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Chick embryo fibroblasts (CEFs) infected with three strains of Marek's disease virus serotype 1 (MDV1), GA, Md5 and JM, were subjected to indirect immunofluorescence assay with monoclonal antibodies (MAbs) against MDV1 homolog of glycoprotein D (MDV1 gD) of herpes simplex virus. By the MAbs, a number of MDV1 gD-positive cells were detected in CEFs infected with GA, whereas only a few and no positive cells were detected in CEFs infected with Md5 and JM, respectively. The MDV1 gD in GA-infected CEFs was recognized as the band of 64 kDa in immunoblot analysis using one of the MAbs. This is the first report that the MDV1 gD was detected in MDV1-infected cell cultures.

Unique transcriptional transactivation by the human immunodeficiency virus type 1 (HIV-1) Tat protein of long terminal repeat (LTR)-driven RNA expression, in the absence of the transactivator responsive element (TAR), was previously demonstrated in central nervous system (CNS)-derived astrocytic cell-lines, including U87MG. In the present study, RNase protection assays were utilized to reveal the molecular mechanism(s) underlying transactivation of the HIV-1-LTR in these cells. Short transcripts, which represent abortive HIV-1 transcription, could not be detected either in the absence or presence of Tat, and no differences in transcript levels were detected using 5' probes, as compared to 3' probes, in the experiments. Thus, the transactivational effects of Tat, in U87MG cells, were potentially based on the increase of transcriptional initiation, both in TAR-dependent and -independent states. Further, by using newly established stable cellular transformant, containing HIV-1-LTR-reporter gene constructs, TAR-independent transactivation was demonstrated to efficiently function primarily in transiently-transfected U87MG cells. U87MG cells, stably-transfected with the intact HIV-1 proviral genome, produced very low levels of virus after long-term culture, as previously reported in other astrocytic cells. These cells demonstrated profoundly restricted transcription of the HIV-1 genome, with no detectable levels of HIV-1-specific RNA by Northern blotting, indicating that the restriction of viral production in these cells is principally due to the low level of overall transcription from the 5' HIV-1-LTR. Transcription of HIV-1 RNA in this cell could not be significantly up-regulated by various stimulators, such as phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-alpha (TNF-alpha) and sodium butyrate. These data suggest that the restriction of HIV-1 transcription in these cells may be controlled by different mechanism(s) from those in lymphocytic or monocytic cells.

Glycoproteins B (MDV1-gB) and C (MDV1-gC) of Marek's disease virus type 1 (MDV 1) expressed by baculovirus recombinants [rAcNPV(s)] were investigated for their ability to confer protective immunity in chickens against virulent MDV1 challenge. Immunization of chickens with rAcNPV-expressed MDV1-gB induced antibody against MDV1 and resulted in strong protection against the lethal challenge with MDV1. In contrast, immunization of chickens with rAcNPV-expressed MDV1-gC induced antibody against MDV1, but failed to protect against challenge with the virulent MDV1. Co-immunization with both rAcNPV-expressed MDV1-gB and MDV1-gC seemed to show no synergistic effect.

The gene of Marek's disease virus (MDV) serotype 2 (MDV2) homologous to glycoprotein D (gD) of herpes simplex virus (HSV) was identified and characterized by its nucleotide and predicted amino acid sequences. The MDV2 gD homologous gene contains an open reading frame capable of specifying a polypeptide of 385 amino acids, which include N- and C-terminal hydrophobic domains consistent with signal and anchor regions, respectively, and two potential N-linked glycosylation sites, one of which was located in a highly conserved region when compared to MDV serotype 1 (MDV1) and herpesvirus of turkeys (HVT). By northern blot analysis using a MDV2 gD-specific DNA probe, two highly abundant polycistronic 6.0 and 4.2 kb transcripts were detected in MDV2-infected cells. The genes encoding MDV2 protein kinase (PK), gD, and glycoprotein I (gI) homologues are transcribed to form 3' coterminal mRNAs of 6.0 kb (encoding PK, gD and gI) and 4.2 kb (encoding gD and gI), respectively. By using rapid amplification cDNA end (RACE) method, several RNA start sites, to be thought those of the 4.2 kb mRNA, were detected in the upstream of MDV2 gD homologue.

