A CELL CULTURE VACCINE AGAINST BOVINE EPHEMERAL FEVER

SUMMARY A vaccine was prepared from cell culture fluids harvested from the twelfth passage of the 919 strain of bovine ephemeral fever (BEF) virus in Vero cell cultures. Cattle were vaccinated subcutaneously with various combinations of strain 919 virus and adjuvants. Neutralising antibodies were assayed at various times after vaccination and some cattle were challenged by intravenous inoculation with the virulent 417WBC strain of BEF virus. Strain 919 virus of the third and twelfth passage levels in Vero cells produced neither fever, clinical illness nor detectable viraemia in 5 calves inoculated intravenously. Nor could viraemia be detected in 5 heifers receiving vaccine subcutaneously. When the vaccine was administered mixed with aluminium hydroxide adjuvant, the production of neutralising antibodies increased with an increase in the volume of vaccine from 2.5 ml to 10 ml and the response to 2 injections was significantly better than the response to a single injection. The neutralising antibody response was decreased when vaccine was diluted in phosphate buffered saline. The neutralising antibody response following 2 subcutaneous vaccinations with strain 919 virus mixed with aluminium hydroxide adjuvant was higher than that following intravenous inoculation with virulent virus. The vaccine-induced antibodies persisted for at least 12 months, and revaccination at this time led to an increase in the titre of neutralising antibody. Antibodies induced by a single subcutaneous administration of strain 919 virus mixed with Freund's complete adjuvant persisted for at least 40 weeks; those induced by vaccine containing Freund's incomplete adjuvant had virtually disappeared within 16 weeks. All these calves responded to vaccination with aluminium hydroxide-containing vaccine with increases in levels of neutralising antibodies. Of 26 vaccinated calves challenged with virulent BEF virus, 24 remained clinically normal. Two developed brief periods of pyrexia on the seventh day after challenge, but no other clinical signs. One of these calves had a viraemia that was demonstrated only by intravenous inoculation of a susceptible calf. The remaining calf had no detectable viraemia. All of 7 unvaccinated calves developed severe clinical BEF within 5 days of challenge. No disease attributable to the 919 virus occurred in 24 vaccinated pregnant heifers or their newborn calves.     

Comparative studies on the growth of Australian bluetongue virus serotypes in continuous cell lines and embryonated chicken eggs

The replication of cell culture passaged Australian bluetongue virus (BTV) isolates, serotypes 20 (CSIRO19) and 1 (CSIRO156), and an untyped BTV (CSIRO154) was assessed in eight continuous cell lines (one derived from baby hamster kidney cells, BHK-21; three derived from monkey kidney cells, Vero, LLC-MK2 and CV-1P; a foetal ovine lung and a mouse fibroblast cell line, CSL503 and L929, respectively, a Super-Vero-Porcine stable cell line, SVP; and a mosquito cell line, Aedes albopictus cells) and in 11-day-old embryonated chicken eggs (ECE) at different multiplicities of infection. All three viruses replicated in the cell lines tested, maximum extracellular virus yields being attained from BHK-21 cells at high multiplicities of infection (approximately 10 PFU per cell). Also BHK-21 cells produced much higher yields of virus than the other cell lines tested when low multiplicities of infection were used (approximately 10(-4) PFU per cell). All BTV serotypes multiplied in Singh's Aedes albopictus cells with no cytopathogenic effects over the 4 day period tested. The viruses also replicated in 11-day-old ECE; however, the sensitivity of ECE for growth of the Australian serotypes was not as high as has been reported for BTV isolates in other countries. In all cell culture systems and in ECE, BTV1 and BTV20 replicated more efficiently than did CSIRO154 virus.     

Comparison of the effect of various chemical stabilizers and lyophilization cycles on the thermostability of a Vero cell-adapted rinderpest vaccine

The thermostability of a rinderpest vaccine produced on Vero cells was evaluated using a variety of chemical stabilizers and lyophilization protocols. Three stabilizer preparations and three lyophilization schedules were examined using accelerated stability testing at 37 degrees C. The vaccine preparation exhibiting the greatest stability at 37 degrees C was tested at three additional temperatures, 42, 45 and 56 degrees C, and an Arrhenius plot was constructed from the data. The stability of the reconstituted vaccine produced with the two most efficacious stabilizers was examined using three different diluent preparations. The stabilization method and high Vero cell virus batch titers resulted in a lyophilized vaccine which maintained the minimum required dose of log10 2.5 TCID50 tissue culture infectious dose for more than 20 weeks at 37 degrees C.     

