RESUMO
Four dogs were experimentally infected with Anaplasma platys to determine changes in real-time TaqMan PCR detection in blood and tissue, microscopically detectable parasitemia, and platelet concentrations during the first 28 days of infection. Buffy-coat blood cells were PCR positive for A. platys DNA at 4 days after inoculation and remained positive in all dogs until day 14. Marked thrombocytopenia and low parasitemia occurred in dogs during that initial period. During 17 and 28 days post-inoculation, the PCR results on buffy-coat blood cells were intermittently negative in each dog with marked thrombocytopenia and no microscopic evidence of parasitemia. Bone marrow and splenic aspirates collected from the A. platys-infected dogs were tested by real-time TaqMan PCR. Two dogs were PCR positive in spleen and marrow at 28 days post-inoculation, when PCR results for buffy-coat blood cells were negative. Spleen and/or bone marrow samples should be considered as additional samples for PCR testing of dogs, particularly when blood samples are PCR negative during the acute phase of A. platys infection.
Assuntos
Anaplasma/isolamento & purificação , Anaplasmose/microbiologia , Bacteriemia/veterinária , DNA Bacteriano/análise , Reação em Cadeia da Polimerase/veterinária , Doença Aguda , Anaplasma/genética , Anaplasma/imunologia , Animais , Anticorpos Antibacterianos/sangue , Bacteriemia/microbiologia , Plaquetas/microbiologia , Plaquetas/patologia , Medula Óssea/microbiologia , DNA Bacteriano/sangue , Cães , Feminino , Reação em Cadeia da Polimerase/métodos , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Baço/microbiologia , Taq Polimerase , Trombocitopenia/microbiologiaRESUMO
Cats have been shown to be infected with Bartonella henselae genotype I, B. henselae genotype II, and B. clarridgeiae. Feline bartonellosis infections and the strains involved in these infections are important in both veterinary and human medicine. Nucleic acid amplification methods such as polymerase chain reaction (PCR) are being used in both research and diagnostics as tools for understanding many infectious diseases. Bartonella bacteremia in cats is detected by blood culture; however, because of the limitations of culture (delayed turnaround time and sensitivity limits), PCR may be a more efficient method for identifying infected cats. Three distinct PCR assays that could differentiate among B. henselae genotype I, B. henselae genotype II. and B. clarridgeiae were developed and used to detect as few as 3.2 organisms. Fourteen cats experimentally infected with B. henselae genotype I and B. henselae genotype II were followed by bacterial culture and PCR through the course of infection, including periods of primary and relapsing bacteremia. The PCR assay was positive in 11 of the 14 cats for periods of 1-9 weeks after culture became negative. Of the 223 blood specimens that were culture negative, the PCR assay was positive in 38 (17%) of the specimens. Two of the 14 cats developed relapsing bacteremia. The 2 B. henselae genotypes were amplified in the cats and the bacteremic phase of these infections as determined by PCR lasted for a longer period than previously determined by culture. Using laboratory assays such as PCR to understand the strains involved in feline bartonellosis and the course of the infection is important in the understanding of these zoonotic agents.
Assuntos
Bartonella henselae/genética , Doença da Arranhadura de Gato/genética , Reação em Cadeia da Polimerase/veterinária , Animais , Bacteriemia/veterinária , Bartonella henselae/patogenicidade , Sequência de Bases , Doença da Arranhadura de Gato/diagnóstico , Gatos , Primers do DNA , DNA Bacteriano/análise , Genótipo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
Cat scratch disease (CSD) has been difficult to diagnose in animals because of the protracted clinical course of infection and the quiescent phases when the microbial culprit lies dormant. The causative agent in CSD appears to be multiple species and strains of Bartonella. Using polymerase chain reaction (PCR) techniques for amplification of highly variable regions of the 16S ribosomal RNA (rRNA) gene sequence, a very sensitive species- and strain-specific assay for CSD-causing Bartonella species was developed. PCR primers were designed to specifically amplify the 16S rRNA gene of Bartonella species but not of other microbial pathogens. This initial PCR was multiplexed with a universal primer set, based on conserved sequence regions in the 16S rRNA gene, that provides a 162-bp fragment in all species tested. Subsequently, 3 distinct nested PCR primer sets enabled the individual amplification and specific detection of Bartonella henselae type 1, B. henselae type II, and B. clarridgeae. Thus, this 2-step PCR procedure enabled the sensitive detection and identification of these species and the B. henselae genotype by exploiting minor sequences differences. Verification of these results were demonstrated with both sequencing and ligase chain reaction techniques. The diagnostic usefulness of this CSD test has been demonstrated by the analysis of specimens from control and infected cats. The diagnosis was confirmed and the specific B. henselae strain was correctly identified in peripheral blood specimens obtained from control and strain-specific CSD-infected cats. Such an accurate and sensitive diagnostic tool for the detection and identification of CSD causative agents should be a useful for the medical, veterinary, and scientific communities.