A recombinant baculovirus, the genome of which contains DNA encoding Marek's disease virus serotype 1 (MDV1) homolog of glycoprotein D (gD) of herpes simplex virus under the polyhedrin promoter was constructed and designated rAcMDV1gD. Five monoclonal antibodies (MAbs) which recognize the MDV1 homolog of gD (MDV1 gD) in Spodoptera frugiperda cells infected with rAcMDV1gD were prepared. The MAbs reacted with proteins ranging from 52 to 49 kDa in rAcMDV1gD-infected cell lysates by immunoblot analysis. These molecular weights were coincident with molecular weights predicted from the open reading frame of MDV1 gD. By ELISA additivity test, the 5 MAbs were divided into 3 groups which seemed to recognize 3 different epitopes. In addition, all of the 5 MAbs were reactive with chick embryo fibroblasts (CEFs) expressing MDV1 gD. The MAbs are considered to be useful to study the role of MDV1 gD in MDV1 infection.

We constructed the recombinant baculovirus expressing the gene of non-pathogenic Marek's disease virus (MDV) serotype 2 (MDV2) which encodes a polypeptide with partial homology to MDV serotype 1 (MDV1) pp38, an antigen associated with transformed cells. The recombinant MDV2 protein was detected as a band of 32 kDa in immunoblot analysis with MDV2-infected chicken serum. Mouse serum against insect Spodoptera frugiperda cells infected with the recombinant baculovirus immunoprecipitated a 38 kDa molecule from the lysate of MDV2-infected chicken embryo fibroblasts (CEFs) but did not immunoprecipitate the MDV1 pp38 from the lysate of MDV1-infected CEFs. This result indicates that the recombinant MDV2 protein has no epitopes shared with the MDV1 pp38.

Variable levels of viral DNA have been demonstrated within human immunodeficiency virus type 1 (HIV-1) virions purified from cell cultures. In the present studies, it is demonstrated that DNase-resistant viral DNA is associated with HIV-1 virions purified from the peripheral blood plasma of both symptomatic and asymptomatic HIV-1-infected individuals. The differences in viral DNA copy numbers, detected by quantitative PCR in various regions of the HIV-1 genome, indicated that the intravirion HIV-1 DNA is frequently, but perhaps not totally, the result of partial reverse transcription. These in vivo data suggest that it may be valuable to further investigate the impact of virion-associated viral DNA upon the efficiency of intra- and interhost HIV-1 transmission modes.

Phosphorylated protein pp38 is the only protein that is detected in the Marek's disease (MD) lymphoma caused by MD virus serotype 1 (MDV-1) and lymphoblastoid cell lines. In this study, a recombinant protein coded for by the almost entire open reading frame of the MDV-1 pp38 cDNA was produced in E. coli and purified by affinity chromatography. Prior to the expression and purification of the protein, cDNA containing the entire coding region for pp38 was cloned and its nucleotide sequence was determined. Immunoblot analysis indicated that the expressed recombinant protein electrophoresed close to that of the pp38 in infected cells. The difference in mobility of the purified recombinant and the pp38 in infected cells corresponded to a fusion peptide. The recombinant pp38 may be of interest for function analyses and the diagnotic use of pp38.

Recombinant baculoviruses carrying cDNAs of hemagglutinin-neuraminidase (HN) and fusion (F) glycoprotein genes of virulent and avirulent strains of Newcastle disease virus (NDV) were constructed to examine the contribution of the individual proteins in cell fusion. F proteins of both virulent and avirulent strains expressed by the recombinant viruses were glycosylated and translocated onto the cell surfaces and only the F protein of the virulent origin was proteolytically cleaved into F1 and F2 subunits and bound intermolecularly by disulfide bonds. HN proteins of virulent and avirulent strains expressed by the recombinant viruses showed both hemadsorption and neuraminidase activities. Single infection of the recombinant baculoviruses could not induce cell fusion; however, co-infection with the recombinant viruses of the F protein of virulent strain and HN protein of both strains gave clear syncytia in insect cells. The syncytium formation was much clearer in the cells co-infected with the recombinants of F protein of virulent strain and the HN protein of avirulent strain in comparison with those co-infected with F and HN recombinant viruses of virulent origin. The cell fusion was completely blocked by monoclonal antibodies against the F protein but not by those to the HN protein.

