Feline Coronavirus Serotypes 1 and 2: Seroprevalence and Association with Disease in Switzerland

To determine the prevalence of antibodies to feline coronavirus (FCoV) serotypes 1 and 2 in Switzerland and their association with different disease manifestations, a serological study based on immunofluorescence tests was conducted with Swiss field cats using transmissible gastroenteritis virus (TGEV), FCoV type 1 and FCoV type 2 as antigens. A total of 639 serum samples collected in the context of different studies from naturally infected cats were tested. The current study revealed that, with an apparent prevalence of 83%, FCoV serotype 1 is the most prevalent serotype in Switzerland. FCoV type 1 viruses induced higher antibody titers than FCoV type 2, and were more frequently associated with clinical signs and/or feline infectious peritonitis. The antibody development in seven cats experimentally infected with FCoV type 1 revealed that, with progressing duration of infection, antibodies to FCoV type 1 significantly increased over those to FCoV type 2. There was a significant relationship between antibody titers against TGEV, FCoV 1, and FCoV 2 and TGEV antigen detected the highest proportion of seropositive cats. We conclude that a vaccine against FCoV should be based on FCoV type 1-related antigens and that for serodiagnosis of FCoV infection TGEV should be used to attain the highest diagnostic efficiency. When serology is used in addition to clinical signs, hematology, and clinical chemistry results as an aid to diagnose clinical FIP, TGEV shows a diagnostic efficiency equal to that of a FCoV antigen.     

Seroprevalence of Bartonella henselae infection and correlation with disease status in cats in Switzerland.

The prevalence of infection with Bartonella henselae was investigated in cats from different areas of Switzerland. Serum samples of 728 cats were examined for antibodies to B. henselae by immunofluorescent antibody testing, and the results were analyzed with a view to a possible correlation between a positive titer and signalment, clinical signs, infection with feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), feline coronavirus (FCoV), or feline spumavirus (FeSFV), and the living environments of the cats. The seroprevalence in all cats was 8.3%. No significantly different prevalence was found in sick versus healthy cats (9.2 versus 7.2%); however, in sick cats seropositive for B. henselae, there was an increased frequency of stomatitis and a variety of diseases of the kidneys and the urinary tract. There was an increased prevalence of B. henselae in cats positive for FCoV (P = 0.0185) or FeSFV (P = 0.0235) and no statistically significant increased prevalence in cats infected with FeLV or FIV. There was no correlation between a positive titer and sex or breed. The same prevalence of B. henselae antibodies was found in cats with and without access to the outdoors and in cats from single- and multicat households. The seroprevalence was increased in cats living south of the Alps (12.1%); however, this difference was not significant (P = 0.0616).     

Production and Characterisation of Murine Monoclonal Antibodies to Feline Erythrocyte A and B Antigens

Anti-A antiserum from blood type B cats, the current reagent used to detect blood type A cats, is expensive, labour intensive to produce, and can vary in sensitivity between preparations. In contrast, monoclonal antibodies are produced easily in large quantities and pure form. We produced six IgM class murine monoclonal antibodies, four specific for feline blood type A and two that detect feline blood type B, by injection of mice with liposomes incorporating type A or B erythrocyte membrane antigens. Specificities of each monoclonal antibody were characterised by high performance thin layer chromatography of feline erythrocyte membrane glycolipids and by immunoblotting of feline erythrocyte membrane proteins separated by SDS-PAGE. The anti-A monoclonal antibodies specifically detected feline blood type A by direct agglutination of blood-typed samples from many cats. Each anti-A monoclonal antibody agglutinated some, but not all, feline blood type AB samples. Two anti-A monoclonal antibodies appeared identical and recognised [NeuGc]₂G_D3, the major glycolipid antigen of type A blood. The other two also appeared identical to each other and recognised a slower migrating glycolipid band, which may be [NeuGc]G_T3. The two anti-B monoclonal antibodies detected feline blood type B by direct agglutination and both recognised [NeuAc]₂G_D3, the major glycolipid antigen of type B blood. None of the monoclonal antibodies recognised erythrocyte membrane glycoproteins specific for either feline type A or type B blood. The ability of the anti-A monoclonal antibodies produced in this study to specifically detect feline blood type A makes them useful replacements for anti-A antiserum for blood typing of cats. The inability of each anti-A antibody to agglutinate blood from every type AB cat suggests a difference between the A antigen of some type A and some type AB cats.     

