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.

Foot-and-mouth disease in camelids: a review.

Foot-and-mouth disease (FMD) in South American camelids, in dromedaries and Bactrians is reviewed. Recent well-executed experimental studies in New World camels indicate that, although the llama and alpaca can be infected with FMD virus (FMDV) by direct contact, they are not very susceptible and do not pose a risk in transmitting FMD to susceptible animal species. They do not become FMDV carriers. Reports on FMD in dromedaries are, however, conflicting. Serological investigations in Africa and the United Arab Emirates (UAE) on thousands of camel sera were negative and experimental infections have been conducted on only a few dromedaries with one serotype and in one country. The design and execution of most of these experiments were poor and therefore the conclusions are questionable. From these investigations, it seems that dromedaries can contract the disease after experimental infection and through close contact with FMD diseased livestock, but do not present a risk in transmitting FMD to susceptible animals. They do not become FMDV carriers. Recent reports from Mongolia describe similar FMD lesions in Bactrian camels. However, so far no samples have tested positive for FMD. To clarify the situation in Bactrians, samples from suspected clinical cases should be tested because other viral vesicular diseases cannot be distinguished from FMD. Thus, further research on the epidemiology of FMD in camelids is necessary. This would include large-scale serological investigations and experimental infections with different FMD serotypes in connection with susceptible contact animals. The Office International des Epizooties (OIE) Code chapter on FMD includes camelids as being susceptible species to FMD, giving the impression that they are similar to cattle, sheep, goats and pigs in their potential involvement in the epidemiology of FMD. This is clearly not the case, and this issue should be re-addressed by the relevant authorities.

Development of a monoclonal blocking ELISA for the detection of antibodies against fowlpox virus.

To provide a fast and easy method to detect antibodies against fowlpox virus (FWPV) particularly in high numbers of chicken sera we established a monoclonal blocking enzyme-linked immunosorbent assay (ELISA). We chose two different monoclonal antibodies (mAb), anti-FWPV 3D9/2B3 and anti-FWPV 8F3/2E11, which are both directed against the 39-kDa protein of FWPV strain HP-1. The blocking ELISA depends on the blocking of mAb binding to solid-phase antigen in the presence of positive serum. For an epidemiological study a total of 184 serum samples from Gambian chicken flocks were analysed against each of the mAbs. Four of the sera were shown to contain FWPV antibodies. These four sera showed a positive cut-off value of more than 50% inhibition exclusively in the test against the mAb anti-FWPV 8F3/2E11. This phenomenon can be explained by the binding of the mAbs to distinct epitopes on the same protein.

Recent developments in the epidemiology of virus diseases.

There is continual variation in viral epidemics regarding clinical symptoms, duration and disappearance, and the emergence of new diseases. This can be observed in both human and animal diseases. This evolution of virus diseases is mainly related to three factors: aetiological agent, host and environment. As far as genetic alterations of the virus are concerned, two major mechanisms are involved: mutations such as recombination and reassortment; and selection for resistance or susceptibility. This review focuses on the epidemiology of newly emerged virus diseases in man and animals, such as acquired immunodeficiency syndrome, haemorraghic fevers, bovine spongiform encephalopathy, canine haemorraghic disease and respiratory syndrome in horses.

Equine herpesvirus type 1 (EHV-1) myeloencephalopathy: a case report.

An outbreak of neurological disease occurred in a well-managed riding school. Ataxia and paresis were observed in several horses, five of which became recumbent and were euthanized. Post-mortem analysis revealed scattered haemorrhages along the spinal cord, that were reflected by multiple haemorrhagic foci on formalin-fixed sections, with the thoracic and lumbar segments being the most affected. Pathohistologically, perivascular mononuclear cuffing and axonal swelling, especially in the white matter, were evident. Parallel to the course of disease, alterations in myelin sheets and activation of astrocytes and microglial cells were also observed. Virological findings confirmed an acute equine herpesvirus type 1 infection and virus was isolated from the spinal cord of a 26-year-old mare.

Phylogenetic analysis of the 5'-untranslated region of german BVDV type II isolates.

On the basis of genetic differences, bovine viral diarrhoea viruses (BVDV) are subclassified into two distinct genotypes, BVDV type I and BVDV type II. We selected German BVDV type II isolates using the BVDV type I-specific monoclonal antibody WB160 and flow cytometric analysis for further characterization. For molecular characterization, a 288-bp fragment of the 5'-untranslated region (5'-UTR) of the selected isolates was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. Sequence comparisons of the partial 5'-UTR sequences and their phylogenetic analyses demonstrated that the 18 German BVDV type II isolates all belong to either subtype IIa (10 isolates) or subtype IIc (eight isolates). Nevertheless, the German BVDV type II isolates were genetically different (89.9-94.3% sequence identity) from the standard BVDV type II strain 890 from North America, which was recently classified as BVDV type IIa. Furthermore, a clear subdivision of the German BVDV type II isolates into two distinct subtypes (BVDV IIa Germany and BVDV IIc Germany) is shown. Viruses of both subgroups differed in the analysed 5'-UTR fragment from each other (91.6-95.2% sequence identity), but were highly conserved within the same German subtype (97.2-100% sequence identity). These findings are discussed in the context of BVDV type II origin, possible introduction into Germany, its epidemiology and impact for vaccine development.