Recombinant hemagglutinin-neuraminidase (rHN) and fusion (rF) glycoproteins of virulent and avirulent strains of Newcastle disease virus (NDV) expressed by using baculovirus expression system were used to investigate their protective immunization effects in chickens. The efficacy of immunization with these recombinant proteins was evaluated by challenge infection. The chickens immunized with either rHN or rF protein of a virulent strain or rHN protein of an avirulent strain were completely protected from the lethal infection of virulent NDV. On the other hand, the rF protein of an avirulent strain, in which precursor F protein was not cleaved, showed lower protective effects. Significant levels of specific antibodies against respective proteins were detected in sera from survivors, whereas relatively lower levels of antibodies were found in chickens which were killed by challenge infection. These data indicate that either HN or F protein alone could induce protective immune responses and the cleavage of F protein might be important for its immunological potential.

Marek's disease (MD) virus (MDV) serotype 1 (MDV1) pp38, an antigen associated with MD-transformed cells, has been identified as an MDV1 serotype-specific antigen by analyses using specific monoclonal antibodies (MAbs). In the present study, we determined the region in the MDV serotype 2 (MDV2) genome that has homology with the MDV1 pp38 gene. As a result of sequence analysis, it was determined that an open reading frame (ORF) existed in this region. Furthermore, Northern blot analysis and reverse transcriptase polymerase chain reaction confirmed the existence of an RNA transcript related to the ORF in MDV2-infected cells. Computer analyses revealed that MDV1 pp38 could be divided into two parts; one part was highly homologous and the other part was not homologous with the peptides deduced from the ORF of MDV2, indicating that the epitope recognized by a MDV1 pp38-specific MAb is located in the latter part.

The sequence analysis of the genomic region of Marek's disease virus (MDV) serotype 2 (MDV2) which had homology with the gene encoding MDV serotype 1 (MDV1) homologue of glycoprotein C (gC) of herpes simplex virus revealed that there was a single open reading frame (ORF) capable of encoding 479 amino acids. The predicted protein from this ORF had strong hydrophobic regions in both the amino and carboxyl termini and several potential N-linked glycosylation sites, as seen in those from the ORFs of MDV1 and MDV serotype 3 (MDV3 = herpesvirus of turkeys (HVT)) gC homologues. By northern-blot analysis using a DNA fragment located in the MDV2 gC homologue ORF as a probe, 3.6 and 1.9 kb RNA transcripts were identified in MDV2-infected cells. When the alignment of amino acid sequences of the gC homologous genes of three MDV serotypes were compared, the homology between MDV1 and MDV2 and that between MDV1 and HVT showed about 76% identity, whereas that between MDV2 and HVT was about 70% identity. These results suggested a possibility that the evolutionary distance between MDV1 and MDV2 and between MDV1 and HVT is shorter than that between MDV2 and HVT.

The Swarm rat chondrosarcoma is a well established model system for the biochemical studies of cartilage proteoglycans. In this study, we observed the ultrastructure of mitotic chondrocytes in this tumor to confirm whether it is also useful as a morphological model system to examine the mitosis of chondrocytes. As a result, the mitotic chondrocytes in this tumor contained fairly prominent cell organelles, and the endoplasmic reticulum was mainly vacuolized and the Golgi stacks were replaced by the cluster of small vesicles during the later stage of the mitotic cycle. Although the cytoplasmic division began at the telophase, the cytoplasmic cleavage furrow was so narrow that dividing chondrocytes looked as if they were partitioned by a slit. These findings are almost consistent with the changes that occurred in the mitotic chondrocytes of the tibial growth plate, although the degree of disorganization in the Golgi apparatus is slightly different. Therefore, this tumor is useful as a convenient morphological model system to examine the mitosis of chondrocytes because of the facility for taking specimens.

Mitotic chondrocytes containing fairly prominent cell organelles were observed in the proliferative zone of the rat tibial growth plate by electron microscopy. During the later stage of mitosis, the endoplasmic reticulum was mostly vacuolated, and the Golgi stacks were replaced by spherical and cylindrical vacuoles and small vesicles. The cytoplasmic division began at early telophase. However, the cytoplasmic cleavage furrow was so narrow that dividing chondrocytes looked as if they were partitioned by a slit. This is probably one reason for the appearance of the binuclear cells that are occasionally observed in the tibial growth plate.