Equine rhinopneumonitis virus (herpesvirus type 1): attenuation in stable monkey cell line.

An isolate of virulent equine herpesvirus (EHV) type 1 was adapted to Vero stable cell line by 13 serial passages at 37 C and 50 serial passages at 26 C. Characteristics of the attenuated EHV-1 were found to be avirulent, but immunogenic in horses if injected intramuscularly. The attenuated virus was regularly isolated from peripheral leukocytes in inoculated horses, but was not recovered from nasal turbinate tissues. A mild leukopenia was noticed. The attenuated virus produced characteristic large syncytia on primary isolation in rabbit kidney (RK13) or Vero cells at 37 C in contrast to cell rounding observed with virulent EHV-1. The syncytial marker was stable through 20 serial passages in Vero cells at 37 C. New application of double immunodiffusion test for distinguishing between EHV-1 and EHV-2 also is described.     

Establishment of an attenuated strain of bovine respiratory syncytial virus for live virus vaccine

To develop a live virus vaccine for the prevention of bovine respiratory syncytial (BRS) virus infection in calves, an attempt was made to produce an attenuated virus. The RS-52 strain of BRS virus, isolated from the nasal secretions of a naturally infected calf, was subjected to serial passages in adult hamster lung established (HAL) cells at 30 degrees C and the attenuated rs-52 strain as a live virus vaccine was established. The rs-52 strain multiplied better at 30 degrees C than at 34 or 37 degrees C in HAL cells. The differences in the highest virus titers of this strain between the culture temperature of 30 degrees C and that of 34 or 37 degrees C were more than 2.25 log TCID50. Colostrum-deprived newborn calves and 2 approximately 4 months old calves inoculated with the rs-52 strain manifested no abnormal clinical sings at all. However, all inoculated calves produced serum neutralization antibody. When the colostrum-deprived newborn calves immunized with the rs-52 strain were challenged with the virulent NMK7 strain of BRS virus, they exhibited no pyrexia or other abnormal clinical signs at all. An attempt was made to recover the virus from nasal secretions of these calves, but in vain. On the other hand, a nonimmunized control colostrum-deprived newborn calf developed slight fever, mild cough, and slight serous nasal discharge after challenge exposure. The virus was recovered from nasal secretions of this calf. From these results, it was considered that the rs-52 strain could be used as an attenuated live virus vaccine for prevention of BRS virus infection.     

LABORATORY AND FIELD STUDIES WITH A BOVINE EPHEMERAL FEVER VACCINE

Bovine ephemeral fever (BEF) virus vaccines, prepared from the brains of suckling mice infected with strain 525 BEF virus, were evaluated in housed cattle and in the field. The virus in lyophilised preparations was stable for 6 months at -50 degrees C. Thirty-four calves, 5 to 18 months old, were used in laboratory vaccination trials. An increase in serum neutralising antibody was detected in 13 of 14 calves initially free of serum antibody, and all 13 failed to develop clinical illness following challenge with virulent BEF virus. Vaccination resulted in no detectable serum antibody increase in 4 calves, 5 months old, with pre-existing antibody of presumed maternal origin. Seven animals, 18 months of age with serum antibody presumed due to previous BEF infection, developed increased antibody titres following vaccination. In 3 animals vaccinated but not challenged, vaccine-induced antibodies decreased to low levels over 5 months. In contrast, the antibody titres following infection with virulent virus in 2 calves were maintained over 5 months. Field trials, involving 236 animals initially free of serum antibody, were conducted on 5 properties near Mackay and 4 properties near Brisbane. Most of 164 animals were vaccinated with a single dose of lyophilised vaccine containing aluminium hydroxide adjuvant. Only 4 animals failed to develop serum antibody and no adverse reactions to vaccination were reported. Natural infection with BEF occurred in 4 herds at Mackay and clinically mild BEF occurred in 3 of 109 vaccinated and 3 of 46 control animals. On the basis of measured serum antibody titres it was assumed that 8 of 53 animals receiving full vaccine volume, 20 of 40 animals receiving half vaccine volume and 18 of 40 control animals became infected with BEF virus. Two dairy herds in Brisbane became naturally infected with virulent BEF virus 7 months after vaccination. Clinical BEF was observed in 8 of 11 control animals and in 3 of 26 animals which received 2 doses of vaccine. Two strains of BEF virus were isolated from unvaccinated animals that developed clinically mild BEF in the field. These strains either failed to infect, or produced subclinical or very mild BEF, when inoculated intravenously into susceptible calves. The anitbody response to natural infection with apparently mild viruses was short-lived, similar to that produced by vaccination.     

