Subramaniam Srikumaran

Significance: Contrary to the paradigm, the signal peptide of CD18, an adhesive protein expressed on the surface of leukocytes of cattle and other ruminants, is not cleaved. Intriguingly, leukotoxin produced byMannheimia haemolyticabinds to the intact signal peptide and causes lysis of ruminant leukocytes, resulting in acute pneumonia. In this proof-of-principle study, we used precise gene editing to induce a single amino acid substitution in CD18, which resulted in the cleavage of the signal peptide and abrogation of leukotoxin-induced cytolysis of leukocytes of the gene-edited bovine fetus. This report demonstrates the feasibility of developing lines of cattle genetically resistant toM. haemolytica-caused pneumonia, which will have significant impact on the sustainability of food-animal production in the United States and elsewhere.

Fusobacterium necrophorum has been detected in pneumonic bighorn sheep (BHS; Ovis canadensis) lungs, in addition to the aerobic respiratory pathogens Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida, and Mycoplasma ovipneumoniae. Similar to M. haemolytica, F. necrophorum produces a leukotoxin. Leukotoxin-induced lysis and degranulation of polymorphonuclear leukocytes (PMNs) and macrophages are responsible for acute inflammation and lung tissue damage characteristic of M. haemolytica-caused pneumonia. As one approach in elucidating the role of F. necrophorum in BHS pneumonia, we determined the frequency of the presence of F. necrophorum in archived pneumonic BHS lung tissues, and susceptibility of BHS leukocytes to F. necrophorum leukotoxin. A species-specific PCR assay detected F. necrophorum in 37% of pneumonic BHS lung tissues (total tested n=70). Sequences of PCR amplicons were similar to the less virulent F. necrophorum subsp. funduliforme. Fusobacterium necrophorum leuko...

In the absence of livestock contact, recurring lamb mortality in bighorn sheep (Ovis canadensis) populations previously exposed to pneumonia indicates the likely presence of carriers of pneumonia-causing pathogens, and possibly inadequate maternally derived immunity. To investigate this problem we commingled naïve, pregnant ewes (n=3) with previously exposed rams (n=2). Post-commingling, all ewes and lambs born to them acquired pneumonia-causing pathogens (leukotoxin-producing Pasteurellaceae and Mycoplasma ovipneumoniae), with subsequent lamb mortality between 4-9 weeks of age. Infected ewes became carriers for two subsequent years and lambs born to them succumbed to pneumonia. In another experiment, we attempted to suppress the carriage of leukotoxin-producing Pasteurellaceae by administering an antibiotic to carrier ewes, and evaluated lamb survival. Lambs born to both treatment and control ewes (n=4 each) acquired pneumonia and died. Antibody titers against leukotoxin-producing Pasteurellaceae in all eight ewes were ‘protective’ (>1:800 and no apparent respiratory disease); however their lambs were either born with comparatively low titers, or with high (but non-protective) titers that declined rapidly within 2-8 weeks of age, rendering them susceptible to fatal disease. Thus, exposure to pneumonia-causing pathogens from carrier ewes, and inadequate titers of maternally derived protective antibodies, are likely to render bighorn lambs susceptible to fatal pneumonia.

Mannheimia haemolytica causes pneumonia in domestic and wild ruminants. Leukotoxin (Lkt) is the most important virulence factor of the bacterium. It is encoded within the four-gene lktCABD operon: lkt A encodes the structural protoxin, and lktC encodes a trans -acylase that adds fatty acid chains to internal lysine residues in the protoxin, which is then secreted from the cell by a type 1 secretion system apparatus encoded by lktB and lktD . It has been reported that LktC-mediated acylation is necessary for the biological effects of the toxin. However, an LktC mutant that we developed previously was only partially attenuated in its virulence for cattle. The objective of this study was to elucidate the role of LktC-mediated acylation in Lkt-induced cytotoxicity. We performed this study in bighorn sheep ( Ovis canadensis ) (BHS), since they are highly susceptible to M. haemolytica infection. The LktC mutant caused fatal pneumonia in 40% of inoculated BHS. On necropsy, a large number of necrotic polymorphonuclear leukocytes (PMNs) were observed in the lungs. Lkt from the mutant was cytotoxic to BHS PMNs in an in vitro cytotoxicity assay. Flow cytometric analysis of mutant Lkt-treated PMNs revealed the induction of necrosis. Scanning electron microscopic analysis revealed the presence of pores and blebs on mutant-Lkt-treated PMNs. Mass spectrometric analysis confirmed that the mutant secreted an unacylated Lkt. Taken together, these results suggest that acylation is not necessary for the cytotoxic activity of M. haemolytica Lkt but that it enhances the potency of the toxin.

Mannheimia haemolytica,Pasteurella multocida, andBibersteinia trehalosihave been identified in the lungs of pneumonic bighorn sheep (BHS;Ovis canadensis). Of these pathogens,M. haemolyticahas been shown to consistently cause fatal pneumonia in BHS under experimental conditions. However,M. haemolyticahas been isolated by culture less frequently than the other bacteria. We hypothesized that the growth ofM. haemolyticais inhibited by other bacteria in the lungs of BHS. The objective of this study was to determine whetherP. multocidainhibits the growth ofM. haemolytica. Although in monoculture both bacteria exhibited similar growth characteristics, in coculture withP. multocidathere was a clear inhibition of growth ofM. haemolytica. The inhibition was detected at mid-log phase and continued through the stationary phase. When cultured in the same medium, the growth ofM. haemolyticawas inhibited when both bacteria were separated by a membrane that allowed contact (pore size, 8.0 μm) but not when they were separated by a membrane that limited contact (pore size, 0.4 μm). Lytic bacteriophages or bactericidal compounds could not be detected in the culture supernatant fluid from monocultures ofP. multocidaor fromP. multocida-M. haemolyticacocultures. These results indicate thatP. multocidainhibits the growth ofM. haemolyticaby a contact- or proximity-dependent mechanism. If the inhibition of growth ofM. haemolyticabyP. multocidaoccursin vivoas well, it could explain the inconsistent isolation ofM. haemolyticafrom the lungs of pneumonic BHS.