Isolation of Pasteurella haemolytica from tonsillar biopsies of Rocky Mountain bighorn sheep

Isolations of Pasteurella haemolytica were compared from tonsillar biopsies versus nasal passages for 29 free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from central Idaho. Overall, P. haemolytica was isolated from 11 (38%) of 29 sheep. Two (18%) of the 11 positive samples were from only nasal passages compared to eight (73%) from tonsillar biopsies. Pasteurella haemolytica biotype T was isolated from tonsils of nine sheep and from nasal biopsies. Pasteurella haemolytica biotype T was isolated from tonsils of nine sheep and from nasal passages of only one sheep. Two sheep were positive for P. haemolytica biotype A from nasal passages. Culturing tonsillar biopsies as compared to nasal swab samples was a more reliable technique in detecting P. haemolytica, especially biotype T, in bighorn sheep.     

Experimental contact transmission of Pasteurella haemolytica from clinically normal domestic sheep causing pneumonia in Rocky Mountain bighorn sheep

Two Rocky Mountain bighorn lambs (Ovis canadensis canadensis) were held in captivity for 120 days before being housed with two domestic sheep. The lambs were clinically normal and had no Pasteurella spp. on nasal swab cultures. The domestic sheep were known to carry Pasteurella haemolytica biotype A in the nasal passages. After being in close contact for 19 days. P. haemolytica biotype A was cultured from nasal swabs of one of the bighorn lambs. By 26 days, both bighorn sheep developed coughs, were anorectic and became lethargic and nasal swabs yielded P. haemolytica biotype T, serotype 10. Twenty-nine days after contact, the lambs were necropsied and found to have extensive fibrinous bronchopneumonia. From affected tissues pure cultures of beta-hemolytic P. haemolytica biotype T, serotype 10 were grown. Both domestic sheep remained clinically normal and had no gross or microscopic lesions, but they carried the same P. haemolytica serotype in their tonsils. Behavioural observations gave no indication of stress in the bighorn lambs.     

Immunologic responses of domestic and bighorn sheep to a multivalent Pasteurella haemolytica vaccine

The efficacy of a Pasteurella haemolytica vaccine (serotypes A1, A2, and T10) to induce humoral antibodies and alter colonization of the upper respiratory tract by related P. haemolytica spp. strains was evaluated in 10 bighorn (Ovis canadensis canadensis) and 10 domestic (Ovis aries) sheep. Sheep of each species were divided into five pairs based on age and history of respiratory disease. One sheep in each pair was vaccinated twice 2 wk apart with 2 ml of vaccine (VAC group) and the remaining animals (NV group) were injected with 2 ml of sterile saline. Mild, transient lameness was the only observed adverse effect. Blood sera from the sheep were tested for agglutinating antibodies against whole cells of A1, A2, and T10 and for leukotoxin neutralizing antibodies. Antibody titers were expressed as the reciprocal log2 of the highest reactive dilutions. Domestic sheep > 1-yr-old and two bighorn sheep with a history of A1 infection had higher titers throughout the study against A1 cells than domestic sheep < 1-yr-old and bighorns without a history of A1 infection. Both domestic and bighorn sheep had log2 titers of 8 to 12 against A2 cells and 6 to 12 against T10 cells during this time. Bighorn sheep in the VAC group had 2 to 32 fold titer increases for A1 cells by 2 wk post-vaccination (PV) compared to 0 to 2 fold increases in VAC domestic sheep. Two to 16 and 0 to 8 fold increases in antibodies titers to A2 and T10 cells, respectively, were detected in sera of both VAC groups. Sera of bighorn sheep with a history of respiratory disease and all domestic sheep had log2 leukotoxin neutralizing antibody titers of 4 to 14 in contrast to < or = 2 in sera of bighorn sheep without a history of respiratory disease. Neutralizing antibody titers of two bighorns without a history of respiratory disease in the VAC group increased from log2 0 to 5 in one and from 0 to 9 in the other 2 wk PV. Antibody increases in these animals were no longer evident at 16 wk PV while titers of animals with histories of disease remained relatively stable. The types and numbers of Pasteurella spp. isolated from nasal and pharyngeal swabs varied throughout the study without conclusive evidence of suppression of colonization. Although the animals were not experimentally challenged to determine the efficacy of the vaccine, one VAC and one NV bighorn sheep died following introduction of an A2 P. haemolytica strain when leukotoxin neutralizing antibodies had returned to pre-vaccination levels. This vaccine appeared to be safe for use in bighorn sheep and stimulated moderate but transient increases in antibody levels which should provide some protection against naturally occurring disease. A vaccine which would induce production of high and maintained antibodies against multiple strains of P. haemolytica would be valuable for use in bighorn sheep maintained in captivity or when captured for relocation.     