Isolation and characterization of epizootic hemorrhagic disease virus from white-tailed deer (Odocoileus virginianus) in eastern Washington.

A virus was isolated from the spleen of a white-tailed deer (Odocoileus virginianus) that had died during an epizootic in Washington state in 1967. Inoculation of a 10% spleen suspension from the deer caused hemorrhagic disease in normal white-tailed deer. Studies were conducted on the biological, physicochemical, and serologic properties of the Washington isolate. An in vitro assay system, utilizing a cultured primary of white-tailed deer fetal cells from an entire fetus, was employed for isolation and propagation of the virus. Cytopathic effect was characterized by focal development of rounded and clumped cells. Propagation was unsuccessful in suckling mice, BHK-21, and Vero cell cultures. The virus was resistant to treatment with ether, sodium deoxycholate, trypsin, oxytetracycline hydrochloride, and was sensitive to chloroform. Virus yield was not affected when infected cultures were treated with 5-iodo-2'-deoxyuridine, but dactinomycin (actinomycin D) treatment of infected cultures reduced virus yield. The virus was inactivated when heated at 70 C for 5 minutes or when exposed to pH 5 for 18 hours at 4 C. The virus was completely excluded from the filtrate by a 0.10- micronm (APD) membrane filter. Staining of infected cells with acridine orange indicated the presence of double-standard nucleic acid in the cytoplasm. Serum-neutralization tests with antiserums against the homologous virus and the New Jersey and Alberta strains of epizootic hemorrhagic disease virus resulted in neutralization of the Washington isolate. The Washington virus was not neutralized by bluetongue virus antiserum. Cells infected with the Washington isolate exhibited intracytoplasmic fluorescence by the indirect fluorescent antibody method with New Jersey and Alberta epizootic hemorrhagic disease antiserums but not with bluetongue antiserum.     

Serological studies of two additional Australian blue-tongue virus isolates CSIRO 154 and CSIRO 156

Serological surveys revealed that some cattle in northern Australia possessed bluetongue virus (BTV) group-reactive (agar gel diffusion precipitin, AGDP, and complement-fixing, CF) antibodies, but not serum neutralizing (SN) antibodies, to BTV20, a new type previously found in Australia. Attempts were made during 1979 to isolate viruses causing these reactions. There was one isolate of a virus (CSIRO 154) and eight isolates of another virus (CSIRO 156) made from the blood of healthy cattle in the Northern Territory. These viruses could not be distinguished from BTV20 by AGDP, CF or fluorescent-abtibody tests and hence were designated members of the bluetongue serogroup. Serotyping was carried out using the plaque-inhibition and plaque-reduction SN tests. CSIRO 156 virus could not be distinguished from BTV1 by any of the SN tests and it was concluded that it was an Australian isolate of the BTV1 serotype. CSIRO 154 virus was found to be related to, but not identical with, BTV6. It is probably not one of the known 20 BTV serotypes and may represent a new BTV serotype. None of the three Australian BTV isolates is known to cause clinical disease in sheep or cattle under natural conditions, and biochemical comparisons with the African BTV serotypes may show differences not revealed by these serological studies.     

Profiles of toxin production by thermophilic Campylobacter of animal origin.

Seventy-five strains of Campylobacter jejuni and C. coli, which were isolated from a variety of animal species, primarily poultry, were examined for production of toxin. Polymyxin extracts were tested in in vitro assays using CHO-KI, FCL (foetal calf lung), Vero, HeLa and CEF (chicken embryo fibroblast) cells. The toxic effects observed were cell rounding and death. Extracts from almost all C. jejuni and C. coli strains were toxic to both CHO-KI and FCL cells and 69.0% of C. jejuni isolates and 75% of C. coli isolates were also toxic to CEF cells. 50.7% of C. jejuni extracts were toxic to Vero cells and 46.5% toxic to HeLa cells. None of the C. coli isolates were toxic to either of these cell lines. None of the strains tested produced cytotonic enterotoxin. No differences in toxigenicity patterns were evident between Campylobacter isolated from different sources.     