To investigate whether the feline CD 4 (fCD4) molecules are involved in infections of highly lymphotropic feline immunodeficiency virus (FIV) isolates, we expressed fCD4 stably on Crandell feline kidney cells and Felis catus whole foetus 4 cells by transfection of a cDNA encoding the fCD4 glycoprotein, and then infected them with TM 1 and TM 2 strains of FIV, which are unable to infect these cells productively. In spite of fCD 4 being expressed on these cells, no virus production was observed. This result indicates that fCD 4 expression alone cannot induce a productive infection of the FIV TM 1 and TM 2 strains.

A BamHI, EcoRI, and XhoI restriction endonuclease map of Marek's disease virus (MDV) serotype 2 (MDV2) DNA was constructed by double-digest analyses of 28 cloned BamHI and 11 cloned EcoRI fragments of MDV2 DNA, followed by hybridization tests of these cloned BamHI DNA fragments with electrophoretically separated digests of MDV2-infected cell DNA. On this map, MDV2 genome consisted of two segments which have unique regions inserted between two inverted repeat regions as observed in MDV serotype 1 and 3 genomes. Further, the DNA homology among three serotypes of MDV was examined by hybridization under less stringent conditions using cloned BamHI fragments of MDV2 DNA. Most of the MDV2 fragments located within the unique regions hybridized with MDV serotype 1 and 3 DNAs, indicating the presence of the collinear relationship among three serotypes. In addition, MDV2 DNA fragments which hybridized with the DNA fragments encoding MDV1 gp57-65 (or A antigen) or MDV1 gp100, gp60, gp49 (or B antigen) were identified and these fragments of serotypes 1 and 2 found to be collinear.

A gene encoding a homolog of glycoprotein B of herpes simplex virus (gB homolog) has been identified on the Marek's disease virus (MDV) genome (L. J. N. Ross, M. Sanderson, S. D. Scott, M. M. Binns, T. Doel, and B. Milne, J. Gen. Virol. 70:1789-1804, 1989); however, the molecular and immunological characteristics of the gene product(s) are still not clear. In the present study, the gB homolog of MDV was expressed in insect cells by a recombinant baculovirus, and it was characterized to determine its molecular and antigenic properties. The expressed recombinant protein had three molecular sizes (88 to 110, 58, and 49 kDa) and was recognized by antisera from chickens inoculated with each of the three serotypes of MDV. By immunofluorescence analysis, it was shown that the protein was expressed in the cytoplasm and on the surface of the recombinant baculovirus-infected cells. The gB homolog of MDV was processed similarly to pseudorabies virus and varicella-zoster virus with respect to cleavage and the intramolecular disulfide bond between the cleaved products. Interestingly, the expressed protein reacted with monoclonal antibody M51, specific to the B antigen (gp100, gp60, gp49) of MDV, although the locations of the gene encoding the B antigen and of the gene encoding the gB homolog were reported to be different. Moreover, competitive experiments revealed that anti-gB homolog serum and monoclonal antibody M51 recognized the same molecules. From these results, the gB homolog and the B antigen of MDV seem to be the same glycoprotein.

The nucleotide sequence of the glycoprotein B (gB) homologous gene of feline herpesvirus type 1 (FHV-1) was determined. The gene was found to be located within a 9.6 kbp SalI fragment by Southern-blot hybridization with a probe derived from the herpes simplex virus type 1 (HSV-1) gB DNA sequence. Furthermore, the predominant portion of the coding sequences was mapped to a 1.9 kbp Hin cII-EcoRI and its flanking 2.7 kbp Eco RI-Eco RI subfragments in the 9.6 kbp SalI fragment. The entire nucleotide sequence revealed that the FHV-1 gB homologous gene is capable of encoding a polypeptide of 948 amino acids. The predicted precursor polypeptide derived from this open reading frame could have a calculated M(r) of 106 kDa in unglycosylated form and contains ten potential N-linked glycosylation sites and a probable internal proteolytic cleavage site. By Northern-blot analysis using portions of the open reading frame as a probe, 3.9 and 3.3 kb RNA transcripts were identified in FHV-1 infected cells. The alignment of the amino acid sequence of the FHV-1 gB homologue with those of 14 other herpesviruses revealed that 10 cysteine residues were completely conserved. Meanwhile, when evolutionary trees were generated among these herpesvirus gB counterparts, the FHV-1 gB homologous nucleotide sequence seems to be closely related to equine herpesvirus type 4 and its amino acid sequence to pseudorabies virus.