Escherichia coli isolates from young calves in Bavaria: in vitro susceptibilities to 14 anti-microbial agents.

During the occasional testing of Escherichia coli from faecal samples of young calves we observed multi-resistant isolates. Because of the significance of E. coli as an indicator bacterium for resistance trends we tested E. coli populations of young calves over a longer period. Here we present the results of a retrospective study comparing isolates from 1998 to 2000. Moreover, we compared, in a clinical study, the resistance rates of E. coli populations from 67 hospitalized calves both before and after hospitalization (with or without anti-microbial therapy), and with their anamnestic data of antibiotic usage. The highest resistance rates were found to be more than 80% for tetracyclines, ampicillin, sulfonamide/trimethoprim combinations, and chloramphenicol. A significant increase or decrease over the years was not observed. In analysing the data of hospitalized calves, an increase of resistance to some anti-microbials had to be registered that seemed to be connected with the selective pressure due to agents used in the clinic. In comparing anamnestic data and resistance rates it became obvious that reliable data are not easily available and that a number of potential anti-microbial influence factors have to be taken into account.

The equine herpesvirus 1 UL45 homolog encodes a glycosylated type II transmembrane protein and is involved in virus egress.

Experiments to analyze the product of the equine herpesvirus type 1 (EHV-1) UL45 homolog were conducted. Using an antiserum generated against the carboxylterminal 114 amino acids of the EHV-1 UL45 protein, proteins of M(r) 32,000, 40,000, and 43,000 were detected specifically in EHV-1-infected cells. Neither form of the protein was located in purified virions of EHV-1 wild-type strain RacL22 or the modified live vaccine strain RacH, but UL45 was demonstrated to be expressed as a late (gamma-2) protein. Fractionation of infected cells and deglycosylation experiments demonstrated that the EHV-1 UL45 protein represents a type II membrane glycoprotein. Deletion of the UL45 gene in RacL22 and RacH (LDelta45 and HDelta45) showed that UL45 is nonessential for EHV-1 growth in vitro, but that deletion reduced the viruses' replication efficiency. A marked reduction of virus release was observed although no significant influence was noticed either on plaque size or on the syncytial phenotype of the EHV-1 strain RacH.

A universal 'one-tube' RT-PCR protocol for amplifying isolates of bovine viral diarrhoea virus.

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5'-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, ERNs, E1 and E2 genes: (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10(6) infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.

A new inactivated BVDV genotype I and II vaccine. An immunisation and challenge study with BVDV genotype I.

An inactivated vaccine containing BVDV I and II strains (PT810; BVDV I, and 890; BVDV II) and using different adjuvants and antigen dosages was tested in a cattle challenge model. Groups of six healthy, seronegative cattle were vaccinated twice with a low dose (10(6.6) TCID(50) PT810 and 10(7.2) TCID(50) 890) vaccine with the adjuvant Bay R1005 or a high dose (10(7.8) TCID(50) PT810 and 10(8. 2) TCID(50) 890) vaccine with two different adjuvants (Bay R1005 or Polygen). Thirty-eight days after the second vaccination, immunised animals (n=18) and non-vaccinated control animals (n=3) were challenged intranasally with 10(6) TCID(50) BVDV strain PT810. For a period of 16 days, virus was isolated from blood leukocytes and nasal swabs, and neutralising antibody titres were determined.The induction of antibodies following immunisation was strongly dependent on the antigen dosage in the vaccine. The high dose formulation induced high serum neutralising antibody titres against both genotypes of up to 32000 after the second immunisation. Animals with neutralising antibody titres >512 (n=14) did not show any marked leukopenia after challenge and only very little or no virus could be isolated from blood leukocytes and/or nasal swabs when compared to control cattle. Furthermore, some of these animals did not show any boost of neutralising or even NS3-specific antibodies, which renders viral replication unlikely and thus would prevent infection of the fetus. Both adjuvants (Bay R1005 or Polygen) were similarly efficient and induced nearly identical antibody responses. In contrast, four of the six low dosage vaccinates had a marked leukopenia and viraemia as well as detectable nasal virus shedding for several days. We conclude that the selected strains and the system of vaccine preparation with high BVDV antigen dosages and highly efficient new adjuvants provide an effective means of protection against BVDV I infections. Investigations to demonstrate the protection against BVDV II infections, the duration of immunity and the ability of fetal protection by using the high dose vaccine in a fetal challenge model will follow.

Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses.

Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of eastern, western, and Venezuelan equine encephalitis viruses (EEE, WEE, and VEE, respectively). Tests for specificity included all known alphavirus species. The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a subsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximately 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and IV), were also amplified. However, the latter viruses showed slightly smaller fragments of about 290 and 310 bp, respectively. A subsequent seminested PCR amplified a 195-bp fragment only from the 10 tested strains of WEE from North and South America, rendering this assay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegenerate primers, this assay was confined to the medically important and closely related VEE subtypes IAB, IC, ID, IE, and II. The RT-PCR-seminested PCR combination specifically amplified 342- and 194-bp fragments of the region covering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype IAB virus and 70 RNA molecules for subtype IE virus. In addition to the subtypes mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR. The practicability of the latter assay was tested with human sera gathered as part of the febrile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos. All of the nine tested VEE-positive sera showed the expected 194-bp amplicon of the VEE-specific RT-PCR-seminested PCR.

Recent developments in the epidemiology of virus diseases and BSE.

There is a continuous change in viral epidemics with respect to clinical symptoms, their duration or disappearance and the emergence of new diseases. This can be observed both in human and animal diseases. This evolution of virus diseases is mainly related to three factors: etiological agent, host and environment. As far as genetic alterations of the virus are concerned, two major mechanisms are involved: 1) mutations such as recombination and reassortment; 2) selection for resistance or susceptibility. The epidemiology of newly emerged virus diseases in man and animals, such as AIDS and hemorrhagic fevers, and bovine spongiform encephalopathy (BSE), canine hemorrhagic gastroenteritis or respiratory syndrome in horses will be discussed.

The equine herpesvirus 1 IR6 protein that colocalizes with nuclear lamins is involved in nucleocapsid egress and migrates from cell to cell independently of virus infection.

The equine herpesvirus 1 (EHV-1) IR6 protein forms typical rod-like structures in infected cells, influences virus growth at elevated temperatures, and determines the virulence of EHV-1 Rac strains (Osterrieder et al., Virology 226:243-251, 1996). Experiments to further elucidate the functions and properties of the IR6 protein were conducted. It was shown that the IR6 protein of wild-type RacL11 virus colocalizes with nuclear lamins very late in infection as demonstrated by confocal laser scan microscopy and coimmunoprecipitation experiments. In contrast, the mutated IR6 protein encoded by the RacM24 strain did not colocalize with the lamin proteins at any time postinfection (p.i.). Electron microscopical examinations of ultrathin sections were performed on cells infected at 37 and 40 degreesC, the latter being a temperature at which the IR6-negative RacH virus and the RacM24 virus are greatly impaired in virus replication. These analyses revealed that nucleocapsid formation is efficient at 40 degreesC irrespective of the virus strain. However, whereas cytoplasmic virus particles were readily observed at 16 h p.i. in cells infected with the wild-type EHV-1 RacL11 or an IR6-recombinant RacH virus (HIR6-1) at 40 degreesC, virtually no capsid translocation to the cytoplasm was obvious in RacH- or RacM24-infected cells at the elevated temperature, demonstrating that the IR6 protein is involved in nucleocapsid egress. Transient transfection assays using RacL11 or RacM24 IR6 plasmid DNA and COS7 or Rk13 cells, infection studies using a gB-negative RacL11 mutant (L11DeltagB) which is deficient in direct cell-to-cell spread, and studies using lysates of IR6-transfected cells demonstrated that the wild-type IR6 protein is transported from cell to cell in the absence of virus infection and can enter cells by a yet unknown mechanism.

Detection, cDNA cloning and sequencing of canine interleukin 12.

In canine peripheral blood mononuclear cells (PBMC) the mRNAs coding for both subunits of canine interleukin 12 (IL-12) were identified using reverse transcription polymerase chain reaction (RT-PCR). Stimulation of canine PBMC with Staphylococcus aureus strain Cowan plus Concanavalin A for 5 h resulted in significant mRNA synthesis. Likewise, inactivated vaccinia virus induced IL-12 mRNA synthesis, however with different kinetics. The complete nucleotide sequence for both IL-12 subunits was determined using rapid amplification of cDNA ends (RACE)-PCR and cloning of amplified specific cDNAs. Computer-aided amino acid (aa) sequence comparison of both canine IL-12 subunits revealed more than 80% identity with the amino acid sequences of six other mammalian species. Closest relationship was found to human, porcine, bovine and cervine IL-12. However, no reactivity was found with antibodies directed against human IL-12, when supernatants of stimulated canine PBMC were tested. Supernatants of canine PBMC stimulated for IL-12 release also induced interferon gamma (IFN-gamma) mRNA as detectable by RT-PCR; however, it was not clear whether IFN-gamma mRNA synthesis was due to an IL-12 specific effect or other stimuli. As to the stimulating effect of IL-12 on canine IFN-gamma mRNA synthesis, recombinant human IL-12 was found to be a good inducer. Since IL-12 is regarded a major regulatory molecule of T-cell-mediated immune response and cell growth our work on the cloning and sequencing of this cytokine from dogs lays the basis for future investigations on the biological and possible therapeutic role of canine IL-12.