Immunofluorescence with the PI-3 virus--study of various working conditions for the detection of viral infection using immunofluorescence on various types of cell cultures]

We used the fluorescence method for the investigation of the sensitivity of several kinds of cell cultures to the infection with the parainfluenza virus 3 (PI-3). Cultures from calf kidneys were the most sensitive while we did not determine any differences between primary cultures and cultures of the first and second subpassages and/or freshly cultivated or incubated cultures over seven days at a temperature of 37 degrees C. Equal values of infection titres like on the cultures of calf kidneys were determined by immunofluorescence also on the kidney cells of lambs though the presence of the infection was not accompanied by cytopathic changes. Infection of pig kidney cells appeared only after the inoculation of 10(3) TKID50 and higher doses of the virus, the infection having a very slow course of development without detectable cytopathic changes. Fluorescent findings were identical in different tissues. Antibodies present in the culture medium stopped the spreading of the infection by neutralizing the virus released from the cells, however, not the primary infection. The increase in the content of antibodies in the medium led - by inhibiting the intercellular virus - to the slowing down of the growth of primary fluorescent lesions.     

Disinfectant tests at 20 and 10 degrees C to determine the virucidal activity against circoviruses

To evaluate virucidal activity against porcine circovirus type 2 (PCV2), four disinfectants were tested under laboratory conditions. As basis to perform the testing the "Guidelines for testing chemical disinfectants" of the German Veterinary Association (DVG-guidelines) were applied. For simulation of field conditions, the tests were carried out in virus carrier tests, at 20 and 10 degrees C, and under protein load (40% foetal calf serum (FCS) in virus suspension). For disinfection of PCV2 at 20 degrees C an exposure time of 120 min in 2% Disinfectant 1 (20% glutaraldehyde, 12% 2-propenal, polymer with formaldehyde) or Disinfectant 2 (55% formic acid, 7% glyoxylic acid) was necessary. 1% of Disinfectant 3 (Component 1: Potassium peroxomonosulphate. Component 2: Active detergents) disinfected PCV2 on carriers within 180 min. After a reaction time of 120 min with 1% and 60 min with 2% Disinfectant 4 (21% glutaraldehyde, 17% formaldehyde) there could not be detected any virus. Reduction on effectivity through temperature reduction to 10 degrees C were more significant for aldehyde containing preparations Disinfectant 1 and Disinfectant 4 than for Disinfectant 2 and Disinfectant 3. These losses on effectivity could be corrected through extension of exposure time or increase of concentration.     

An experimental inactivated virus vaccine against bovine ephemeral fever 1. Studies of the virus

Studies were carried out to find methods for obtaining optimum yields of bovine ephemeral fever (BEF) virus and for concentrating the virus in order to develop inactivated virus vaccines. Cells from the SVP cell line, which was derived from the pig kidney PS cell line, were most satisfactory for growing and assaying BEF virus. BHK 21 and Vero cells also gave similar yields of virus but were not as useful for virus assay. A plaque assay in SVP cells, in which there was 0.1 μg of actinomycin D per ml of overlay, produced reproducible clear plaques and was slightly more sensitive than assays in BHK 21 cell roller tubes. High multiplicities of infection (MOI), around 1 PFU/cell, produced low yields of infectious virus, whereas decreasing the MOI approximately 100-fold led to an increase in virus yield of up to four logs. BEF virus could be concentrated using zinc acetate or ammonium sulphate but not polyethylene glycol 6000. Ammonium sulphate proved most suitable and produced an easily handled precipitate with up to 100% recovery of virus infectively, and 100-fold concentration was possible. This concentrated virus could be rapidly desalted by gel filtration through Sephadex G-75. The virus could be further purified by sucrose density gradient ultracentrifugation provided the gradient contained a protein stabilizer of 0.1% bovine serum albumin. Inactivation kinetics with 0.025% β-propiolactone was similar to that reported for other rhabdoviruses.     

Standardization of large scale production of homologous live attenuated PPR vaccine in India

Live attenuated homologous vaccine against peste des petits ruminants of sheep and goats was produced on a large scale basis in roller culture bottles using seed virus developed at the Indian Veterinary Research Institute, Muktheswar, India. Vero cells between 130–150 passages with six percent foetal calf serum were used for the production of vaccine. The cells were infected with 0.01 multiplicity of infection and harvested when the cytopathic effect was 80%. The vaccine was freezedried in order to maintain the stability of the vaccine. Identity test and titration was performed and the vaccine titre was monitored to be minimum of 10⁵/100 doses. In-house sterility tests and quality control tests using experimental animals and small ruminants were performed. The vacuum and moisture content of the vaccine were also regulated to be within the normal limits.     

