Leukotoxins of gram-negative bacteria

Pasteurella aerogenes is known as a commensal bacterium or as an opportunistic pathogen, as well as a primary pathogen found to be involved in abortion cases of humans, swine, and other mammals. Using broad-range DNA probes for bacterial RTX toxin genes, we cloned and subsequently sequenced a new operon named paxCABD encoding the RTX toxin PaxA in P. aerogenes. The pax operon is organized analogous to the classical RTX operons containing the activator gene paxC upstream of the structural toxin gene paxA, which is followed by the secretion protein genes paxB and paxD. The highest sequence similarity of paxA with known RTX toxin genes is found with apxIIIA (82%). PaxA is structurally similar to ApxIIIA and also shows functional analogy to ApxIIIA, since it shows cohemolytic activity with the sphingomyelinase of Staphylococcus aureus, known as the CAMP effect, but is devoid of direct hemolytic activity. In addition, it shows to some extent immunological cross-reactions with ApxIIIA. P. aerogenes isolated from various specimens showed that the pax operon was present in about one-third of the strains. All of the pax-positive strains were specifically related to swine abortion cases or septicemia of newborn piglets. These strains were also shown to produce the PaxA toxin as determined by the CAMP phenomenon, whereas none of the pax-negative strains did. This indicated that the PaxA toxin is involved in the pathogenic potential of P. aerogenes. The examined P. aerogenes isolates were phylogenetically analyzed by 16S rRNA gene (rrs) sequencing in order to confirm their species. Only a small heterogeneity (<0.5%) was observed between the rrs genes of the strains originating from geographically distant farms and isolated at different times.

Characterization of PaxA and Its Operon: a Cohemolytic RTX Toxin Determinant from Pathogenic Pasteurella aerogenes

Specificity and performance of diagnostic PCR assays.

Lechiguana is a disease of cattle characterized by large, hard, subcutaneous swellings that grow rapidly and result in death after 3-11 months in untreated animals. Cattle treated with antibiotics recover. The disease has been reported from five states in south and southeastern Brazil. Histologically, the lesion consists of focal proliferation of fibrous tissue infiltrated by plasma cells, eosinophils, lymphocytes and sometimes neutrophils. The primary lesion is an eosinophilic lymphangitis, which results in eosinophilic abscesses, with occasional rosettes containing bacteria in their centres. Much experimental and epidemiological evidence, reviewed in this article, supports the suggestion that lechiguana is caused by an association of Pasteurella granulomatis (syn: Mannheimia granutomatis) and Dermatobia hominis.

Lechiguana (focal proliferative fibrogranulomatous panniculitis) in cattle.

Mannheimia granulomatis was isolated for 10 months from the milk of a cow with elevated somatic cell counts. The infection was self-limiting. Phenotypic and molecular characteristics of the isolate were determined.

https://journals.sagepub.com/doi/pdf/10.1177/104063871002200627

Bovine subclinical mastitis caused by Mannheimia granulomatis.

Pasteurella granulomatis (Pg) is a recently identified bacterium associated with proliferative fibrogranulomatous panniculitis (also called "lechiguana") in Brazilian cattle. Recent attempts to experimentally reproduce this disease have only been partially successful. We hypothesized that Pg may produce hemolysin(s) and/or cytotoxin(s) which could contribute to its pathogenicity in susceptible cattle. The objective of this study was to determine the presence and degree of hemolytic and leukotoxic activity of selected isolates of Pg. Either ovine or bovine blood agar plates were streaked with 1 of 7 Pg isolates, incubated at 37 degrees C +/- 1 C for 48 hours, and examined for hemolysis. Two of seven isolates showed hemolytic activity on bovine plates, while all seven showed hemolytic activity on ovine plates. By use of the CAMP reaction, involving simultaneous intersecting cultures of Staphylococcus aureus and Pg, all seven Pg isolates showed enhanced (positive CAMP) hemolysis within 24 hours on bovine blood agar plates. Preliminary results using tetrazolium (MTT) dye reductions with bovine neutrophils showed leukotoxicity in 13 of 16 Pg cultures. Alamar blue tests indicate leukotoxic activity for all 7 Pg isolates. We conclude that some Pg isolates have variable hemolytic and/or leukotoxic properties and that this variability (presence and/or degree) of these 2 properties may affect the relative pathogenicity of Pg in susceptible cattle.

D. J. Wise

Papers

Toxin production by Pasteurella granulomatis.