Production and Characterisation of Murine Monoclonal Antibodies to Feline Erythrocyte A and B Antigens

Anti-A antiserum from blood type B cats, the current reagent used to detect blood type A cats, is expensive, labour intensive to produce, and can vary in sensitivity between preparations. In contrast, monoclonal antibodies are produced easily in large quantities and pure form. We produced six IgM class murine monoclonal antibodies, four specific for feline blood type A and two that detect feline blood type B, by injection of mice with liposomes incorporating type A or B erythrocyte membrane antigens. Specificities of each monoclonal antibody were characterised by high performance thin layer chromatography of feline erythrocyte membrane glycolipids and by immunoblotting of feline erythrocyte membrane proteins separated by SDS-PAGE. The anti-A monoclonal antibodies specifically detected feline blood type A by direct agglutination of blood-typed samples from many cats. Each anti-A monoclonal antibody agglutinated some, but not all, feline blood type AB samples. Two anti-A monoclonal antibodies appeared identical and recognised [NeuGc]₂G_D3, the major glycolipid antigen of type A blood. The other two also appeared identical to each other and recognised a slower migrating glycolipid band, which may be [NeuGc]G_T3. The two anti-B monoclonal antibodies detected feline blood type B by direct agglutination and both recognised [NeuAc]₂G_D3, the major glycolipid antigen of type B blood. None of the monoclonal antibodies recognised erythrocyte membrane glycoproteins specific for either feline type A or type B blood. The ability of the anti-A monoclonal antibodies produced in this study to specifically detect feline blood type A makes them useful replacements for anti-A antiserum for blood typing of cats. The inability of each anti-A antibody to agglutinate blood from every type AB cat suggests a difference between the A antigen of some type A and some type AB cats.     

Prevalence of antibodies to feline parvovirus, calicivirus, herpesvirus, coronavirus, and immunodeficiency virus and of feline leukemia virus antigen and the interrelationship of these viral infections in free-ranging lions in east Africa.

While viral infections and their impact are well studied in domestic cats, only limited information is available on their occurrence in free-ranging lions. The goals of the present study were (i) to investigate the prevalence of antibodies to feline calicivirus (FCV), herpesvirus (FHV), coronavirus (FCoV), parvovirus (FPV), and immunodeficiency virus (FIV) and of feline leukemia virus (FeLV) antigen in 311 serum samples collected between 1984 and 1991 from lions inhabiting Tanzania's national parks and (ii) to evaluate the possible biological importance and the interrelationship of these viral infections. Antibodies to FCV, never reported previously in free-ranging lions, were detected in 70% of the sera. In addition, a much higher prevalence of antibodies to FCoV (57%) was found than was previously reported in Etosha National Park and Kruger National Park. Titers ranged from 25 to 400. FeLV antigen was not detectable in any of the serum samples. FCoV, FCV, FHV, and FIV were endemic in the Serengeti, while a transient elevation of FPV titers pointed to an outbreak of FPV infection between 1985 and 1987. Antibody titers to FPV and FCV were highly prevalent in the Serengeti (FPV, 75%; FCV, 67%) but not in Ngorongoro Crater (FPV, 27%; FCV, 2%). These differences could be explained by the different habitats and biological histories of the two populations and by the well-documented absence of immigration of lions from the Serengeti plains into Ngorongoro Crater after 1965. These observations indicate that, although the pathological potential of these viral infections seemed not to be very high in free-ranging lions, relocation of seropositive animals by humans to seronegative lion populations must be considered very carefully.     

Full genome analysis of a novel type II feline coronavirus NTU156

Infections by type II feline coronaviruses (FCoVs) have been shown to be significantly correlated with fatal feline infectious peritonitis (FIP). Despite nearly six decades having passed since its first emergence, different studies have shown that type II FCoV represents only a small portion of the total FCoV seropositivity in cats; hence, there is very limited knowledge of the evolution of type II FCoV. To elucidate the correlation between viral emergence and FIP, a local isolate (NTU156) that was derived from a FIP cat was analyzed along with other worldwide strains. Containing an in-frame deletion of 442 nucleotides in open reading frame 3c, the complete genome size of NTU156 (28,897 nucleotides) appears to be the smallest among the known type II feline coronaviruses. Bootscan analysis revealed that NTU156 evolved from two crossover events between type I FCoV and canine coronavirus, with recombination sites located in the RNA-dependent RNA polymerase and M genes. With an exchange of nearly one-third of the genome with other members of alphacoronaviruses, the new emerging virus could gain new antigenicity, posing a threat to cats that either have been infected with a type I virus before or never have been infected with FCoV.     