A recombinant baculovirus containing a cDNA which encodes haemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) was constructed. Spodoptera frugiperda cells infected with this recombinant virus produced a large amount of HN glycoprotein similar to the authentic HN in size. The recombinant HN glycoprotein was localized on the surface of the infected cells and conserved its haemadsorption and neuraminidase activities. The antigenic properties of the recombinant HN glycoprotein seemed to be slightly different from the authentic one, as judging by the reactivity with a panel of monoclonal antibodies specific to the antigenic sites responsible for neutralization of viral infectivity. Chickens inoculated with the cells infected with the recombinant virus developed haemagglutination-inhibition and virus neutralization antibodies, and were completely protected from the NDV challenge.

An avian thymidine kinase deficient (TK-) fibroblast cell line (QTTK-) was established from a Japanese quail cell line, QT35, and characterized the biological properties. QTTK- could grow in the presence of 5-bromo-2'-deoxyuridine (BUdR, 100 micrograms/ml) and not in the growth medium with hypoxanthine-aminopterin-thymidine. Compared to QT35 cells, the 3H-thymidine incorporation of the QTTK- cells and the TK activity of the cell extract significantly decreased to 0.3% and 0.5%, respectively. In the thymidylate synthetase activity, the karyotype, and the sensitivities to either fowlpox virus (FPV) or herpesvirus of turkeys (HVT), OTTK- cells were similar to the parental QT35 cells. Since QTTK- cells were permissive to FPV and HVT infections and these viruses could not grow in the presence of 50 to 75 micrograms/ml of BUdR, QTTK- cells may be useful for the construction of recombinant FPV and HVT that have foreign genes within the TK gene of the virus genome.

The role of immune responses to haemagglutinin-neuraminidase (HN) protein in protection against a Newcastle disease virus (NDV) infection was investigated using a recombinant vaccinia virus expressing HN (HN-RVV). Live HN-RVV replicated in chickens and completely protected them from lethal infection with virulent NDV. Inactivated HN-RVV also protected chickens when administered with adjuvant but not when administered without adjuvant. However, large amounts of the inactivated HN-RVV (100-fold excess) without adjuvant provided protection. Specific antibodies against the HN protein of NDV were detected in sera from survivors but not from dying birds. However, the kinetics of antibody responses in chickens inoculated with live HN-RVV and inactivated HN-RVV were considerably different. These results clearly confirm that immune response(s) solely to the HN protein of NDV can provide chickens with protection against NDV challenge, and show that the presence of antibodies to the HN protein correlates significantly with the protection from NDV infection at least in HN-immunized chickens.

A recombinant baculovirus expressing the A antigen (A Ag) of Marek's disease virus (MDV) was constructed. In Spodoptera fruiperda (Sf) cells infected with the recombinant virus, A Ag expression was localized to the cell surface. Only a small amount of recombinant A Ag was detected in the culture supernatant of infected Sf cells, but authentic A Ag is mainly secreted into the culture supernatant of MDV-infected chicken embryo fibroblasts. Cell surface-associated recombinant A Ag seemed to be slightly larger than authentic A Ag, whereas the secreted recombinant A Ag seemed to be smaller. The recombinant A Ag was shown to be reactive with the sera of MDV-infected chickens by immunodiffusion studies and ELISA. Sera of chickens immunized with recombinant A Ag formed a precipitin line with the culture supernatant of MDV-infected chicken embryo fibroblasts in an immunodiffusion test. These results indicate that the recombinant A Ag expressed by the recombinant baculovirus retains the antigenic and immunogenic properties of the authentic A Ag.

Anti-influenza antibody (Ab) titers were measured in order to elucidate whether there are any disturbances in Ab production in chronic renal failure (CRF) patients. A total of 55 CRF patients plus 15 normal individuals were vaccinated with influenza vaccine twice, 4 weeks apart of the 55 CRF patients, 15 were not on dialysis, 10 were undergoing hemodialysis (HD), and 30 were on continuous ambulatory peritoneal dialysis (CAPD). Of the 30 CAPD patients, 14 had peritonitis. Serum Ab titers were measured by complement fixation (CF) and hemagglutination inhibition (HI) tests, and IgG and IgM class specific antibodies by ELISA. All groups responded to immunization, but CAPD patients with peritonitis and CRF patients not yet on dialysis did not show a significant elevation in IgM class Ab titers. The number of CAPD patients with peritonitis who achieved positive titers was significantly lower in HI (p less than 0.01) and IgG class antibodies (p less than 0.05) compared with normal controls. Two patients with frequent peritonitis did not show any response to vaccination. It was concluded that patients with renal dysfunction have some abnormalities in Ab production against influenza vaccine, the effects of which were more pronounced in the CAPD patients with frequent peritonitis.

