PCR detection of Anaplasma platys in blood and tissue of dogs during acute phase of experimental infection.

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.

Doxycycline clearance of experimentally induced chronic Ehrlichia canis infection in dogs.

BACKGROUND: Ineffective clearance of Ehrlichia canis after doxycycline administration has been reported despite the fact that the recommended treatment for canine ehrlichiosis is doxycycline. The effectiveness of doxycycline in clearing E canis infection from the blood and tissues of dogs requires additional evaluation. HYPOTHESIS: Doxycycline (5 mg/kg PO q12h), administered for 4 weeks, will eliminate E canis infection from the blood and tissues of experimentally infected dogs. ANIMALS: Fifteen Walker hound-mixed breed dogs were inoculated subcutaneously with E canis-infected canine histiocytic cells 4 months before doxycycline treatment. METHODS: Four dogs were treated with doxycycline (5 mg/kg PO q12h for 3 weeks), 5 dogs were treated with doxycycline at the same dosage for 4 weeks, and 5 control dogs were not treated. Dexamethasone (0.4 mg/kg i.v.) was given after treatment to precipitate recrudescence of any remaining E canis organisms. Platelet counts, anti-E canis immunofluorescent antibodies, and polymerase chain reaction (PCR) detection of E canis deoxyribonucleic acid (DNA) in blood and tissues were evaluated. RESULTS: E canis DNA was not detected in the blood and tissues of doxycycline-treated dogs after treatment. Platelet counts were within reference intervals, and E canis antibodies decreased. Spontaneous clearance of E canis infection occurred in 2 of 5 control dogs. Three control dogs had E canis DNA detected in blood and tissues, platelet counts remained low or within the reference interval, and E canis antibodies remained high. CONCLUSIONS AND CLINICAL IMPORTANCE: As administered in this study, doxycycline cleared E canis from the blood and tissues of experimentally infected dogs.

Failure of imidocarb dipropionate to clear experimentally induced Ehrlichia canis infection in dogs.

The recommended treatment for canine ehrlichiosis is tetracycline or its analog doxycycline, although recent reports have documented ineffective clearing of Erchlichia canis after doxycycline administration. Imidocarb dipropionate is used as an alternative treatment to tetracycline or is used in conjunction with doxycycline. The effectiveness of imidocarb dipropionate in clearing Ehrlichia species from the blood and tissues of dogs with E. canis infection has not been thoroughly evaluated. Fifteen dogs were experimentally infected with E. canis. Ten dogs were treated with imidocarb dipropionate (6.6 mg/kg, IM, 2 injections given 2 weeks apart). Five infected control dogs were not treated. Blood samples from all 15 dogs were E. canis DNA positive by PCR assay by 3 weeks after inoculation (PI), and E. canis antibodies were detected by IFA assay by 1 week PI. Blood platelet counts in all dogs were below the reference interval by 4 weeks PI. E. canis DNA was detected in bone marrow and splenic aspirates by PCR assay 4 weeks PI but not before infection. Bone marrow aspirates were E. canis DNA positive by PCR assay in 14/15 dogs, and splenic aspirates were E. canis DNA positive by PCR assay in 13/15 dogs. Blood samples from all treated and control dogs remained positive for E. canis DNA by PCR assay, and platelet counts remained below preinoculation values 13 weeks PI (6 weeks after 2nd treatment). As administered in this study, imidocarb dipropionate did not clear experimental E. canis infection in dogs.

Differential detection of Bartonella species and strains in cat scratch disease diagnostics by polymerase chain reaction amplification of 16S ribosomal RNA gene.

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.

Evaluation and use of a nested polymerase chain reaction assay in cats experimentally infected with Bartonella henselae genotype I and Bartonella henselae genotype II.

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.

Immunodiagnosis of Ehrlichia canis infection with recombinant proteins.

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.

Primary and anamnestic responses of bovine bronchoalveolar and peripheral blood lymphocyte subsets to aerosolized Pasteurella haemolytica A1.

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.

Antibody responses to Pasteurella haemolytica 1:A and three of its outer membrane proteins in serum, nasal secretions, and bronchoalveolar lavage fluid from calves.

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.

Memory and CD8+ are the predominant bovine bronchoalveolar lymphocyte phenotypes.

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.

Fingerprinting of Ehrlichia species by repetitive element polymerase chain reaction.

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.

Characterization of a new isolate of Ehrlichia platys (Order Rickettsiales) using electron microscopy and polymerase chain reaction.

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.

PCR detection of acute Ehrlichia canis infection in dogs.

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.

Isolation of Ehrlichia canis from dogs following subcutaneous inoculation.

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.

Characterization of bovine pulmonary and serum antibody responses after parenteral or intrapulmonary vaccination with live Pasteurella haemolytica.

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.

Induction of acute bronchopneumonia in mice by intrabronchial inoculation of Pasteurella haemolytica serotype 1.

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.

Purification of a Pasteurella haemolytica serotype 1-specific polysaccharide epitope by use of monoclonal antibody immunoaffinity.

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.

Detection of antigenemia by enzyme-linked immunosorbent assay in horses with experimental Ehrlichia risticii infection.

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.

Detection of humoral antigen and antibody by enzyme-linked immunosorbent assay in horses with experimentally induced Ehrlichia equi infection.

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.

A characterization of monoclonal antibodies prepared against Pasteurella haemolytica serotype 1 surface antigens.

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.