Bartonella henselae prevalence in domestic cats in California: risk factors and association between bacteremia and antibody titers.

The isolation of Bartonella henselae, the agent of cat scratch disease, from the blood of naturally infected domestic cats and the demonstration that cats remain bacteremic for several months suggest that cats play a major role as a reservoir for this bacterium. A convenience sample of 205 cats from northern California was selected between 1992 and 1994 to evaluate the B. henselae antibody and bacteremia prevalences and to determine the risk factors and associations between bacteremia and antibody titers. B. henselae was isolated from the blood of 81 cats (39.5%). Forty-two (52%) of these bacteremic cats were found to be infected with > or = 1,000 CFU/ml of blood. Impounded or former stray cats were 2.86 (95% confidence interval [CI] = 1.94, 4.22) times more likely to be bacteremic than the pet cats. Young cats ( < 1 year old) were more likely than adult cats to be bacteremic (relative risk = 1.64; (95% CI = 1.19, 2.28). Bacteremic cats were more likely than nonbacteremic cats to be infested with fleas (relative risk = 1.64; 95% CI = 1.38, 1.96). No association between B. henselae infection and feline immunodeficiency virus antibody prevalence was observed. Eighty-one percent of the cats (166 of 205) tested positive for B. henselae antibodies, and titers were higher in bacteremic than in nonbacteremic cats. Multiple logistic regression analysis indicated that younger age and seropositivity for B. henselae antibodies were associated with bacteremia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sarcoma virus-induced transformation specific antigen: presence of antibodies in cats that were naturally exposed to leukemia virus.

Healthy cats that were naturally exposed to feline leukemia virus (FeLV) were examined for the presence of antibodies to FOCMA-S, a transformation-specific antigen believed to be encoded by the feline sarcoma virus (FeSV). AntiFOCMA sera obtained from cats which were viremic with FeLV had analogous endpoint titers whether tested on FeLV-transformed nonproducer mink fibroblasts or cat lymphoma cell lines productively infected with the FeLV. These results suggest that FOCMA-S shares at least one major antigenic determinant with FOCMA-L, which is expressed on lymphoma cells.     

Epitope-specific antibody responses to virulent and avirulent feline infectious peritonitis virus isolates.

Feline infectious peritonitis virus (FIPV) has been isolated several times from infected cats. Some of these isolates vary markedly in their ability to cause disease. Specific-pathogen-free cats were inoculated with the avirulent FIPV-UCD-2 isolate or the extremely virulent FIPV-79-1146 isolate or both. After 1 month, cats which had received FIPV-79-1146 were either dead or showed clinical signs of FIP. All cats which received only FIPV-UCD-2 remained healthy up to 6 months after inoculation. Antibody-mediated immune enhancement of disease was not observed in cats which received FIPV-UCD-2 before inoculation with FIPV-79-1146. Monoclonal antibodies which recognized type-specific epitopes on each of the structural polypeptides of these two viruses were used in competitive-inhibition enzyme-linked immunosorbent assays to analyze the humoral immune responses of the cats. All cats produced antibodies to epitopes found on the homologous virus. In addition, cats inoculated with FIPV-79-1146 also produced antibodies which inhibited the binding of the anti-FIPV-UCD-2 E1 monoclonal antibody. One cat inoculated twice with FIPV-UCD-2 produced antibodies which inhibited the binding of the anti-FIPV-79-1146 N- and E1-specific monoclonal antibodies. Competitive enzyme-linked immunosorbent assays may prove useful in distinguishing cats which are infected with virulent FIPV isolates from cats infected with avirulent feline coronaviruses.     

Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR)

Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale.     

Enzyme-linked immunosorbent assay for immunoglobulin G antibody to Pasteurella multocida in rabbits.