In situ hybridization was applied to detect fowlpox virus (FPV) DNA in formalin-fixed paraffin-embedded sections of the skin from infected chickens by using a biotinylated probe and a streptavidin-alkalinephosphatase conjugate. The immunohistochemical examination was applied to compare the distribution of the FPV DNA to that of related antigenic protein in serial sections. In the infected epithelial cells, FPV DNA was detected in cytoplasmic inclusion bodies and in the rest of cytoplasm. Likewise, immunohistochemical examination revealed the virus antigen in cytoplasm. Ultrastructurally, virions were observed in the cytoplasmic inclusion bodies, and immature virus particles were in the rest of the cytoplasm. The study proved restricted distribution of FPV DNA in the cytoplasm.

The antigens and the protein profiles of virulent and avirulent clones of Leptospira interrogans serovar copenhageni, strain Shibaura were compared by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and radioimmunoprecipitation. An antigen specific to the virulent clone was detectable only in the virulent clone by Western blotting using anti-virulent clone but not anti-avirulent clone antisera. The molecular weight of the antigen was estimated to be approximately 32000 by SDS-PAGE, and the antigen was found to be thermostable. A 43000 dalton antigenic molecule was found to be specific for the virulent clone, and the 41000 dalton antigenic molecule was found to be specific for the avirulent clone by Western blotting using both anti-virulent and anti-avirulent clones antisera. Therefore, these antigenic molecules seemed to contain at least one antigenic determinant which is common to the virulent and the avirulent clones. No difference between the virulent and avirulent clones was observed in the comparison of protein profiles on SDS-PAGE and surface exposed antigenic protein profiles on radioimmunoprecipitation using radioiodinated leptospiral surface exposed proteins. Therefore, the antigens which were different between the virulent and avirulent clones were suspected not to be protein antigens.

High-performance liquid chromatography using spherical aggregates of strontium-phosphate hydroxyapatite(SrHA) micro-crystals as adsorbent has been developed; preliminary performance tests were carried out by using several types of protein. It can be deduced that, in parallel with the case of usual calcium-phosphate hydroxyapatite(CaHA), with SrHA also, two types of effective surface, vector a (or vector b) and vector c surfaces, appear on the crystal: the same protein molecular generally shows slightly different chromatographic behaviors between the CaHA and the SrHA packed column. Combining the SrHA and the CaHA packed column would lead to an efficient fractionation of a particular molecule from an assembly of molecules with subtle structural differences from one another.

The cytotoxic effects of acyclovir, which is a purine nucleoside analogue and is known as an antibiotic substance, were examined on three lines of rat skin fibroblast FR cells; normal FR cells, FRtk- cells which are deficient in the activity of thymidine kinase (tk) and FRtk-HSVtk+ cells which were prepared by introducing herpes simplex virus' tk gene to FRtk- cells. When FRtk-HSVtk+ cells growing exponentially were incubated in the presence of acyclovir for 4 h, the surviving fractions of the cells decreased in a concentration-dependent manner. Whereas, decrease of the surviving fractions was almost indiscernible in both FR cells and FRtk- cells at the whole ranges of drug-concentrations tested. These results indicate that acyclovir is phosphorylated by the herpes simplex virus' tk and becomes toxic to FRtk-HSVtk+ cells. This also means that FRtk-HSVtk+ cells are useful for the investigation of the biological activity of nucleoside analogues.

Each virulent strain of copenhageni, canicola and pomona of Leptospira interrogans attached effectively to MDCK cells and primary dog kidney cells, while the avirulent or less virulent line of the same strain and avirulent strains belonging to the same serovars and the avirulent reference strains of other serovars did not. Inhibition of the attachment of the virulent copenhageni to MDCK cells was found in the presence of the homologous immunoglobulin G Fab fragment. Strains of L. biflexa attached to the animal cells, but they differed from those of virulent L. interrogans in their capability to attach to glass.