Fusobacterium necrophorum in North American Bighorn Sheep (Ovis canadensis) Pneumonia

Pneumonia is a major factor in the drastic decline of bighorn sheep (BHS; Ovis canadensis) populations in North America (Miller 2001). The respiratory pathogens commonly detected in pneumonic BHS lungs include Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida, and Mycoplasma ovipneumoniae (Miller 2001; Besser et al. 2008). Mycoplasma ovipneumoniae can predispose BHS to M. haemolytica infection (Dassanayake et al. 2010). Lysis and degranulation of polymorphonuclear leukocytes (PMNs) and macrophages by M. haemolytica leukotoxin are responsible for the acute inflammation and lung injury characteristic of M. haemolytica-caused pneumonia (Slocombe et al. 1985). Several anaerobic bacteria have also been reported in pneumonic BHS lungs (Besser et al. 2008, 2012). The role of these anaerobic bacteria, including Fusobacterium necrophorum in BHS pneumonia is not clear. Fusobacterium necrophorum is a gram-negative, nonmotile, nonspore-forming, strictly anaerobic organism that causes a variety of suppurative and necrotic infections, generally called necrobacillosis (Nagaraja et al. 2005). It is one of the common anaerobic bacteria isolated from abscesses, respiratory tract infections, and other necrotizing infections in domestic livestock, wild mammals, and humans (Nagaraja et al. 2005). It is commonly found in foot rot in domestic sheep (Ovis aries; Roberts and Egerton 1969). Fusobacterium necrophorum produces several virulence factors which include leukotoxin, endotoxin, hemolysin, and hemagglutinin (Tan et al. 1996). Of these, leukotoxin is considered to be the major virulence determinant (Nagaraja et al. 2005). Leukotoxin is cytotoxic to leukocytes, hepatocytes, and ruminal epithelial cells of cattle and domestic sheep (Ishii et al. 1988; Tan et al. 1992). Herein we determine the presence of F. necrophorum in archived pneumonic BHS lung tissues and the susceptibility of BHS leukocytes to F. necrophorum leukotoxin.

For the study we obtained 70 pneumonic lung tissue samples from dead or euthanized BHS submitted to, and archived in, Washington Animal Disease Diagnostic Laboratory (Pullman, Washington, USA). Normal lung tissue samples from five BHS that did not show signs of pneumonia and lacked gross or microscopic lesions distinctive of pneumonia on necropsy were used as negative controls. Pharyngeal swabs from 42 apparently healthy BHS were also examined. Genomic DNA was extracted from pneumonic and normal BHS lung tissues and healthy BHS pharyngeal swabs using QIAamp DNA Mini Kit (Qiagen, Valencia, California, USA). To detect F. necrophorum, we performed a PCR assay specific for F. necrophorum RNA polymerase beta subunit gene (rpoB) using the forward primer (TP1F 5′-TCTACGTATGCCTCACGGAT-3′) and the reverse primer (TP2R 5′-AGGAATATGAGGATGAGGAT-3′; Narongwanichgarn et al. 2003). Using PCR, we detected F. necrophorum in 37% of 70 pneumonic lung tissues, 40% of five nonpneumonic lung tissues, and 31% of 42 pharyngeal swabs.

There are two subspecies of F. necrophorum: subsp. necrophorum and subsp. funduliforme (Shinjo et al. 1991). Although both cause illnesses, subsp. necrophorum is considered more pathogenic than funduliforme (Lechtenberg et al. 1988). The difference in virulence has been attributed to differences in the amount of leukotoxin produced, and quantity and composition of lipopolysaccharide between the two subspecies (Scanlan et al. 1986). Subsp. necrophorum has been more frequently encountered in infections than subsp. funduliforme (Lechtenberg et al. 1988). To determine the subspecies of F. necrophorum, the 925 base-pair PCR amplicons from the lung tissue (n=26) and pharyngeal samples (n=13), were sequenced. The sequences of PCR amplicons were either identical to, or bearing a single base pair difference from, F. necrophorum subsp. funduliforme (GenBank accession AJSY01000008), but differing from subsp. necrophorum by 13 or 14 base pairs.

The presence of F. necrophorum in 37% of pneumonic BHS lungs raises the question as to whether it is a significant pathogen in BHS pneumonia. We previously tested these 70 samples for Pasteurellaceae species, particularly M. haemolytica (Shanthalingam et al. 2014). The PCR analysis in that study revealed that 77% of these samples carried M. haemolytica. Presence of F. necrophorum in only 37% of pneumonic lung tissues, 40% of healthy lung tissues, and 31% of pharyngeal swabs from healthy BHS suggests that this bacterium is not a major pathogen in BHS pneumonia. The detection of subsp. funduliforme in 100% of the F. necrophorum-positive samples tested is in agreement with previous observations that subsp. funduliforme occurs more frequently in mixed infections (Lechtenberg et al. 1988; Tan et al. 1996).