Using ribosomal RNA gene restriction patterns in distinguishing isolates of Pasteurella haemolytica from bighorn sheep (Ovis canadensis).

Pasteurella haemolytica isolates (n = 31) from two isolated captive herds of Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were characterized and compared phenotypically (biotype, serotype, hemolytic activity) and by a genomic fingerprinting method known as ribotyping. Seven to nine distinct phenotypes were observed. Depending on the method used for serotyping, one to three phenotypes were common to both herds. Eighteen isolates, recovered from both herds, were non-hemolytic, biotype T, indirect hemagglutination assay serotype 4. Ribotyping, a method for highlighting genetically conserved deoxyribonucleic acid restriction site heterogeneity with a 32P-labelled Escherichia coli ribosomal ribonucleic acid probe, produced six to eight distinct ribotype pattern groups within the 31 P. haemolytica isolates, depending on the restriction enzyme used. In contrast to phenotypes, ribotypes appeared unique to each herd, and ribotyping helped to further differentiate some isolates of the same biotype and serotype. In addition, ribotyping provided an alternative means for evaluating relationships between isolates differing in hemolytic activity but which were otherwise phenotypically identical. We propose that ribotyping may be a useful adjunct to other bacterial characterization methods in studying the epizootiology of pasteurellosis in bighorn sheep.     

Detecting nonhemolytic Pasteurella haemolytica infections in healthy Rocky Mountain bighorn sheep (Ovis canadensis canadensis): influences of sample site and handling

Effects of sampling procedures on ability to culture Pasteurella spp. from Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were examined experimentally. Sample site influenced (P less than 0.0001) recovery of P. haemolytica in adult bighorn sheep. We isolated nonhemolytic P. haemolytica from 18 of 19 tonsillar swabs and 18 of 19 tonsillar biopsies from adult sheep, yet only four of 19 nasal swabs yielded isolates. Sample handling also affected (P less than 0.0001) recovery of P. haemolytica. Nonhemolytic P. haemolytica was cultured from 14 of 19 tonsillar swabs plated directly onto blood agar, but from only two of 19 swabs stored for 24 hr in modified Stuart's medium. We detected nonhemolytic P. haemolytica at least once in bronchial aspirates from four and in nasal swabs from three of six bighorn lambs. Based on direct cultures of tonsillar swabs and/or biopsies, all 26 bighorn sheep (seven lambs, 19 adults) sampled were infected with nonhemolytic P. haemolytica; only two lambs developed pneumonia during the study period. Thirty-four of 37 nonhemolytic P. haemolytica isolates tested were biotype T; three were biotype A. Serotypes 3; 4; 3, 4 and 3, 4, 10 were identified in a subsample of 17 isolates. Our data suggest tonsillar swabs or biopsies plated directly onto blood agar and incubated immediately offer the greatest probability of recovering nonhemolytic P. haemolytica from health bighorn sheep.     

Characterization of Pasteurella multocida associated with pneumonia in bighorn sheep

Pasteurella multocida is a highly diverse group of bacteria recognized as important pathogens. Although P. multocida is not ordinarily associated with disease in Rocky Mountain bighorn sheep (Ovis canadensis canadensis), numerous isolates were cultured in high numbers from free-ranging bighorn sheep in the Hells Canyon area of Idaho, Washington, and Oregon (USA) during the winter of 1995-96. Animals captured in Hells Canyon and held in captivity, and their offspring, also harbored P. multocida. Biochemical utilization tests on 90 isolates identified three subspecies: P. multocida multocida a (n = 54); P. multocida multocida b (n = 13); and P. multocida gallicida (n = 15); and a non-speciated biotype, U6 (n = 8). Genomic DNA digestion with restriction endonuclease Hha I separated the isolates into 62 unique restriction fragment length polymorphism profiles. Capsular type A was predominant (72% of isolates). Only one isolate type, which may have been transmitted from a feral goat, was capsular type D, possessed the structural gene, toxA, for dermonecrotoxin detected by polymerase chain reaction, and produced toxin as determined by monoclonal antibody immunoblot. In conclusion, bighorn sheep in this study carried diverse types of generally non-toxigenic P. multocida associated with epizootic pneumonia.     