Mechanisms contributing to the virus persistence in Aleutian disease

In this review published results and further studies concerning the persistence of Aleutian disease virus (ADV) isolate SL3 are presented. By Southern blot and in situ hybridization with strand-specific RNA probes focal replication of ADV-DNA was demonstrated in spleen, mesenteric lymph nodes, sporadically in mononuclear cells of the peripheral blood and bone marrow cells. These findings further support the concept of the lymphotropism of ADV. All cell culture-adapted ADV strains appear to have a ts-defect. Our in vitro studies indicate that the ADV isolate G(orham) induced the synthesis of comparable amounts of viral replicative DNA and viral proteins VP1 and VP2 at the non-permissive temperature of 37 degrees C. However, the viral progeny DNA synthesis was about threefold less at 37 degrees C compared to the permissive temperature of 32 degrees C. These findings suggest that the reduced level of viral progeny DNA at 37 degrees C accounts for the reduced production of infectious ADV. Finally, we provided experimental evidence that the apparent lack of neutralizing antibodies in AD is due to the masking of critical viral epitopes by cellular phospholipids.     

Aerosol stability of bovine parainfluenza type 3 virus.

Aerosols of bovine parainfluenza type 3 virus were generated with a Devilbiss 40 nebulizer from Eagle's minimum essential medium and nasal secretion from a non-infected calf and stored in a rotating drum at temperatures of 6 degrees C or 32 degrees C and relative humidities of 30% or 90%. The aerosols were sampled at seven minutes, one, two and three hours after the start of generation with an all glass impinger (AGI-30) and titrated for infectivity in cell cultures. Physical decay was determined by a rhodamine tracer technique. Media, temperature or relative humidity had little effect on the survival of parainfluenza type 3 virus during spraying (zero to seven minutes). During aging of aerosols at 32 degrees C and 30% relative humidity, parainfluenza type 3 virus was less stable in Eagle's minimum essential medium than in nasal secretion from a noninfected calf, but at 6 degrees C and 30% relative humidity, the virus was more stable in Eagle's minimum essential medium. At 32 degrees C, the virus was less stable during aging at 90% relative humidity than at 30% relative humidity. The virus was consistently more stable during aging of aerosols at 6 degrees C than at 32 degrees C.     

Akabane virus infection in the pregnant ewe. 1. Growth of virus in the foetus and the development of the foetal immune response

Three different pools of the CSIRO 16 strain of Akabane virus differing in their laboratory passage histories were used to inoculate 39 ewes between 32 and 36 days pregnant; 22 pregnant ewes received inocula containing no virus. There was no difference in the development, duration and titre of the viraemia and neutralising antibody response between the three infected groups of ewes. Both infected and control ewes had 141% foetuses when autopsied at 69 to 105 days gestation. Of the 55 foetuses from infected ewes 44 (80%) had gross developmental abnormalities.At autopsy of the dams Akabane virus was isolated only from the uterine caruncle. From foetal samples virus was isolated from a wide range of tissues, from one foetus at 69 days and from the blood of four foetuses at 95 to 106 days gestation. Virus was also isolated from 24 of the choriolllantoic fluid samples and from 37 placentomes of the 44 foetuses with developmental defects, in concentrations ranging from 10² to 10^5.5 TCID₅₀/ml or/g. No virus was isolated from the tissues of the control ewes or their foetuses.Neutralising antibody to Akabane virus was detected in 78% of the foetal sera from the infected group, titres ranging from 2 to 64. IgM and IgG₁ and neutralising antibody were detected in sera of 40 foetuses with developmental abnormalities including three that were of 76 to 78 days gestation. Neutralising antibody was detected only in serum that contained IgG₁ but may also have been associated with IgM in infected foetuses. IgM was detected in the serum of most foetuses including the non-infected controls, but sera from the control foetuses did not contain IgG₁ or neutralising antibody to Akabane virus. No IgG₂ or IgA were detected in any foetal serum.     

