infektsiia po teletata chrez reaktsiia za svurzvane na komplementa.

Studied was tentatively the occurrence of a respiratory syncytial virus infection on some farms with records of the disease. Investigations were carried out on the base of the complement-fixation test (CFT) specially worked out and on the specific complement-fixing antigen obtained from a bovine respiratory syncytial virus (BRSV). Tested were several methods for the production of a complement-fixing antigen. Most appropriate proved the one obtained by the extraction of an infected cell monolayer in glycerine buffer saline. It was stated that CFT was a proper test for the rapid herd diagnosis of BRSV infection. The study of double serum samples from 240 calves involved in 40 enzootics revealed that CFT could successfully be employed in 18 per cent of the cases of respiratory infections in calves in as many as 17 enzootics. However, the etiologic part played by the virus was dominating in some of these enzootics only. In the remaining ones the participation of the virus was either partial or sporadic.

[Enzyme-linked immunosorbent assay (ELISA) in rotavirus detection]. / Vurkhu enzimsvurzaniia imunosorbenten metod (ELISA) za dokazvane na rotavirusi.

Reagents were obtained by means of which the method of the enzyme-linked immunosorbent assay (ELISA) was worked out, based on the double sandwich principle, for the demonstration of rotavirus antigen presence in the fecal samples. The reaction makes use of polyvinylchloride plaques that are employed in tablet packing, and enables the rapid quantitative diagnosis of rotaviral enteritis in both animals and humans. The specificity of ELISA has been demonstrated through the comparative investigation of various viruses and bacterial antigens as well as by means of the periodic study of experimentally infected calves. The investigation of a total of 207 fecal samples from diseased calves showed that 28.5 per cent reacted positively for rotaviruses by the immunodiffusion test, and 42 per cent--by ELISA. Rotavirus enterites showed an expressed age-associated curve from the first to the fifteenth day of age with a peak between the fourth and the seventh day.

[Purification of coronaviruses from contaminated cattle feces]. / Predchistvane na koronavirusi ot kontaminirani govezhdi fekali.

A scheme was worked out for the obtaining of purified Corona viruses from fecal extracts, which include: centrifugation in an discontinued gradient of sucrose 20--45 per cent, gel filtration through a Sephadex 4B column, and centrifugation in a linear gradient of sucrose 10--50 per cent. By means of such purification the virions remain morphologically intact in sucrose density of 1.11--1.14 gr/cm3, containing no ballast proteins. As many as five polypeptides are demonstrated in polyacrylamide electrophoresis.

[Isolation of the bovine herpesvirus-3 from sick calves]. / Izolirane na govezhdi kherpesvirus-3 ot bolni teleta.

Three herpes strains identified as herpes virus-3 were isolated in revolving test-tubes with cell cultures of nasal samples and lung parenchyma from diseased calves at the time of a respiratory enzooty. Large crystalloid aggregates of herpes virus virions were observed in the nucleus and in the cytoplasma by the electron-microscopic investigation performed on infected cell cultures. The experimental infection of calves with the isolated strain produced slight symptoms of respiratory disease in single calves only. The infected calves were susceptible to a following infection with cattle herpes virus-1.

[Production and study of the immunogenic properties of a bivalent inactivated vaccine against mucosal disease (bovine viral diarrhea and infectious rhinotracheitis)]. / Poluchavane i prouchvane imunogennite kachestva na bivalentna inaktivirana vaksina protiv mukozna bolest (virusna diariia i zarazen rinotrakheit po govedata).

Bivalent inactivated vaccine against mucous disease (MD) and infectious rhinotracheitis (IR) in cattle was produced from cell cultural MD and IR virus suspensions. The vaccine was concentrated on aluminium hydroxide, inactivated by ethanol and is without residual virus. Saponine in final 1:1500 dilution is added as supplementary adjuvant. Immunogeneity of the vaccine was tested on 10-month-old calves, which had shown full resistance against experimental infection with virulent strains of both viruses. Testing on calves for harmlessness by use of a five-fold higher vaccine dose indicated complete tolerance of the vaccine. The prophylactic effect of the vaccine applied in practical work to directly threatened with immediate MD and IR infection cows, including pregnant ones, consisted in reduced number of cases of abortion, of inborn malformations, in lower neonatal calf death-rate, etc. No disturbances were observed following two-fold vaccination of the animals, a fact proving its harmlessness. The positive results of the studied vaccine allow its further application in the combined prophylaxis of MD and IR in calf fattening and breeding complexes.