Assuntos
Bartonella henselae/genética , Doenças do Gato/diagnóstico , Doença da Arranhadura de Gato/diagnóstico , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico 16S/genética , Animais , Bartonella henselae/patogenicidade , Sequência de Bases , Doenças do Gato/genética , Doenças do Gato/microbiologia , Doença da Arranhadura de Gato/genética , Gatos , Diagnóstico Diferencial , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
Ehrlichia canis causes a potentially fatal rickettsial disease of dogs that requires rapid and accurate diagnosis in order to initiate appropriate therapy leading to a favorable prognosis. We recently reported the cloning of two immunoreactive E. canis proteins, P28 and P140, that were applicable for serodiagnosis of the disease. In the present study we cloned a new immunoreactive E. canis surface protein gene of 1,170 bp, which encodes a protein with a predicted molecular mass of 42.6 kDa (P43). The P43 gene was not detected in E. chaffeensis DNA by Southern blot, and antisera against recombinant P43 (rP43) did not react with E. chaffeensis as detected by indirect fluorescent antibody (IFA) assay. Forty-two dogs exhibiting signs and/or hematologic abnormalities associated with canine ehrlichiosis were tested by IFA assay and by recombinant Western immunoblot. Among the 22 samples that were IFA positive for E. canis, 100% reacted with rP43, 96% reacted with rP28, and 96% reacted with rP140. The specificity of the recombinant proteins compared to the IFAs was 96% for rP28, 88% for P43 and 63% for P140. The results of this study demonstrate that the rP43 and rP28 are sensitive and reliable serodiagnostic antigens for E. canis infections.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Doenças do Cão/diagnóstico , Ehrlichia/imunologia , Ehrlichiose/veterinária , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Sequência de Bases , Southern Blotting , Western Blotting/métodos , Clonagem Molecular , Doenças do Cão/microbiologia , Cães , Ehrlichiose/diagnóstico , Ehrlichiose/microbiologia , Técnica Indireta de Fluorescência para Anticorpo/métodos , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Análise de Sequência de DNAAssuntos
Anti-Infecciosos/uso terapêutico , Doenças do Cão/tratamento farmacológico , Ehrlichia/fisiologia , Ehrlichiose/veterinária , Fluoroquinolonas , Quinolonas/uso terapêutico , Animais , Anticorpos Antibacterianos/sangue , DNA Bacteriano/sangue , Cães , Ehrlichia/genética , Ehrlichia/imunologia , Ehrlichiose/tratamento farmacológico , Enrofloxacina , Imunofluorescência/veterinária , Reação em Cadeia da Polimerase/veterinária , Falha de TratamentoRESUMO
Site-specific responses of bronchoalveolar and peripheral blood lymphocyte subsets were compared during primary and anamnestic immune responses against live Pasteurella haemolytica A1 (Ph1). Eight 1-year old calves were sequentially exposed intrabronchially with aerosolized Ph1 on days 0, 14, and 21, and two calves were sham exposed. Bronchoalveolar and peripheral blood lymphocytes were analyzed before each Ph1 exposure, and on days 3 and 7 post exposure using single and two-color flow cytometry to identify CD2+, CD4+, CD8+, CD21+, CD45R+, CD25+ and gammadelta lymphocyte subsets. Significant differences (p < 0.05) in bronchoalveolar and peripheral blood lymphocyte subsets were observed before Ph1 exposure. Subsequent aerosol exposures, resulted in significant (p < 0.05) changes in bronchoalveolar lymphocyte subsets and the CD4:CD8 bronchoalveolar lymphocyte ratio, but concomitant changes were not observed in peripheral blood lymphocytes. Expression of CD2, CD4 and CD8 lymphocyte differentiation antigens was consistently lower and more heterogeneous on bronchoalveolar lymphocytes. Differential analysis of bronchoalveolar leukocytes revealed a significant increase in bronchoalveolar lymphocytes and neutrophils during anamnestic responses.
