George L. Murphy

A molecular and serologic survey of Ehrlichia canis, E. chaffeensis, and E. ewingii in dogs and ticks from Oklahoma.

Polymerase chain reaction and Southern hybridization were used to survey for the presence of Ehrlichia canis, Ehrlichia chaffeensis, and Ehrlichia ewingii in blood samples of 65 dogs that harbored ticks from northcentral and northeastern Oklahoma. Dog blood samples were also examined for antibodies against E. canis and E. chaffeensis, using an immunofluorescent antibody test. Ten of 65 dogs (15.4%) examined were positive for Ehrlichia spp. by PCR. Four (6.2%) were positive for E. ewingii, 2 (3.1%) for E. canis, and 4 (6.2%) for E. chaffeensis. Seven dogs (10.8%) were seropositive for E. canis or E. chaffeensis. Ticks collected from PCR-positive dogs were examined by PCR for the presence of Ehrlichia DNA. Several groups of ticks were PCR-positive for E. ewingii or E. canis. E. canis was detected in Rhipicephalus sanguineus, which is considered the major vector for that organism. E. ewingii was detected in a larger variety of ticks, including the only known vector Amblyomma americanum, as well as in Dermacentor variabilis and R. sanguineus. Results suggest that Ehrlichia spp. which are canine and human pathogens circulate in dogs in Oklahoma and in several tick species that feed on dogs.

Cats Surviving Natural Infection with Cytauxzoon felis: 18 Cases (1997–1998)

Eighteen cats surviving natural infection with Cytauxzoon felis were identified. All cats came from a limited geographic area in northwestern Arkansas and northeastern Oklahoma. Clinical signs in most cats were similar to those described for cytauxzoonosis; however, 4 cats were asymptomatic. All cases were initially diagnosed by microscopic identification of signet ring-shaped piroplasms in erythrocytes of peripheral blood smears. Four of 4 cats tested had detectable serum antibodies to C felis. Four different cats were positive by polymerase chain reaction (PCR). Partial sequencing of the PCR product from 1 cat revealed >99% homology with the reported sequence of C felis. Repeated examination of blood smears from 12 cats revealed that the erythroparasitemia was generally persistent for the duration of follow-up (3–154 days). Survival did not seem dependent on treatment, as only 1 cat was treated with a drug with potential antiprotozoal activity (imidocarb dipropionate), and 4 cats received no treatment. The findings of this study may indicate the existence of a less virulent strain of C felis.

Identification of immunogenic, surface-exposed outer membrane proteins of Pasteurella haemolytica serotype 1

Pasteurella haemolytica serotype 1 (S1) is the bacterium most frequently recovered from the lungs of cattle that have succumbed to shipping fever pneumonia. P. haemolytica outer membrane proteins (OMPs) are important immunogens in the development of resistance to pneumonic pasteurellosis. The purpose of this study was to identify the repertoire of immunogenic, surface-exposed P. haemolytica (S1) OMPs, that could be important in the development of protective immunity. We determined surface exposure of OMPs by (1) their susceptibility to protease treatment and (2) their ability to adsorb out antibodies from bovine immune sera. For a comprehensive identification of immunogenic, surface-exposed OMPs, we used bovine antisera from calves that were resistant to experimental P. haemolytica challenge after (1) natural exposure to P. haemolytica, (2) vaccination with live P. haemolytica, or (3) vaccination with P. haemolytica OMPs. We identified 21 immunogenic, surface-exposed P. haemolytica OMPs. Most were recognized by all three immune sera. However, some were recognized by one or two of the three antisera. Our analyses identified surface-exposed, immunogenic proteins that were not identified in previous studies.

Outer membrane proteins of bovine Pasteurella multocida serogroup A isolates

The outer membrane proteins (OMPs) of P. multocida serotypes A3 (7 isolates), A4 (2 isolates), A3,4 and A2 (one isolate each) obtained from pneumonic cattle (10 isolates) and from one pig isolate were investigated to identify potential immunogens. SDS-PAGE of P. multocida OM isolated by SDG centrifugation of spheroplasts revealed eight major OMPs. Outer membranes isolated by sarcosyl extraction or SDG had similar protein composition on Coomassie blue-stained SDS-PA gel and on immunoblots. Two major OMPs (M(r)s of 35 and 46 kDa at 100 degrees C) demonstrated heat modifiability with apparent M(r)s of 30 and 34 kDa at 37 degrees C, respectively. The N-terminal aa sequences of these heat modifiable proteins revealed homology with E. coli OmpA and Hib P1 proteins, respectively. Protease treatment of whole cells followed by western immunoblots using bovine convalescent sera identified several immunogenic, surface-exposed and conserved OMPs among the eleven P. multocida isolates examined. The whole organism SDS-PAGE profiles of the eleven P. multocida isolates differed such that six patterns were seen. These patterns could potentially be used as a typing system for P. multocida bovine isolates based on the molecular weights of whole cell proteins. The above observations have potentially important implications relative to the immunity to infection.

A Genotypically Unique Babesia gibsoni-like Parasite Recovered From a Dog in Oklahoma

A small Babesia gibsoni-like parasite was identified and isolated as the cause of clinical babesiosis in a dog from Oklahoma. Because this was potentially the first documented case of B. gibsoni infection in Oklahoma, further characterization was warranted, and the 18S nuclear small subunit ribosomal RNA gene was sequenced. Sequence comparison with other piroplasms from dogs showed significant nucleotide sequence differences between this isolate and both B. canis and B. gibsoni. These findings demonstrate that in domestic dogs in North America there are at least 2 “small” B. gibsoni-like organisms with distinct nucleotide sequences and that the geographic distribution of the “small” canine Babesia species may be wider than previously recognized.