Effects of delivery method on serological responses of bighorn sheep to a multivalent Pasteurella haemolytica supernatant vaccine

The safety and efficacy of a remotely delivered multivalent Pasteurella haemolytica supernatant vaccine (serotypes A2 and T10) were examined in captive Rocky, Mountain bighorn sheep (Ovis canadensis canadensis). Twenty bighorn sheep were grouped according to baseline leukotoxin neutralizing antibody titers (< or =2 or >2 log2(-1)) and vaccination history (previously vaccinated or unvaccinated). Within these groups, animals were randomly assigned to one of two delivery treatments: hand injection (control) or biobullet implantation. All bighorns received a single dose from the same lot of vaccine (n = 10/treatment); four additional animals were injected intramuscularly with 0.9% saline as unvaccinated sentinels. Mild, transient lameness one day after hand injection or biobullet implantation was the only adverse effect. Serum neutralizing antibody titers to P. haemolytica leukotoxin differed between delivery treatments (P = 0.009) and among baseline titer/vaccination history groups (P = 0.013). Neutralizing titers were higher among hand-injected bighorns. Although neutralizing titers were lower among implanted bighorns than hand-injected controls at 1 wk (P = 0.002) and 2 wk (P = 0.021) after vaccination, seroconversion rates in response to implantation (6/10) and hand injection (9/10) did not differ (P = 0.303). Agglutinating antibody titers to T10 were high and did not vary over time or between delivery treatments. Agglutinating antibody titers to A2 in the hand-injected controls were not different (P > or = 0.07) than those in bighorns vaccinated with biobullet implantation. These data demonstrate that although hand injection elicits higher absolute titers, biobullet implantation may also stimulate effective antibody responses to P. haemolytica supernatant vaccine. Further evaluation of biobullet vaccination against pneumonic pasteurellosis in free-ranging populations of wild bighorn sheep is warranted.     

Distinct plasmid profiles of Pasteurella haemolytica serotypes and the characterization and amplification in Escherichia coli of ampicillin-resistance plasmids encoding ROB-1 beta-lactamase.

Thirty-five isolates of Pasteurella haemolytica from cattle or sheep were screened for the presence of plasmids and for resistance to a range of antibiotics. Eight strains (four of serotype A1, three of serotype A2 and one untypable) contained plasmid DNA and isolates of the same serotype had similar plasmid profiles, which were different from those of the other serotypes. All but one of the plasmid-bearing strains were isolated from pneumonic animals or from animals in contact with pneumonic cattle or sheep. In A2 and untypable strains, there was no obvious correlation between antibiotic resistance and the presence of a specific plasmid. In contrast, all plasmid-bearing A1 strains exhibited ampicillin resistance (ApR), which was shown by transfer studies to be plasmid-mediated. Plasmid DNA prepared from E. coli transformants was not routinely detected on ethidium-bromide-stained agarose gels, but could be amplified to detectable levels by treatment of cultures with chloramphenicol (Cm) or by modifying the growth conditions. The ApR plasmids from P. haemolytica were identical by restriction enzyme analysis. Restriction analysis and hybridization data indicated that these plasmids were closely related to the prototype ROB-1 beta-lactamase-encoding plasmid, originally isolated from Haemophilus influenzae. From substrate profiles and isoelectric focusing data, the beta-lactamases encoded by the P. haemolytica plasmids were indistinguishable from the ROB-1 beta-lactamase.     

