Immunofluorescence with PI-3 virus--preparation and evaluation of conjugates from immune sera of various animal species]

Immune sera with a high content of antibodies to para-influenza-3 (PI-3) virus suitable for production of specific conjugated immunoglobulins were prepared on conventionally reared calves, rabbits, and guinea-pigs, applying antigen in different ways. When processing the sera of the above mentioned animal species to conjugates, the sera differed in pure IgG yield, in completed conjugate, and molar F/P ratios, but their ability to demonstrate antigens of the PI-3 virus in the dilution was approximately the same. The serum prepared on a colostrum deprived calf showed the lowest antibody content and the lowest pure IgG yield, the colouring ability of the conjugate prepared being as well somewhat lower.     

Isolation and detection of avian adenoviruses on cell cultures]

It is important, from the hygienic and epizootological view-point, to detect the presence of various microbiological agents in birds. The authors present the results of the virological examination of poultry, avian adenoviruses being the main object of investigation. Ninety-eight virus strains were isolated from chickens of different age categories and were included in the group of avian adenoviruses according to the form of cytopathic changes on the cell cultures of hen kidneys, according to the need for adaptation passages, chloroform, thermal, and pH resistance, character of nucleic acid, formation of intranuclear inclusions, and joint precipitation antigen, shared with the reference virus.     

A versatile flow cytometry-based immunofluorescence inhibition assay for the detection of bovine viral diarrhea virus-specific antibodies

A fast, sensitive and reliable flow cytometry-based (FACS = fluorescence activated cell sorting) immunofluorescence inhibition assay (FACS-IFI) for the detection of virus-specific antibodies in sera is described. The method was evaluated using sera from cattle experimentally infected with bovine viral diarrhea virus (BVDV). Virus-infected cells, which were fixed and permeabilized, were incubated with diluted sera from immunized or control animals. Monoclonal antibodies (mabs) against different viral proteins were added, and detected with ALEXA488-conjugated goat-antimouse antibodies. The fluorescence signals were detected by flow cytometry and determined as mean channel values. Results were expressed as percent fluorescence inhibition compared to standardized negative sera. The FACS-IFI test with sera from experimentally infected animals was highly sensitive and specific. Comparison of the FACS-IFI results with a commercially available blocking ELISA, an indirect ELISA and the standard serum neutralization test showed a strong correlation. Furthermore, the detection of protein-specific antibodies was possible using the FACS-IFI test.     

Spontaneous infection of cell lines with mycoplasmas and their detection using the fluorescence method

The continual cellular lines of bovine kidneys MDBK and AUBEK and of monkey kidneys VERO were infected spontaneously in the course of long-range cultivation by the mycoplasmas M. arginini and A. laidlawii. The mycoplasmic infection reduced significantly the growth activity of the cells and influenced negatively their morphology. The method of an indirect proof of mycoplasmas by bisbenzimid Hoechst suited well to demonstrate the infection; it had been modified to a simple and rapid test. The starting infection could be demonstrated already at the time when the amount of infected cells in the culture was relatively low.     

Multiplication of the IBR, PI-3 and BVD-MD viruses in cell and organ cultures of bovine fetuses after experimental intrauterine infections

The intrauterine infection of four- to nine-month-old bovine foetuses with the PI-3, BVD-MD viruses, performed 7 to 72 days prior to their delivery, did not exert any significant influence upon the susceptibility of primary cell cultures from foetal organs and tissues to further viral infection in vitro. The BVD-MD and IBR viruses multiplied in the primary cell cultures from the organs of a foetus infected with the PI-3 virus seven days before delivery even in the presence of endogenous PI-3 virus. Persisting infection with the PI-3 virus also failed to influence the susceptibility of foetal organ cultures to infection with the IBR and PI-3 viruses in vitro. The IBR virus and endogenous PI-3 virus multiplied simultaneously to high titres in the organ cultures of thymus and lungs whereas in the organ cultures of kidneys, spleen and testes the multiplication of endogenous PI-3 virus was suppressed.     

Indirect immunofluorescence test using polyclonal antibodies for the detection of Taylorella equigenitalis

Contagious equine metritis is a horse disease that causes endometrial inflammation due to Taylorella equigenitalis. Since Taylorella asinigenitalis was characterized, genital swab culture has proved to be an insufficient method for distinguishing between the two Taylorella species. Here, we developed an indirect immunofluorescence (IIF) test using polyclonal antibodies. Specificity, sensitivity, and detection limit were assessed using isolated bacteria (55 T. equigenitalis strains, 46 T. asinigenitalis strains and 18 other bacterial species), experimental and genital swabs in comparison to bacterial culture and polymerase chain reaction (PCR) testing. Our results indicated that IIF using polyclonal antibodies allows T. equigenitalis to be discriminated from T. asinigenitalis. This test constitutes a rapid, sensitive and specific tool for confirming presumptive colonies of T. equigenitalis.     