Three antigen preparations of Pasteurella multocida, lipopolysaccharide antigen, boiled-cell extract antigen, and boiled whole-bacterium antigen, were used in an enzyme-linked immunosorbent assay (ELISA) to detect rabbit immunoglobulin G antibody to P. multocida. The sensitivity of each antigen preparation was compared by using sera from P. multocida-infected and uninfected rabbits and sera from two rabbits immunized with different serotypes of P. multocida. In the ELISA, all three antigen preparations detected high titers of antibodies in infected rabbits and markedly lower levels in uninfected rabbits. When whole-bacterium or boiled-cell extract antigens were used, the ELISA detected antibodies in sera from both immunized rabbits, but with lipopolysaccharide antigen, only antibody to the homologous serotype was detected. Sera absorbed with P. multocida and Bordetella bronchiseptica, another respiratory pathogen of rabbits, revealed that antibodies detected in the ELISA did not cross-react. Since the lipopolysaccharide antigen was more difficult to prepare and may be type specific, and since the whole-bacterium antigen was the least sensitive, the boiled-cell extract was chosen as the best antigen preparation to use in the ELISA.     

Monoclonal antibodies to a virulent strain of transmissible gastroenteritis virus: comparison of reactivity with virulent and attenuated virus

Summary Twelve hybridomas secreting monoclonal antibodies (MAbs) against Miller virulent strain of transmissible gastroenteritis virus (TGEV) were generated and characterized. In a cell culture immunofluorescence (CCIF) assay, three MAbs directed against peplomer protein (E 2) had perinuclear fluorescence and four unclassified MAbs showed cell membrane fluorescence. Six of these seven MAbs neutralized both attenuated and virulent TGEV, and the seventh (an unclassified MAb) neutralized only the latter virus. Two MAbs able to bind the cell membrane of infected cells had low neutralizing antibody titers (8 to 72) but were able to distinguish between virulent and attenuated TGEV (9- to 72-fold differences in neutralizing titers). Two E 2-specific MAbs had higher neutralizing antibody titers (782 to 34,117) and showed 4- to 13-fold differences in titers against the attenuated and virulent TGEV strains. Five MAbs which were specific for nucleocapsid (N) protein had cytoplasmic, particulate fluorescence in CCIF, and did not neutralize TGEV. Comparison of CCIF antibody titers of MAbs to the virulent and attenuated strains of TGEV indicated that differences existed in titers of most E 2 and all N-specific MAbs, with titers consistently higher against virulent TGEV (homologous strain).Hyperimmune antisera prepared in gnotobiotic pigs against the attenuated, virulent and a recent isolate of TGEV immunoprecipitated the 3 major structural proteins of both the attenuated and virulent TGEV strains. Relative mol. wt. differences in the E 1 and E 2 proteins between the two virus strains were revealed using either the hyperimmune pig sera or MAbs. In addition to the 48 K N protein, a 44 K protein was coimmunoprecipitated by the hyperimmune sera and MAbs, but mainly from lysates of attenuated TGEV.     

Detection and prevalence of serotypes of feline syncytial spumaviruses

Summary Three serotypes of feline syncytial virus (FSV) were detected by neutralisation tests: 906, a serotype of low prevalence and 702 and 951 which were serotypes of higher prevalence, between which a minor one-way antigenic difference was detected. Serum antibody in naturally-infected cats in some cases neutralised 951 but not 702 or 906 which suggested that 951 could be considered as a major distinct serotype. An increase in prevalence of antibody to FSV in cats over a 5 year period from 1977–1981 was detected by neutralisation, agar gel immunodiffusion, and fluorescent antibody techniques. Over the 5 year period the prevalence of antibody to the 951 serotype increased and the overall increase in prevalence of antibody to FSV during this period appeared to relate to dissemination of the 951 serotype.     

Feline immunodeficiency virus: quantification in peripheral blood mononuclear cells and isolation from plasma of infected cats

Summary The titer of feline immunodeficiency virus in peripheral blood mononuclear cells (PBMC) and the presence of infectious virus in plasma was investigated over 20 week period in 8 experimentally infected cats, 3 uninfected cats and 2 naturally infected cats by end point dilution cultures using a feline T-lymphoblastoid cell line (MYA-1). FIV was isolated from PBMC of all infected cats, but not from the uninfected cats. FIV was also isolated consistently from 100 µl plasma from most of the experimentally infected cats, but not from the 2 naturally infected cats. The virus titer in PBMCs in both experimentally and naturally infected cats was comparatively high, ranging from 10 TCID/10⁶ PBMC to 14,286 TCID/10⁶ PBMC. The titers in PBMC of individual cats remained unchanged or varied only slightly over the 20 week period. In contrast, the titers varied substantially between cats, with significantly higher titers in the youngest litter (4 cats) than in the oldest litter (3 cats). This suggests that there is an age-related factor influencing the level of PBMC virus titers in experimental infection with FIV. A similar age-related susceptibility has been shown with feline leukemia virus. More importantly, the sustained titers in the experimentally infected cats bear close resemblance to infection of children with human immunodeficiency virus. These data reinforce suggestions that age and immune maturity have a fundamental influence on PBMC and plasma titers in lentivirus infections.     

Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type IV, V, and VI antigens, group 3,4 antigen, and an epitope common to all Shigella flexneri and Shigella dysenteriae type 1 stains.

Monoclonal antibodies reactive with Shigella flexneri O antigens were generated in both mouse and rat systems. Antibody-producing hybridomas were screened in an enzyme-linked immunosorbent assay using chemically defined lipopolysaccharides as antigens, and the epitope specificities were determined with a panel of lipopolysaccharides and synthetic O-antigen-specific glycoconjugates as antigens. To verify the specificity seen in the enzyme-linked immunosorbent assay, the antibodies were used in agglutination against a large number of S. flexneri strains. Monoclonal antibodies with the following specificities were identified: type, antigen IV (reactive with serotype 4a and 4b bacteria); type antigen V (reactive with serotype 5a and 5b bacteria); type antigen VI (reactive with serotype 6 bacteria); group antigen 3,4(reactive with serotype 1a, 2a, 3b, 4a, 5a, and Y bacteria); and group antigen 1 (reactive with an epitope present on all S. flexneri and Shigella dysenteriae type 1 bacteria). Furthermore, a monoclonal antibody defining a new O-antigenic epitope present on some S. flexneri strains of serotypes 4a, X, and Y was characterized (4X). The monoclonal antibodies analyzed in this study define epitopes described by polyclonal antisera (type antigens IV, V, and VI), define a hitherto uncharacterized epitope (group antigen 1), and finally identify new epitopes in what has previously been considered as one epitope (group antigen 3,4 and type antigen IV). These immunochemically characterized monoclonal antibodies may have a powerful potential in studies of the importance of humoral immunity in shigellosis.     

Antibodies to Surface Antigens of Herpesvirus Type 1- and Type 2-Infected Cells Among Women with Cervical Cancer and Control Women

Cells infected with herpesvirus type 1 or type 2 develop surface antigens which can be detected by immunofluorescence. Using the indirect immunofluorescence technique, we determined antibody titers to the surface antigens in 20 sera from persons with known type 1 virus infections, 20 sera from persons with known type 2 virus infections, 30 sera from women with cervical cancer, and 30 sera from matched control women. There was a good correlation between antibody activity to the surface antigens and neutralizing antibody activity for both herpesvirus type 1 and herpesvirus type 2 in all groups of sera examined. Women with cervical cancer did not have unusually high or low titers of antibody activity to the surface antigens. In the selected sera examined, analysis of antibodies to the surface antigens was not superior to the microneutralization test in distinguishing women with cervical cancer from control women.     

Detection of salivary antibodies in cats infected with feline immunodeficiency virus.

The saliva of cats infected with feline immunodeficiency virus was examined for total immunoglobulin content and antiviral antibodies. Seropositive cats showed an increase in salivary immunoglobulin G levels, which was only partly attributable to the enhanced prevalence of oral inflammatory lesions, compared with the levels in seronegative cats. Immunoglobulin G, but not immunoglobulin M, levels in serum were also increased. Salivary antibodies were determined by indirect immunofluorescence and Western blot (immunoblot) analysis. All but 1 of the 16 seropositive cats examined were positive, while all 16 control cats were negative. The presence of oral lesions was not a prerequisite for antibody detection in saliva. It was concluded that salivary antibody might be usefully exploited for diagnostic and epidemiologic purposes.     

Immune responses to the R4 protein antigen of group B streptococci and its relationship to other streptococcal R4 proteins.