Infectious keratoconjunctivitis in bighorn sheep, Silver Bell Mountains, Arizona, USA

An infectious keratoconjunctivitis (IKC) epizootic in bighorn sheep (Ovis canadensis) occurred in the Silver Bell Mountains, Arizona, USA, from 1 December 2003 to 31 March 2004. We used standard culture methods and polymerase chain reaction (PCR) amplification of the 16S rRNA gene to test for the causative agents of IKC and other diseases reported to be associated with bighorn sheep populations. All bighorn sheep and domestic goat test results were negative except for Mycoplasma spp. and Branhamella spp. The culture and PCR results differed. Conjunctival swabs from four of 19 IKC-affected bighorn sheep tested by culture were positive for Mycoplasma spp., whereas 22 of 22 bighorn sheep samples tested by PCR were positive for Mycoplasma spp. None of 13 domestic goats tested positive by culture for Mycoplasma spp., whereas five of 16 tested positive by PCR. Three of 16 domestic goats and seven of 24 IKC-affected bighorn sheep tested positive for Branhamella spp. by culture. Bighorn sheep began showing clinical signs of IKC between 21 and 28 days following initial detection of domestic goats in bighorn sheep habitat. The IKC epizootic lasted 122 days, and individual bighorn sheep were blind for an average of 38.4 days. Given the clear potential for disease transmission to bighorn sheep, we recommend that land managers not allow the pasturing of domestic goats near occupied bighorn sheep habitat.     

Epidemic pasteurellosis in a bighorn sheep population coinciding with the appearance of a domestic sheep

A pneumonia epidemic reduced bighorn sheep (Ovis canadensis) survival and recruitment during 1997-2000 in a population comprised of three interconnected wintering herds (Kenosha Mountains, Sugarloaf Mountain, Twin Eagles) that inhabited the Kenosha and Tarryall Mountain ranges in central Colorado, USA. The onset of this epidemic coincided temporally and spatially with the appearance of a single domestic sheep (Ovis aires) on the Sugarloaf Mountain herd's winter range in December 1997. Although only bighorns in the Sugarloaf Mountain herd were affected in 1997-98, cases also occurred during 1998-99 in the other two wintering herds, likely after the epidemic spread via established seasonal movements of male bighorns. In all, we located 86 bighorn carcasses during 1997-2000. Three species of Pasteurella were isolated in various combinations from affected lung tissues from 20 bighorn carcasses where tissues were available and suitable for diagnostic evaluation; with one exception, beta-hemolytic mannheimia (Pasteurella) haemolytica (primarily reported as biogroup 1(G) or 1(alphaG)) was isolated from lung tissues of cases evaluated during winter 1997-98. The epidemic dramatically lowered adult bighorn monthly survival in all three herds; a model that included an acute epidemic effect, differing between sexes and with vaccination status, that diminished linearly over the next 12 mo best represented field data. In addition to the direct mortality associated with epidemics in these three herds, lamb recruitment in years following the pneumonia epidemic also was depressed as compared to years prior to the epidemic. Based on observations presented here, pasteurellosis epidemics in free-ranging bighorn sheep can arise through incursion of domestic sheep onto native ranges, and thus minimizing contact between domestic and bighorn sheep appears to be a logical principle for bighorn sheep conservation.     

A bighorn sheep die-off in southern Colorado involving a Pasteurellaceae strain that may have originated from syntopic cattle

We investigated a pasteurellosis epizootic in free-ranging bighorn sheep (Ovis canadensis) wherein a Pasteurellaceae strain carried by syntopic cattle (Bos taurus) under severe winter conditions appeared to contribute to pneumonia in affected bighorns. Twenty-one moribund or dead bighorn sheep were found on the "Fossil Ridge" herd's winter range, Colorado, USA, between 13 December 2007 and 29 February 2008. Eight carcasses examined showed gross or microscopic evidence of acute to subacute fibrinous bronchopneumonia. All eight carcasses yielded at least one β-hemolytic Mannheimia haemolytica biogroup 1(±(G)) strain, and seven also yielded a β-hemolytic Bibersteinia trehalosi biogroup 4 (CDS) strain; evidence of Pasteurella multocida, Mycoplasma ovipneumoniae, and parainfluenza 3 and bovine respiratory syncytial viruses was also detected. Isolates of β-hemolytic Manneimia haemolytica biogroup 1(G) from a bighorn carcass and a syntopic cow showed 99.5% similarity in genetic fingerprints; B. trehalosi biogroup 4(CDS) isolates were ≥94.9% similar to an isolate from a nearby bighorn herd. Field and laboratory observations suggested that pneumonia in affected bighorns may have been caused by a combination of pathogens including two pathogenic Pasteurellaceae strains--one likely of cattle origin and one likely of bighorn origin--with infections in some cases perhaps exacerbated by other respiratory pathogens and severe weather conditions. Our and others' findings suggest that intimate interactions between wild sheep and cattle should be discouraged as part of a comprehensive approach to health management and conservation of North American wild sheep species.     

