Lung lesions in SCID-bo and SCID-bg mice after intratracheal inoculation with wild-type or leucotoxin-deficient mutant strains of Mannheimia haemolytica serotype 1.

The purpose of this study was to investigate SCID-bg mice engrafted with bovine haematolymphoid tissues (SCID-bo) as a model for studying bovine Mannheimia haemolytica serotype 1- induced pneumonia, in which leucotoxin (LKT) plays a major role. In experiment A, SCID-bo and SCID-bg mice were inoculated intratracheally with either (1) phosphate-buffered saline (PBS), (2) M. haemolytica wild-type strain 89010807N ("LKT(+)WT"), (3) a M. haemolytica leucotoxin-deficient mutant of strain 89010807N ("LKT(-)mutant"), or (4) the M. haemolytica wild-type Oklahoma strain. Mice were killed for examination at intervals between 20 and 44h after inoculation. Lung lesions consisted of thickened alveolar septa and neutrophil and macrophage infiltrates in the bronchioles and alveoli. Lung lesion scores in the SCID-bo mice inoculated with LKT(+)WT or LKT(-) mutant were significantly (P<0.05) greater than those of the PBS control group, but the two bacterial strains produced results that did not differ significantly. M. haemolytica was isolated from lung, liver and spleen after inoculation but less frequently as time progressed. In experiment B, SCID-bg mice were inoculated intratracheally with live LKT(+)WT or formalin-killed LKT(+)WT and killed 24, 48 or 96 h later. Lung lesions were histologically similar to those observed in experiment A; however, there were no significant differences in the lung lesion scores between groups. It was concluded that the lesions seen in this study were probably not due to LKT, and that the SCID-bo mouse does not provide a good rodent model for bovine pneumonia.

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.

Naturally occurring Ehrlichia chaffeensis infection in coyotes from Oklahoma.

A nested polymerase chain reaction assay was used to determine the presence of Ehrlichia chaffeensis, E. canis, and E. ewingii DNA in blood samples of free-ranging coyotes from central and northcentral Oklahoma. Of the 21 coyotes examined, 15 (71%) were positive for E. chaffeensis DNA; none was positive for E. canis or E. ewingii. Results suggest that E. chaffeensis infections are common in free-ranging coyotes in Oklahoma and that these wild canids could play a role in the epidemiology of human monocytotropic ehrlichiosis.

Attempted transmission of Ehrlichia chaffeensis among white-tailed deer by Amblyomma maculatum.

A deer was needle-exposed intravenously to Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) in canine macrophage (DH82) cells and 7 days later was infested with laboratory-reared Amblyomma maculatum (Koch) (Acari:Ixodidae) nymphs for acquisition feeding. After molting, the adult ticks were allowed to feed on a naive deer. The organism was reisolated from the needle-exposed deer by cell culture and E. chaffeensis DNA was detected in the deer's blood by PCR. Similar isolation/recovery techniques were used for the tick-exposed deer and no evidence of infection was found. Although these findings must be considered as preliminary owing to inadequate controls, the data suggest that A. maculatum is probably not a suitable vector for E. chaffeensis.

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.

Characterization of a Pasteurella haemolytica A1 mutant deficient in production of three membrane lipoproteins.

OBJECTIVE: To determine whether a Pasteurella haemolytica A1 mutant that is unable to produce membrane lipoproteins has reduced susceptibility to complement-mediated killing, and to characterize the mutant strain. SAMPLE POPULATION: 12 sera from cattle resistant to P haemolytica challenge exposure after vaccination with P haemolytica or its antigens, or after natural exposure. PROCEDURES: Complement-mediated killing assays were performed, using wild-type and mutant strains and, as antibody source, various immune sera from cattle that were resistant to P haemolytica challenge exposure. Antibody response to whole-cell antigens produced by mutant and wild-type strains, production of outer membrane proteins and iron-regulated outer membrane proteins by the 2 strains, and growth of the 2 strains in various media were analyzed. RESULTS: Compared with wild-type P haemolytica, the lipoprotein mutant strain had increased susceptibility to bovine complement-mediated killing. Aside from the lipoproteins that are not produced by the mutant, immunoblot analysis did not reveal differences between immunoreactive antigens that are produced by the 2 strains. Some iron-regulated, outer membrane proteins, which usually are only produced by P haemolytica under iron-deficient conditions, were produced constitutively by the mutant. The mutant grew to a lower final cell density and at a lower rate under conditions likely to reflect those encountered in vivo. CONCLUSIONS: Lack of 3 membrane lipoproteins resulted in enhanced susceptibility to bovine complement-mediated killing. Site-specific mutagenesis of genes encoding P haemolytica membrane lipoproteins alters production of iron-regulated outer membrane proteins by P haemolytica. Growth characteristics of the mutant suggested that it may have reduced capacity for survival in vivo.

Hemolytic activity of the Pasteurella haemolytica leukotoxin.

A Pasteurella haemolytica mutant incapable of producing leukotoxin was created by allelic replacement. Concentrated culture supernatants from wild-type P. haemolytica, but not from the mutant, contained the 102-kDa leukotoxin protein and lysed bovine lymphoma cells and sheep erythrocytes. Wild-type P. haemolytica demonstrated the typical beta-hemolytic phenotype on sheep and rabbit blood agar, whereas the mutant did not.

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.

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.