The R antigen, a trypsin-resistant protein observed in group A, C, F, G, and L streptococci, has also been found in group B streptococci (GBS). Although four species of the R antigen have been described for GBS, the R4 protein is the most prevalent in GBS isolates recovered from humans. This study examined the prevalence of antibodies against the R4 antigen by Western blot (immunoblot) (WB) in sera from 40 mothers colonized with GBS serotype II and III and from 26 noncolonized mothers; 92.5% of the colonized mothers had anti-R4 antibodies, compared with 54% of the noncolonized mothers (P < 0.001). Findings of antibodies in neonatal cord sera (n = 14) were concordant with maternal results by WB analysis for 71% of mother-infant pairs colonized with serotype II and for 57% of pairs colonized with serotype III. Of mothers known to be colonized with type II/R4 or III/R4, 100% (n = 12) had antibody against R4 by WB. This study also evaluated the prevalence of antibody to the GBS R4 antigen in 48 sera from individuals with high and low group A streptococcal anti-DNase B titers. Of those individuals with an anti-DNase B titer of > 640, 64% had a positive WB for anti-R4 antibody, compared with 30% of individuals with low anti-DNase B titers (P < 0.05). The R4 antigen of GBS had immunologic identity to the R4 antigen of group A streptococci. Overall, the findings suggested that antibodies to the streptococcal R4 antigen were commonly present in GBS-colonized mothers and that transplacental passage of these antibodies occurred. The presence of antibody to R4 in non-GBS-colonized individuals may be due to immunologic responses to past exposure to the R antigen present in GBS or other streptococcal groups.     

Serotype specificity and immunogenicity of the capsular polymer of Haemophilus pleuropneumoniae serotype 5.

Serotyping of Haemophilus pleuropneumoniae and serologic assays for detection of serotype-specific antibody are problematic due to the potential cross-reactivity of the crude antigens used for raising immune serum or for serology. The capsular polymer (CP) of H. pleuropneumoniae serotype 5 was investigated for serotype-specific activity with antiserum to whole cells or with antiserum made monospecific to CP by adsorption with a capsule-deficient mutant. When antiserum to whole cells or monospecific antiserum to CP was tested against purified CP from serotypes 1 to 7 by immunodiffusion or enzyme-linked immunosorbent assay, only capsules of serotype 5 were reactive. In addition, only encapsulated serotype 5 cells reacted with serum monospecific to CP in an indirect immunofluorescent-antibody assay. Serotype-specific antibody was completely inhibited in each assay by preincubation of purified CP with the serum. Antiserum to whole cells of H. pleuropneumoniae serotype 5 contained antibodies to proteins and lipopolysaccharide; these antibodies cross-reacted with antigens of heterologous serotypes by dot-blot enzyme-linked immunosorbent assay and immunoblotting. The antigenic activity of CP was stable after heating for at least 30 min at 100 degrees C. High titers of antibody to CP were present in the sera of rabbits immunized intravenously with whole log-phase cells or in the convalescent sera of pigs experimentally infected with H. pleuropneumoniae serotype 5. However, the purified CP was poorly immunogenic in rabbits and swine. Our results indicate that the capsule is the serotype-specific antigen of H. pleuropneumoniae and that a monospecific antiserum to capsule or purified capsule should be used for serotyping or serologic assays, respectively.     

Experimental infection of domestic cats with Bartonella koehlerae and comparison of protein and DNA profiles with those of other Bartonella species infecting felines

Bartonella koehlerae, a recently described feline Bartonella species, was isolated from two naturally infected cats in northern California. We experimentally infected domestic cats with B. koehlerae to establish the microbiological and immunological characteristics of this infection in cats and to compare it to infections with those caused by B. henselae and B. clarridgeiae. Four cats were inoculated intradermally with B. koehlerae (8.6 x 10(7) to 3.84 x 10(8) CFU/ml). None of the cats presented any obvious clinical signs, but all cats developed bacteremia, which peaked at 3.36 x 10(4) to 1.44 x 10(6) CFU/ml of blood between day 14 and day 36 postinoculation. B. koehlerae-inoculated cats had a bacteremia duration (mean, 74 days) shorter than did cats inoculated with B. clarridgeiae (mean, 324 days) (P = 0.03). None of the four cats inoculated with B. koehlerae had bacteremia relapse. As shown by enzyme-linked immunosorbent assay (ELISA) using B. koehlerae outer membrane protein (OMP) antigens, the four cats developed a species-specific antibody response, and ELISA testing using other feline Bartonella OMP antigens showed statistically lower optical density values. All four cats developed similar antibody reactivity patterns to B. koehlerae OMP antigens as seen by Western blotting, each with at least 20 seroreactive protein bands. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein profile differences were observed for both whole-cell lysate and OMPs from B. koehlerae, compared with B. henselae and B. clarridgeiae. B. koehlerae was more closely related to B. henselae than to B. clarridgeiae by protein profile, and this relatedness was also confirmed by analysis of the genomic DNA profiles by pulsed-field gel electrophoresis.