Assuntos
Doenças dos Bovinos/imunologia , Subpopulações de Linfócitos/imunologia , Mannheimia haemolytica/imunologia , Infecções por Pasteurella/veterinária , Alvéolos Pulmonares/imunologia , Aerossóis , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Monoclonais/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Bovinos , Feminino , Citometria de Fluxo , Masculino , Infecções por Pasteurella/imunologia , Distribuição AleatóriaRESUMO
OBJECTIVE: To determine systemic and mucosal antibody responses in calves to Pasteurella haemolytica 1:A and to 2 major outer membrane proteins (OMP) and 1 major iron-regulated OMP of P haemolytica 1:A. ANIMALS: 23 crossbred calves. PROCEDURE: 2 experiments were performed in the first experiment, 6 calves were vaccinated and challenge exposed intranasally with an aerosol of P haemolytica 1:A and 6 calves were only challenge exposed. In the second experiment, 8 calves were vaccinated in the area of the tracheal bifurcation with an aerosol of P haemolytica 1:A and 3 calves were used as controls. Serum, nasal secretions, and bronchoalveolar lavage (BAL) samples were collected, and IgG1, IgG2, IgA, and IgM titers were determined. Nasal secretions and BAL samples were also submitted for bacterial culture. RESULTS: Serum antibody responses in the 2 groups were similar. Antibody titers in nasal secretions and BAL samples increased in calves vaccinated intranasally. In calves vaccinated in the area of the tracheal bifurcation, antibody titers increased in BAL samples but not in nasal secretions. Antibody responses did not correlate with results of bacterial culture. CONCLUSIONS: Results indicated that intranasal administration of P haemolytica 1:A may be a better method for stimulating protective immune responses in the upper portion of the respiratory tract than lung administration. The single dilution ELISA provided a reliable and economical method for determining antibody titers.
Assuntos
Anticorpos Antibacterianos/biossíntese , Proteínas da Membrana Bacteriana Externa/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Doenças dos Bovinos/imunologia , Mannheimia haemolytica/imunologia , Mucosa Nasal/imunologia , Infecções por Pasteurella/veterinária , Animais , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Vacinas Bacterianas , Bovinos , Imunoglobulina A/biossíntese , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Mucosa Nasal/metabolismo , Infecções por Pasteurella/imunologiaRESUMO
Bovine lymphocytes obtained by bronchoalveolar lavage (BAL) of healthy calves were simultaneously analyzed and compared to peripheral blood lymphocytes using monoclonal antibodies specific for bovine leukocyte differentiation antigens. Phenotypic differences were observed between bronchoalveolar and peripheral blood T-lymphocyte subpopulations, demonstrating selective lymphocyte migration to the bovine lung. The bronchoalveolar and peripheral blood T-lymphocyte populations, defined by expression of CD2, were similar, but bronchoalveolar T lymphocytes were predominately CD8+ while peripheral blood T cells were predominately CD4+. In addition, memory lymphocytes, characterized by low expression of CD45R and activated lymphocytes (CD25+), were found in significantly higher proportions in the bronchoalveolar compartment. The proportion of gammadelta T lymphocytes was, however, significantly higher in peripheral blood. B cells were observed in similar proportions in the bronchoalveolar compartment and peripheral blood.
Assuntos
Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Bovinos/imunologia , Subpopulações de Linfócitos/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Bovinos/sangue , Memória Imunológica , Antígenos Comuns de Leucócito/metabolismo , Subpopulações de Linfócitos/citologia , Fenótipo , Receptores de Interleucina-2/metabolismoRESUMO
To facilitate identification of ehrlichial pathogens, we developed a new technique based on fingerprints resulting from repetitive element polymerase chain reaction (rep-PCR). This technique uses consensus tRNA primers to generate amplification products that reflect distance polymorphisms between adjacent tRNA genes. Species-specific fingerprint patterns were obtained for seven Ehrlichia spp., as well as the unnamed causative agent of human granulocytotropic ehrlichiosis. Bands ranged in size from approximately 50 to 1,000 base pairs. Banding patterns varied depending on dilution of template DNA, with lower dilutions giving more complex banding patterns. These preliminary data indicate that repetitive-sequence-based PCR appears to be a useful technique for identifying ehrlichial organisms to the species, and perhaps the strain level. Compared with other conventional molecular-biologic methods, rep-PCR offers the advantages of ease of performance and rapid availability of results.