Virological and serological studies on the role of PI-3 virus, BRSV, BVDV and BHV-1 on respiratory infections of cattle. I. The detection of etiological agents by direct immunofluorescence technique

Nasal cells extracted from nasal swabs obtained from 95 cattle with signs of respiratory disease, out of eleven different herds, were tested for BHV-1, PI-3 virus, BRSV and BVDV using direct immunofluorescence technique. Viral antigen positive samples were detected in seven out of eleven herds examined. Of the 95 individual diseased cattle, 19 were found positive for at least one viral antigen. It was found that especially BHV-1 and PI-3 virus are important causative agents in cattle respiratory disease, both or in combination with other pathogenic agents. Multiple infection in virologically positive herds were observed in six (9.8%) of 61 animals tested. The findings reveal that single or multiple infections of selected viruses may be present in an important range in cattle and that direct immunofluorescence technique as a rapid method, based on the detection of viral antigen in nasal swab samples, is useful to establish the viral aetiology of acute bovine respiratory disease caused by these viruses, particularly in the diagnosis of mixed viral infections.     

Immunofluorescence tests for the detection of antibodies against Corynebacterium pyogenes and streptococci in blood serum and vaginal mucus of cattle]

Bacteriological tests were applied to cattle with endometritis and vaginitis. Included were cervical mucus samples and immunofluorescence tests to detect in that mucus as well as in blood serum antibody to Corynebacterium pyogenes and Streptococcus haemolyticus. The results pointed at intensive contact of the animals with the above pathogens and to their frequent occurrence in cervical mucus of cattle afflicted with endometritis and vaginitis. They also supported the assumption of localised antibody formation in the sexual organs or female cattle.     

Comparison of the sensitivity of laboratory animals and tissue cultures to infection with Aujeszky's disease virus]

In mice and guinea-pigs high susceptibility was demonstrated following i. c. administration of the virus, approximately equalling to that of rabbits and tissue cultures. Also a relatively high susceptibility of guinea-pigs was demonstrated, with very distinct clinical symptoms of the disease, as compared with mice and rats after the other manners of infection. On the basis of the results obtained white mice were utilized for routine diagnostic of Aujeszky disease. After i. c. administration of positive, virus-containing material encephalitis develops with a rapid exitus occurring 12-24 hours earlier than in the rabbits. In brain tissue antigen was always demonstrated by immunofluorescence examination. Along with the biological experiment replicas of pig organs were examined by immunofluorescence method. Maximum of positive yellow-green fluorescence is found in the cytoplasma of epithelial tonsillar cells, sporadically in the cell nuclei. Epithelial cells are deformed according to the infection degree. The amount of antigen in the brain tissue is not so pronounced as in the tonsillar tissue, yet in animals with clinical pathological symptoms the antigen was always demonstrated.     

The growth of attenuated strains of canine parvovirus, mink enteritis virus, feline panleukopenia virus, and rabies virus on various types of cell cultures

The growth characteristics were studied in the attenuated strains of canine parvovirus CPVA-BN 80/82, mink enteritis virus MEVA-BN 63/82 and feline panleucopenia virus FPVA-BN 110/83 on the stable feline kidney cell line FE, and in the attenuated canine distemper virus CDV-F-BN 10/83 on chicken embryo cell cultures (KEB) and cultures of the stable cell line VERO. When the FE cultures were infected with different parvoviruses in cell suspension at MOI 2-4 TKID50 per cell, the first multiplication of the intracellular virus was recorded 20 hours p. i. In the canine parvovirus, the content of intracellular and extracellular virus continued increasing parallelly until the fourth day; then, from the fourth to the sixth day, the content of extracellular virus still increased whereas that of intracellular virus fell rapidly. In the case of the mink enteritis virus the release of the virus into the culture medium continued parallelly with the production of the cellular virus until the sixth day. In the case of the feline panleucopenia virus the values concerning free virus and virus bound to cells were lower, starting from the second day p. i. When KEB or VERO cultures were infected in cell suspension with the canine distemper virus at MOI about 0.004 per 1 cell, the replicated intracellular virus was first recorded in the KEB cultures five hours after infection but in the VERO cultures only 20 hours after infection, with a timely release of the virus into the culture medium in both kinds of tissue. In the KEB and VERO cultures the highest values of infection titres were recorded on the fourth day p. i., the course of virus multiplication on the cells being parallel with its release into the culture medium.