Mannheimia (Pasteurella) haemolytica leukotoxin utilizes CD18 as its receptor on bighorn sheep leukocytes

Pneumonia caused by Mannheimia (Pasteurella) haemolytica is a highly fatal disease of bighorn sheep (Ovis canadensis). Leukotoxin (Lkt), secreted by M. haemolytica, is an important virulence factor of this organism, and is cytolytic to bighorn sheep leukocytes. Previously, we have shown that CD18, the beta subunit of beta2 integrins, serves as the receptor for Lkt on bovine leukocytes. Furthermore, anti-CD18 antibodies inhibit Lkt-induced cytotoxicity of bighorn sheep leukocytes. Therefore, we hypothesized that Lkt utilizes CD18 as its receptor on bighorn sheep leukocytes. Confirmation of bighorn sheep CD18 as a receptor for Lkt requires the demonstration that the recombinant expression of bighorn sheep CD18 in Lkt-nonsusceptible cells renders them susceptible to Lkt. Therefore, we transfected cDNA encoding CD18 of bighorn sheep into a Lkt-nonsusceptible murine cell line. Cell surface expression of bighorn sheep CD18 on the transfectants was tested by flow cytometry with anti-CD18 antibodies. Transfectants stably expressing bighorn sheep CD18 on their surface were subjected to flow cytometric analysis for detection of Lkt binding, and cytotoxicity assays for detection of Lkt-induced cytotoxicity. Leukotoxin bound to the transfectants. More importantly, the transfectants were effectively lysed by Lkt in a concentration-dependent manner, whereas the parent cells were not. These results clearly indicate that M. haemolytica Lkt utilizes CD18 as a receptor on bighorn sheep leukocytes. Identification of CD18 as a receptor for Lkt on bighorn sheep leukocytes should enhance our understanding of the pathogenesis of pneumonia, which in turn should help in the development of control measures against this fatal disease of bighorn sheep.     

Sequence diversity and molecular evolution of the leukotoxin (lktA) gene in bovine and ovine strains of Mannheimia (Pasteurella) haemolytica

The molecular evolution of the leukotoxin structural gene (lktA) of Mannheimia (Pasteurella) haemolytica was investigated by nucleotide sequence comparison of lktA in 31 bovine and ovine strains representing the various evolutionary lineages and serotypes of the species. Eight major allelic variants (1.4 to 15.7% nucleotide divergence) were identified; these have mosaic structures of varying degrees of complexity reflecting a history of horizontal gene transfer and extensive intragenic recombination. The presence of identical alleles in strains of different genetic backgrounds suggests that assortative (entire gene) recombination has also contributed to strain diversification in M. haemolytica. Five allelic variants occur only in ovine strains and consist of recombinant segments derived from as many as four different sources. Four of these alleles consist of DNA (52.8 to 96.7%) derived from the lktA gene of the two related species Mannheimia glucosida and Pasteurella trehalosi, and four contain recombinant segments derived from an allele that is associated exclusively with bovine or bovine-like serotype A2 strains. The two major lineages of ovine serotype A2 strains possess lktA alleles that have very different evolutionary histories and encode divergent leukotoxins (5.3% amino acid divergence), but both contain segments derived from the bovine allele. Homologous segments of donor and recipient alleles are identical or nearly identical, indicating that the recombination events are relatively recent and probably postdate the domestication of cattle and sheep. Our findings suggest that host switching of bovine strains from cattle to sheep, together with inter- and intraspecies recombinational exchanges, has played an important role in generating leukotoxin diversity in ovine strains. In contrast, there is limited allelic diversity of lktA in bovine strains, suggesting that transmission of strains from sheep to cattle has been less important in leukotoxin evolution.     

Paratuberculosis (Johne's disease) in bighorn sheep and a Rocky Mountain goat in Colorado.