Assuntos
Técnicas de Tipagem Bacteriana , Impressões Digitais de DNA , Ehrlichia/classificação , Ehrlichia/genética , Reação em Cadeia da Polimerase/métodos , Primers do DNA , Ehrlichiose/microbiologia , Eletroforese , Humanos , RNA Bacteriano/genética , RNA de Transferência/genética , Sequências Repetitivas de Ácido Nucleico , Moldes GenéticosRESUMO
A mixed-breed pup approximately 3 months old obtained in north central Oklahoma by the Laboratory Animal Resources Unit of Oklahoma State University presented with platelet inclusions. The dog developed severe thrombocytopenia (< 10,000 microliters-1) following the appearance of inclusions. Blood films were monitored daily and when about 75% of platelets had inclusions, samples were collected in EDTA and processed for electron microscopic (EM) studies and polymerase chain reaction (PCR). EM studies on glutaraldehyde-fixed buffy coat revealed rickettsia-like inclusions in numerous platelets. Serologic examination, using Ehrlichia platys antigen, showed high titre suggestive of E. platys infection. PCR primers derived from a highly variable region of the 16S rRNA gene sequence of E. platys were used to specifically amplify that region of the parasite's DNA. Sequencing of the PCR product obtained by general Ehrlichia primers showed one nucleotide difference from the published sequence for E. platys which suggests possible strain variation of this intracellular parasite. Our results indicate that PCR may be a useful tool in the diagnosis of E. platys infection and that, like other Ehrlichia spp., E. platys isolates may vary.
Assuntos
DNA Bacteriano/sangue , Doenças do Cão , Ehrlichia/classificação , Ehrlichiose/veterinária , Animais , Sequência de Bases , Plaquetas/microbiologia , Plaquetas/ultraestrutura , Primers do DNA , Cães , Ehrlichia/isolamento & purificação , Ehrlichia/ultraestrutura , Ehrlichiose/sangue , Ehrlichiose/microbiologia , Microscopia Eletrônica , Dados de Sequência Molecular , Contagem de Plaquetas , Reação em Cadeia da Polimerase/métodos , Trombocitopenia/etiologia , Trombocitopenia/veterináriaRESUMO
A polymerase chain reaction (PCR)-based detection assay that specifically detected Ehrlichia canis in dogs with acute infections was developed. A region of the 16S ribosomal RNA gene of E. canis was targeted for PCR amplification and chemiluminescent hybridization (CH) with a complementary internal 287-base pair (bp) oligonucleotide probe. The CH improved the PCR assay sensitivity 1,000-fold as compared with visualization on ethidium bromide-stained agarose gels. The PCR assay with CH (PCR/CH) detected as little as 30 fg of E. canis genomic DNA, the equivalent of approximately 150 E. canis organisms. The 495-bp product defined by the specific primers was not detected when genomic DNA from E. platys, E. chaffeensis, E. risticii, and E. equi were used in the PCR/CH assay. The PCR/CH assay was tested with unfractionated blood samples collected from 9 dogs experimentally infected with E. canis. The PCR/CH assay had greater detection sensitivity than did cell culture isolation (CCI) from infected blood. PCR/CH detected E. canis 7 days prior to CCI in 4 of 6 experimentally infected dogs. The results obtained with the PCR/CH assay otherwise consistently matched the results obtained by CCI. This PCR/CH assay is a rapid, sensitive, and specific method for E. canis detection with sensitivity comparable to or exceeding that of CCI. A diagnosis of E. canis using this PCR/CH assay can be made in 2 days as compared with 1-4 weeks for CCI. The PCR/CH assay appears to be an acceptable alternative or complement to current diagnostic techniques.