The alphavirus 3'-nontranslated region: size heterogeneity and arrangement of repeated sequence elements.

The 3'-nontranslated region (NTR) of representative strains of all known alphavirus species was amplified by reverse transcription-polymerase chain reaction. For 23 of them, the 3'-NTR sequence was determined. Together with previously published data, this allowed an analysis of the 3'-NTR of the viruses in the genus Alphavirus. The length of the 3'-NTRs varied from 77 nt for Pixuna virus to 609 nt for Bebaru virus. The 19-nt conserved sequence element directly adjacent to the poly(A) tract was found in all viruses, supporting the hypothesis that this region is a cis-acting sequence element during viral replication and essential for virus growth in vitro. Within the 3'-NTR of all alphaviruses, repeated sequence elements of various numbers and lengths were found. Their composition was very consistent in both the Venezuelan equine encephalitis (VEE) and the Sindbis-like viruses, although their number was constant only within the latter group. For the VEE viruses, our data suggested that insertion events rather than deletions from an ancestor with a long 3'-NTR created the various number of repeated sequence elements. Among the remaining viruses, both the number and the composition of repeated sequence elements varied remarkedly.

[Effect of steam application based on microbiological and parasitologic test procedures]. / Untersuchungen zum Effekt der Dampfeinwirkung anhand mikrobiologischer und parasitologischer Prüfverfahren.

In the present study steam application was investigated with regard to microbicidal and parasiticidal effects. The cleaning apparatus used (Uninova Company) works at a boiler pressure of about 5 bar and consequently with a temperature up to 155 degrees C inside the boiler. Whereas the ambient atmosphere working temperature of steam is slightly below 100 degrees C. The tests are based on the DVG guidelines for testing chemical disinfectants (2). Different steaming times and distances were used in germ carrier tests with three different germ carriers (tile, wood, carpet) and three different test germs (Staphyloccocus aureus, Pseudomonas aeruginosa, Candida albicans) in order to determine the optimum conditions for biocidal effects of steam-application. These optimum conditions were additionally tested with two test viruses (ECBO- and Reo-virus) and a parasitological resting form (ascarid worm eggs). Swirling of germs caused by steam turbulence was minimized by covering the steam outlet nozzle with cloth. The experiments showed logarithmical reduction factors of at least 5.0 in the germ count at steaming times of 5 seconds and a steaming distance of 2.5 cm for all three test germs on all three germ carriers (mean of 10 repeated tests). The virological tests showed good disinfection results after a steaming time of only 2 seconds using aseptic gauze as germ carrier and also after 5 seconds using wood as a carrier. Finally in testing vitality of undeveloped Ascarid worm eggs only 2 seconds of steam treatment proved to be sufficient for a 100 percent destruction. According to the present results steam treatment is most likely to become a valuable, ecologically compatible method in controlling hygienic problems, with a potential of partly replacing chemical disinfectants. In particular we see applications in keeping pets and companion animals, provided the above mentioned rules are followed (steaming distance 2.5 cm; steaming time 5 seconds; cloth). In farm animal stables steam disinfection seems harder to achieve because of large, rough surfaces and economical reasons as e.g. expenditure of time and energy.

Genetic and antigenic heterogeneity among feline calicivirus isolates from distinct disease manifestations.

The capsid protein genes of five feline calicivirus (FCV) isolates associated with different disease manifestations were cloned and sequenced. The viruses represented two recent isolates from cats with chronic stomatitis, one recent isolate from a cat with acute stomatitis, one recent isolate each from a cat with acute respiratory symptoms and the classical limping syndrome strain FCV-2280. The amino acid sequences were compared with eight other published sequences and analyzed for their relationships. Phylogenetic analysis of the complete capsid protein sequences or of known antigenic regions of that protein (hypervariable regions A and E) did not group the isolates of different disease manifestations in distinct subclusters. Monoclonal antibodies (MAbs) generated against either a chronic stomatitis isolate or a recent isolate associated with respiratory symptoms were tested against a panel of 11 recent isolates and four "classical' FCV strains, covering all known disease associations. With those MAbs no obvious clustering with respect to disease manifestation could be seen. Four specific sera prepared in rabbits against our prototype isolates also failed to cluster those isolates according to the disease manifestations when examined in neutralization tests. From these antigenic and genetic analyses of the capsid protein the hypothesis of the existence of biotypes of FCV responsible for distinct disease manifestations could not be confirmed.