Growth of bluetongue and epizootic hemorrhagic disease of deer viruses in poikilothermic cell systems

Continuous cell lines from the ticks Dermacentor variabilis, D. parumapertus, D. nitens, Rhipicephalus sanguineus and R. appendiculatus, the mosquitoes Aedes albopictus and Culex quinquefasciatus and the African toad Xenopus laevis were tested for their ability to replicate bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) viruses, and for their sensitivity as potential isolation systems. BT serotype 17 grew to peak titers of 10(4.5)-10(7.5) TCID50 ml-1 in all except one of the tick cell lines, EHD 2 virus attained titers similar to that of BT 17 in the mosquito and toads cells, but failed to replicate in tick cells. Only Aedes albopictus and Xenopus laevis cells were as sensitive to infection with low-passage BT 11 and EHD 2 viruses as control cultures of Vero and BHK cells. At 27 degrees C, persistent infection of Xenopus laevis cells occurred, producing low yields of BT 17 and EHD 2. When shifted to 32 degrees C, these cultures expressed virus in exponential increments. No cytopathic effect (CPE) was seen in any of the tick-virus systems, but infected mosquito and toad cells detached from the monolayer within 3-6 days after inoculation with either virus. In the toad cells, this CPE was presaged by the development of plaques within 48 h after infection. Potential applications of poikilotherm systems in orbivirus research are discussed.     

Use of a serum-free medium to produce in vitro Neospora caninum and Toxoplasma gondii tachyzoites on Vero cells

Neospora caninum and Toxoplasma gondii are cyst-forming coccidian parasites of human and veterinary clinical relevance. In vitro cultivation of the protozoans using Vero cells is usually performed in order to produce antigenic materials. Quantitative and qualitative comparisons of Vero cells grown in RPMI medium supplemented either with foetal calf serum (FCS), horse serum (HS) or a specific serum-free additive (DefCell) were performed. A serum-free cell culture system used to propagate N. caninum (NC-1 isolate) and T. gondii tachyzoites (Rh stain) were compared with the other two cell culture systems. FCS supplemented media was found to be more effective than the others in promoting Vero cells and N. caninum tachyzoites. However, it was found unable to support adequate T. gondii tachyzoite proliferation. Vero cells, T. gondii and N. caninum tachyzoite production gave similar growth patterns with either HS or DefCell supplemented media. Defcell was considered as a good alternative to supplement culture medium.     

Toxicity of Haemophilus pleuropneumoniae to porcine lung macrophages

Viable Haemophilus pleuropneumoniae bacteria were toxic for porcine alveolar macrophages in vitro. This cytotoxic effect proved to be dose-related. A cell-free extract of H. pleuropneumoniae, heat-killed bacteria, and a Pasteurella multocida field strain were nontoxic. When macrophages were cultured with H. pleuropneumoniae bacteria in a ratio of 100 macrophages to six bacteria, ultrastructural signs of cellular degeneration were observed within 1 h. This degeneration was observed in macrophages with or without phagosomes containing H. pleuropneumoniae. A cytotoxic substance was filtered from a H. pleuropneumoniae culture in Eagle's minimal essential medium supplemented with Earle's salts (EMEM) and 10% foetal calf serum that was incubated for 10 h at 37 degrees C. This substance was destroyed by heating at 65 degrees C for 30 min. Macrophages were less susceptible to the toxic effect of H. pleuropneumoniae when serum of convalescent pigs was added.     

Disinfection of caliciviruses at 20 and 10 degrees C

Five disinfectants, Venno FF super, Venno Vet 1 super, Venno Oxygen, M&Enno-Veterin?r B neu und Neopredisan 135-1, were tested to evaluate their efficacy against caliciviruses at 20 and 10 degrees C. As model test virus served feline calicivirus type F9 (FCV F9). All disinfectants were tested according to Guidelines of the German Veterinary Association (DVG). The investigations were performed in suspension tests and germ carrier tests. The suspension tests were carried out without and with protein load. As protein was used foetal calf serum at the concentration of 40%. Venno FF super showed less protein dependence, however a considerable temperature dependence. This matter can be corrected by increase of concentration on 2%. Venno Vet 1 super was without protein especially effective. The losses on the effectiveness through low temperature and protein load can be annulled also here by increase of concentration. Venno Oxygen was more effective in the comparison to that here named both preparations. The effects of temperature can be corrected by extension of reaction time. The most effective preparation was M&Enno Veterin?r B neu. The disinfection occurred at 20 degrees C with 0.5% solution within 120 min and at 10 degrees C with 1.0% solution within 60 min. The fifth disinfectant Neopredisan was in suspension tests without protein load and carrier tests with gauze at 20 and 10 degrees C relative convincing but in germ carrier tests with poplar wood, no complete disinfection could be achieved within tested concentrations and reaction times.