Assuntos
Doenças do Cão , Ehrlichia/isolamento & purificação , Ehrlichiose/veterinária , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Primers do DNA , Cães , Ehrlichia/genética , Ehrlichiose/diagnóstico , Genes Bacterianos , Medições Luminescentes , Masculino , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Sensibilidade e EspecificidadeRESUMO
Subcutaneous inoculation of dogs with Ehrlichia canis was investigated as a more appropriate model of canine ehrlichiosis, which is naturally transmitted by arthropod vectors. A dose-dependent response occurred following subcutaneous inoculation of seven groups of dogs with log concentrations of E. canis-infected canine-origin cells. Ehrlichial infection in dogs was defined as concurrence of an increased titer of anti-E. canis immunoglobulin G (IgG) antibody in serum, a decreased platelet concentration, and isolation of E. canis by blood culture. In dogs administered the two lowest doses, no changes were detected. In seven of nine dogs administered three intermediate doses, the only change detected was a transient and mild increase in the anti-E. canis IgG antibody titer in serum. Only two of nine dogs inoculated with the intermediate doses developed an ehrlichial infection. Five of six dogs administered the two highest dose of E. canis developed an ehrlichial infection. These dogs had the highest IgG antibody titers in serum and the earliest isolation of E. canis from blood. In dogs that developed an ehrlichial infection, thrombocytopenia occurred by 28 days after inoculation, while increased IgG antibody titers in serum and blood cultures positive for E. canis occurred as early as 14 days postinoculation. Thrombocytopenia and seroconversion occurred later in the course of infection than previously reported for ehrlichial infections induced by intravenous inoculation. The route of administration and E. canis inoculum size can influence the course of ehrlichial infection and should be regarded as important variables in experimentally induced canine ehrlichiosis.
Assuntos
Doenças do Cão/etiologia , Ehrlichia/isolamento & purificação , Ehrlichiose/veterinária , Animais , Anticorpos Antibacterianos/sangue , Modelos Animais de Doenças , Doenças do Cão/imunologia , Doenças do Cão/microbiologia , Cães , Ehrlichia/imunologia , Ehrlichia/patogenicidade , Ehrlichiose/etiologia , Ehrlichiose/microbiologia , Imunoglobulina G/sangue , Injeções Subcutâneas , Masculino , Trombocitopenia/etiologia , Fatores de TempoRESUMO
Pulmonary and serum antibody responses were evaluated in eight calves vaccinated [four intrapulmonary-right diaphragmatic lobe (IP) and four subcutaneous (SC)] with Pasteurella haemolytica A1 (Ph-1) impregnated agar beads and eight respective sham-vaccinated calves. Experimental and sham groups were challenged in both diaphragmatic lobes with Ph-1 34-37 d after vaccination (DAV) and necropsied 6 d after challenge (DAC; 40-43 DAV). IgG antibodies contained in fluids from the diaphragmatic lobes of vaccinated calves had different patterns of antigen specificity compared with IgG antibodies in analogous sera. Using ELISA, anti-Ph-1 IgA and IgG antibody concentrations were significantly higher (P < 0.05) in lung lavage fluids from the IP group before and after challenge compared to the SC and sham groups. The IP and SC groups developed IgA, IgG and IgM antibody titers in nonvaccinated lung lobes after vaccination and challenge. The IP and SC groups exhibited significantly (P < 0.05) smaller pulmonary lesions than the sham groups and pulmonary IgG and IgA antibodies were associated with increased protection.
Assuntos
Anticorpos Antibacterianos/metabolismo , Doenças dos Bovinos/imunologia , Mannheimia haemolytica/imunologia , Infecções por Pasteurella/veterinária , Animais , Anticorpos Antibacterianos/sangue , Lavagem Broncoalveolar , Bovinos , Doenças dos Bovinos/patologia , Doenças dos Bovinos/prevenção & controle , Injeções Subcutâneas , Pulmão/imunologia , Pulmão/patologia , Infecções por Pasteurella/imunologia , Infecções por Pasteurella/prevenção & controle , Vacinação/métodos , Vacinação/veterináriaRESUMO
Dose dependent pulmonary lesions of acute bronchopneumonia were induced in male, outbred Swiss Webster mice by intrabronchial inoculation of Pasteurella haemolytica. Five exponential dilutions ranging from 5 x 10(4) to 5 x 10(8) colony forming units per mL (CFU/mL) of Pasteurella haemolytica serotype 1 were inoculated into five groups of mice. Mice were killed by cervical dislocation 24 hours postinoculation. Pulmonary lesions occurred in mice of all five groups, however, 5 x 10(7) CFU/mL was the minimal dose which consistently produced lesions. Focal parenchymal necrosis, suppurative bronchiolitis, and flooding of interalveolar septa and alveoli by edema fluid, fibrin, neutrophils and macrophages, were observed microscopically. We conclude that outbred Swiss Webster mice can be used as a model for the study of selected disease mechanisms of acute lung inflammation and that this model may be used to determine some of the pathogenic mechanisms involved in the development of pulmonary lesions in bovine pneumonic pasteurellosis.