To detect the F. necrophorum leukotoxin gene in lung tissues and pharyngeal swab extracts, we performed a leukotoxin-specific PCR assay using the primers lktA-F 5′-AAATGGTGAAAGAATGACAA-3′ and lktA-R 5′-TGCATAATTTCCTACTCCTG-3′ (Tadepalli et al. 2008). All F. necrophorum-positive pharyngeal swab extracts (n=13) and normal lung tissues (n=2) had the leukotoxin gene as confirmed by sequencing of PCR amplicons. Sixty-nine percent (18/26) of F. necrophorum-containing pneumonic lung tissue samples were positive for the leukotoxin gene. Ludlam et al. (2009) reported that 92% of subsp. funduliforme isolates of bovine origin possessed the leukotoxin gene. Therefore, it is likely that most of the F. necrophorum isolates from BHS also have the leukotoxin gene.

Because F. necrophorum subsp. funduliforme was detected in BHS pneumonic lungs, it was of interest to us to determine the susceptibility of BHS leukocytes to the F. necrophorum leukotoxin. Leukotoxin was produced from an isolate of F. necrophorum subsp. funduliforme and M. haemolytica (Gentry and Srikumaran 1991; Narayanan et al. 2002). We isolated PMNs and peripheral blood mononuclear cells (PBMCs) from peripheral blood of BHS and domestic sheep by density gradient centrifugation and determined cytotoxicity of leukotoxin from F. necrophorum and M. haemolytica by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye-reduction cytotoxicity assay (Gentry and Srikumaran 1991). For analyzing cytotoxicity of F. necrophorum and M. haemolytica leukotoxin to BHS and domestic sheep PMNs and PBMCs, we used a repeated-measures analysis of variance. Results were considered statistically significant at P<0.05.

Bighorn sheep PMNs and PBMCs were susceptible to F. necrophorum leukotoxin-induced cytotoxicity in a dose-dependent manner. Percent cytotoxicity of BHS PMNs caused by F. necrophorum leukotoxin was significantly higher than that of BHS PBMCs at leukotoxin dilutions 1:8 to 1:64. As with M. haemolytica leukotoxin, F. necrophorum leukotoxin was twofold more cytotoxic to BHS PMNs than to PBMCs, as judged by leukotoxin concentrations causing 50% cytotoxicity to PMNs and PBMCs (Fig. 1). The plausible explanation for enhanced cytotoxicity of M. haemolytica leukotoxin to BHS PMNs is that CD18, the receptor for M. haemolytica leukotoxin (Deshpande et al. 2002), is expressed to a higher level on PMNs than on PBMCs. Differential susceptibility of BHS PMNs and PBMCs to F. necrophorum leukotoxin cannot be deciphered until the receptor for this leukotoxin on BHS leukocytes is identified. Similar to M. haemolytica leukotoxin (Silflow and Foreyt 1994), F. necrophorum leukotoxin was fourfold more cytotoxic to BHS PMNs than to domestic sheep PMNs, as judged by leukotoxin concentrations causing 50% cytotoxicity to the PMNs (Fig. 2). Identification of the leukocyte receptor for F. necrophorum leukotoxin will help elucidate the molecular basis for this differential susceptibility.

Various pulmonary lesions have been associated with F. necrophorum in domestic animals. Pulmonary lesions have also been reported in white-tailed deer (Odocoileus virginianus) and pronghorn (Antilocapra americana; Wobeser et al. 1975; Edwards et al. 2001). Lung is a richly oxygenated and a highly defended organ. Therefore, as an anaerobe, F. necrophorum must overcome high oxygen concentrations in the lungs, in addition to the phagocytic mechanisms to survive, proliferate, and produce its virulence factors. It is conceivable that the lysis of alveolar macrophages and PMNs by leukotoxin produced by M. haemolytica abrogates or mitigates the host defense mechanisms. More importantly, killing of PMNs could lead to parenchymal cell damage, abscess formation, and sequestration leading to an anaerobic environment in lungs. Fusobacterium necrophorum is likely to enter the lungs after M. haemolytica enters the lungs and initiates tissue damage, creating a microenvironment more conducive for growth of anaerobic bacteria. The leukotoxin of F. necrophorum would also help protect it from phagocytosis. In conclusion, although Fusobacterium leukotoxin can lyse BHS leukocytes, it is unlikely that this bacterium plays a direct role in the development of pneumonia. Further studies are necessary to definitively ascertain the role of F. necrophorum in BHS pneumonia.

This research was supported by funds from the Wild Sheep Foundation and its State Chapters, Nevada Bighorns Unlimited, Rocky Mountain Bighorn Society, and US Forest service.