Detection of Anaplasma Marginale DNA in Bovine Erythrocytes by Slot-Blot and in Situ Hybridization with a PCR-Mediated Digoxigenin-Labeled DNA Probe

A 409-base pair (bp) DNA fragment derived from the msp-1β gene of Anaplasma marginale was amplified and simultaneously labeled with digoxigenin-11-dUTP by a polymerase chain reaction (PCR) assay. The resulting digoxigenin-labeled 409-bp PCR product was used as a probe for slot-blot and in situ hybridization to detect A. marginale DNA from experimentally infected bovine erythrocytes. The hybrid formation was detected with alkaline phosphatase-conjugated anti-digoxigenin antibody and substrates 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt. In slot-blot hybridizations, the probe detected A. marginale DNA from approximately 1,000-10,000 infected erythrocytes in 1.25 ml of whole blood, which is equivalent to a parasitemia level of 0.00001%. The probe proved to be A. marginale-specific when tested with 17 species of microorganisms. The applicability of the probe for diagnosis was tested by screening A. marginale infections in 2 experimentally infected splenectomized cattle before microscopically detectable parasitemias and after acute infection. After inoculation of infected blood, A. marginale infections were detected with the probe 14 days prior to detection in stained smears. Microscopically inapparent parasitemias were also detected with the probe for 2 months after acute disease. When the probe was used for in situ hybridization on methanol-fixed blood smears, probe reaction could be visualized with light microscopy on A. marginale inclusions within infected erythrocytes. The probe reaction was not observed on leukocytes and uninfected erythrocytes from infected blood smears, on erythrocytes from uninfected blood samples, or on samples infected with A. ovis, Babesia bovis, or B. bigemina. This PCR-mediated nonradioactive probe appears to be a sensitive diagnostic test for A. marginale.

Pathogenesis and Virulence of Pasteurella Haemolytica in Cattle: An Analysis of Current Knowledge and Future Approaches

Pasteurella haemolytica is the cause of a severe fibrinous pleuropneumonia of cattle termed Shipping Fever (Frank, 1989). Serovar Al is isolated most often from bovine pneumonic pasteurellosis. The pathogenesis of pneumonic pasteurellosis relies on several bacterial-host interactions: nasopharyngeal colonization, inhalation, pulmonary alveolar colonization, host response to colonization, and bacterial evasion of host defense (Whiteley et al., 1992).

Genetic and immunological analyses of a 38 kDa surface-exposed lipoprotein of Pasteurella haemolytica A1.

Pasteurella haemolytica serotype A1 is the bacterial pathogen most frequently isolated from the lungs of cattle with bovine respiratory disease. As part of a study to characterize P. haemolytica antigens which are important in eliciting resistance to pneumonic pasteurellosis, we have cloned and sequenced the gene encoding a 38 kDa lipoprotein, Lpp38. The deduced amino acid sequence of Lpp38 is similar to those of the Escherichia coli polyamine transport proteins PotD (70%) and PotF (33%). P. haemolytica Lpp38 is present in both inner membrane and outer membrane fractions of the cell envelope. Susceptibility of Lpp38 to cleavage by extracellular proteases indicates that portions of the protein are surface-exposed. A protein of similar molecular mass in P. haemolytica strains from all 12 serotypes of biotype A and in an untypeable strain was detected by an anti-Lpp38 monoclonal antibody. Lpp38 is recognized by sera from calves resistant to infection after natural exposure to P. haemolytica and by sera from calves protected against infection by vaccination with P. haemolytica A1 outer membranes or with live bacteria. These data suggest a role for this protein in the development of immunity to P. haemolytica infection.

Analysis of tandem, multiple genes encoding 30-kDa membrane proteins in Pasteurella haemolytica A1.

A number of outer membrane proteins (OMPs), including a 30-kDa protein, may be important in eliciting immunity to Pasteurella haemolytica A1, the causative agent of bovine pneumonic pasteurellosis. To better understand the nature of the 30-kDa antigen, several genes encoding this protein were sequenced. Sequence analysis revealed that three separate genes encoding similar, yet distinct, versions of the 30-kDa protein are tandemly arranged on the P. haemolytica A1 chromosome. The genes appear to be transcribed from a single promoter. The deduced amino acid sequences of the proteins encoded by these genes are similar to a 28-kDa inner membrane lipoprotein of Escherichia coli and a 28-kDa membrane protein which may contribute to the virulence of Haemophilus influenzae type b strains.

Construction of isogenic mutants of Pasteurella haemolytica by allelic replacement.

We describe methods for the mutagenesis of cloned Pasteurella haemolytica (Gram-) genes and for the construction of P. haemolytica mutants by allelic exchange. We used these methods to construct isogenic mutants of P. haemolytica which no longer synthesize three membrane lipoproteins (Lpp). A single genetic locus, consisting of three tandemly arranged genes encoding 28-30-kDa membrane Lpp, was replaced with a mutated locus which carries the beta-lactamase-encoding ApR gene from a 4.2-kb P. haemolytica plasmid. The inactivated locus was introduced into P. haemolytica by electroporation of a plasmid which carries the mutated locus, but is incapable of replicating in P. haemolytica. Southern and Western blot analyses indicate that the wild-type locus was replaced by the mutated locus through a double-crossover recombination event and that the membrane Lpp were no longer produced by the mutant strain. These methods should be useful in constructing mutant loci which can be used to analyze the roles for various P. haemolytica proteins in the pathogenesis of bovine pneumonic pasteurellosis.