Assuntos
Broncopneumonia/veterinária , Modelos Animais de Doenças , Mannheimia haemolytica , Camundongos , Pasteurelose Pneumônica/patologia , Doença Aguda , Animais , Broncopneumonia/etiologia , Broncopneumonia/patologia , Bovinos , Pulmão/patologia , Pulmão/ultraestrutura , Masculino , Microscopia Eletrônica , Pasteurelose Pneumônica/etiologiaRESUMO
A murine IgM monoclonal antibody causing bacterial agglutination was used in an immunoaffinity procedure to purify a serotype 1-specific polysaccharide epitope from Pasteurella haemolytica. The P haemolytica serotype 1-specific antibody was precipitated from peritoneal ascitic fluid, dialyzed, and covalently attached to cyanogen bromide-activated Sepharose 4B beads. Retention of purified antibody activity and coupling efficiency were > 99% when evaluated by ELISA, agglutination testing, and protein determination. Potassium thiocyanate was selected as an eluant on the basis of reversible dissociation of bacterial agglutination and was titrated for the lowest effective concentration. Immunobead activity was observed microscopically by immobilization of encapsulated P haemolytica serotype 1 and its reversible dissociation after elution with 0.4M potassium thiocyanate. Specificity of immobilization was visualized, using P haemolytica serotypes 2 and 5, which were not bound, and by blocking serotype-1 binding with homologous capsular material. Saline-extractable capsular material from P haemolytica serotype 1 was used as an antigen source. After elution of the serotype 1-specific polysaccharide epitope, the product was dialyzed and analyzed, using chemical and immunologic methods. The immunoaffinity product contained no detectable protein and greater than half the original hexosamine content. Using defined monoclonal antibodies in ELISA, titration of the original capsular material and the immunoaffinity product revealed specific retention of lipopolysaccharide, a 10- to 30-kd polysaccharide antigen common to all P haemolytica and P multocida serotypes, and serotype 1-specific capsular polysaccharide, indicating possible epitope sharing among polysaccharide antigens of P haemolytica serotype 1.
Assuntos
Epitopos/isolamento & purificação , Mannheimia haemolytica/imunologia , Polissacarídeos Bacterianos/isolamento & purificação , Aglutinação , Animais , Anticorpos Monoclonais , Complexo Antígeno-Anticorpo , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G , Indicadores e Reagentes , Camundongos , Polissacarídeos Bacterianos/imunologiaRESUMO
Four horses were inoculated with Ehrlichia risticii contained in either infected murine P388 D1 cells or heparinized blood from an infected horse. All 4 horses produced serum antibody, plasma antigen, and clinical signs of the disease. An enzyme-linked immunosorbent assay was used to detect antibody in the serum and was also used in conjunction with an anti-E. risticii monoclonal antibody to detect antigenemia. These laboratory and clinical findings were correlated to determine the efficiency of the antigen detection method for discerning E. risticii infection.
Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/sangue , Ehrlichiose/diagnóstico , Doenças dos Cavalos , Animais , Anticorpos Monoclonais , Ehrlichia/crescimento & desenvolvimento , Ehrlichiose/sangue , Ehrlichiose/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Cavalos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Camundongos , Células Tumorais CultivadasRESUMO
An enzyme-linked immunosorbent assay (ELISA) was used to detect antigen in plasma and antibody in serum of 3 horses inoculated with Ehrlichia equi. Clinical signs, including rectal temperature, were correlated with the antigen and antibody detection. ELISA was very efficient in detection of serum antibody. Antigen detection using monoclonal antibodies to E. equi and ELISA should be considered as a diagnostic method.
Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/sangue , Ehrlichiose/veterinária , Doenças dos Cavalos/diagnóstico , Animais , Anticorpos Monoclonais , Ehrlichia/imunologia , Ehrlichiose/sangue , Ehrlichiose/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Doenças dos Cavalos/sangue , Doenças dos Cavalos/imunologia , Cavalos , Imunoglobulina G/sangue , Imunoglobulina M/sangueRESUMO
The production and characterization of monoclonal antibodies against Pasteurella haemolytica serotype 1 is described. Ten monoclonal antibodies were produced and divided, on the basis of their properties, into six different groups. One produced bacteria agglutination only of P. haemolytica serotype 1. Three antibodies bound with P. haemolytica serotypes 1, 5-8 and 12 and the antigen was identified in immunoblots as lipopolysaccharide. Two antibodies bound P. haemolytica serotypes 1, 2, 5-8 and 12 and P. multocida serotypes 1-7, 9, 12, 15 and 16, recognizing an epitope present on a 29 kDa outer membrane protein. One antibody bound all P. haemolytica and P. multocida serotypes. The antigen was a hexosamine less than 30 kDa which contained a formalin sensitive epitope. One antibody bound only to P. haemolytica serotype 1 and the antigen was identified as a 66 kDa outer membrane protein. Two antibodies bound P. haemolytica serotypes 1, 2, 5-9 and 12 and the antigen, while not identified, was localized on the outer membrane. This study identified antigens which contribute to the cross-reactions among P. haemolytica and P. multocida serotypes and the antibodies may be useful in investigating the pathogenesis of pneumonic pasteurellosis.
Assuntos
Anticorpos Monoclonais/biossíntese , Antígenos de Bactérias/imunologia , Mannheimia haemolytica/imunologia , Pasteurelose Pneumônica/microbiologia , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/análise , Antígenos de Superfície/análise , Antígenos de Superfície/imunologia , Bovinos , Ensaio de Imunoadsorção Enzimática , Epitopos/análise , Epitopos/imunologia , Hibridomas , Immunoblotting , Imuno-Histoquímica , Mannheimia haemolytica/ultraestrutura , Microscopia EletrônicaRESUMO
Systemic and pulmonary antibody responses of calves to Pasteurella haemolytica were evaluated by measuring immunoglobulin production in blood for 9 days and in pulmonary lavage fluid for 7 days after intrapulmonary inoculation. Clinical signs, pulmonary lesions, pulmonary and systemic inflammatory response, and amount of antigen in lavage fluid were used to evaluate the response of calves to challenge with P haemolytica. The pulmonary response consisted of production of IgG, IgE, and IgM antibodies to P haemolytica antigens and a 17- to 68-fold increase of cells in lavage fluid 8 hours after inoculation, with a gradual decrease toward normal. Antibodies of the IgM isotype to P haemolytica were demonstrated as early as 8 hours through 7 days after inoculation in 3 of 3 calves. Of the anti-P haemolytica isotypes, IgM was found in the highest concentration. In all of the inoculated calves, IgE was found 1 to 2 days after inoculation, and IgG was found in 2 of 3 inoculated calves from day 1 through 7 after inoculation. Detection of IgG correlated with smaller pulmonary lesions. Immunoglobulin A was not detected in lavage fluid. Serum was evaluated for IgG and IgM antibody response to P haemolytica. Specific IgM was detectable 5 days after inoculation, and IgG was detectable 7 days after inoculation. Pasteurella haemolytica antigens were not detected in serum or plasma. A transient increase in neutrophil count was found 8 hours after inoculation, with return to baseline values by 24 hours after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Pulmão/imunologia , Mannheimia haemolytica/imunologia , Pasteurelose Pneumônica/imunologia , Animais , Vacinas Bacterianas/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , Bovinos , Contagem de Células , Pulmão/microbiologia , Mannheimia haemolytica/isolamento & purificaçãoRESUMO
A model of bovine pneumonic pasteurellosis, using an indwelling bronchial catheter for inoculation and subsequent lavage of a single main stem bronchus of the lung, was evaluated in a preliminary efficacy trial of an experimental therapeutic compound. Inoculation of 10(7) Pasteurella haemolytica organisms into the bronchus consistently induced a focal pneumonic lesion with typical morphology of pneumonic pasteurellosis in the left or right caudal lung lobe. The experimental treatment caused significant (P less than 0.05) reduction in lung lesion volume, compared with that of a saline-treated control. It also caused significant (P less than 0.05) reduction in lavage fluid bacterial counts at 48 hours after inoculation, compared with counts in the controls. The inflammatory cell count and the percentage of neutrophils increased markedly in lavage fluids 8 hours after inoculation, but differences were not detected between treatments. Significant differences between treatments were not found in clinical signs, rectal temperature, or histologic changes. This model appears to be a sensitive indicator of treatment efficacy and has the advantage over previous models of pneumonic pasteurellosis of allowing sequential monitoring of the primary lesion site.