Between May, 1972 and February, 1978, six cases of paratuberculosis (Johne's Disease) caused by Mycobacterium paratuberculosis were diagnosed in free-ranging Rocky Mountain bighorn sheep (Ovis canadensis) and one Rocky Mountain goat (Oreamnos americanus) on or near Mt. Evans in Colorado. Diagnosis of paratuberculosis was based on gross and histopathologic examination of the animals and by isolation of M. paratuberculosis from three sheep and the goat. The clinical signs and pathologic changes seen in the bighorn sheep resembled those described in cattle, while the lesions in the goat were similar to those described for domestic sheep and goats.     

Mosaic Structure and Molecular Evolution of the Leukotoxin Operon (lktCABD) in Mannheimia (Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi

The mosaic structure and molecular evolution of the leukotoxin operon (lktCABD) was investigated by nucleotide sequence comparison of the lktC, lktB, and lktD genes in 23 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains. Sequence variation in the lktA gene has been described previously (R. L. Davies et al., J. Bacteriol. 183:1394-1404, 2001). The leukotoxin operon of M. haemolytica has a complex mosaic structure and has been derived by extensive inter- and intraspecies horizontal DNA transfer and intragenic recombination events. However, the pattern of recombination varies throughout the operon and among the different evolutionary lineages of M. haemolytica. The lktA and lktB genes have the most complex mosaic structures with segments derived from up to four different sources, including M. glucosida and P. trehalosi. In contrast, the lktD gene is highly conserved in M. haemolytica. The lktC, lktA, and lktB genes of strains representing the major ovine lineages contain recombinant segments derived from bovine or bovine-like serotype A2 strains. These findings support the previous conclusion that host switching of bovine A2 strains from cattle to sheep has played a major role in the evolution of the leukotoxin operon in ovine strains of M. haemolytica. Homologous segments of donor and recipient alleles are identical, or nearly identical, indicating that the recombinational exchanges occurred relatively recent in evolutionary terms. The 5' and 3' ends of the operon are highly conserved in M. haemolytica, which suggests that multiple horizontal exchanges of the complete operon have occurred by a common mechanism such as transduction. Although the lktA and lktB genes both have complex mosaic structures and high nucleotide substitution rates, the amino acid diversity of LktB is significantly lower than that of LktA due to a higher degree of evolutionary constraint against amino acid replacement. The recombinational exchanges within the leukotoxin operon have had greatest effect on LktA and probably provide an adaptive advantage against the host antibody response by generating novel antigenic variation at surface-exposed sites.     

Comparative aspects of biochemical polymorphism in the blood of Caprinae species and their hybrids

Biochemical variation at 14 blood loci was reviewed, and specific features compared experimentally in sheep Ovis aries, mouflon Ovis musimon, goat Capra hircus, aoudad Ammotragus lervia and in 2 stillborn aoudad × goat hybrids. Variation at 3 loci was also studied in dall sheep Ovis dalli, bighorn sheep Ovis canadensis and rocky mountain goat Oreamnos americanus. Haemoglobin C production in an anaemic Hb AB mouflon and in mouflon × sheep hybrids was examined. Mouflon differ from domestic sheep in that synthesis of both Hb β^AHb β^Bchains is switched off during Hb C production. The mouflon × sheep hybrids switched off one or both chains depending on whether they had inherited sheep or mouflon Hb β chain genes. In general aoudad showed a closer affinity to goats than to sheep.     

Proximity-Dependent Inhibition of Growth of Mannheimia haemolytica by Pasteurella multocida

Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia trehalosi have been identified in the lungs of pneumonic bighorn sheep (BHS; Ovis canadensis). Of these pathogens, M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under experimental conditions. However, M. haemolytica has been isolated by culture less frequently than the other bacteria. We hypothesized that the growth of M. haemolytica is inhibited by other bacteria in the lungs of BHS. The objective of this study was to determine whether P. multocida inhibits the growth of M. haemolytica. Although in monoculture both bacteria exhibited similar growth characteristics, in coculture with P. multocida there was a clear inhibition of growth of M. haemolytica. The inhibition was detected at mid-log phase and continued through the stationary phase. When cultured in the same medium, the growth of M. haemolytica was 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 of P. multocida or from P. multocida-M. haemolytica cocultures. These results indicate that P. multocida inhibits the growth of M. haemolytica by a contact- or proximity-dependent mechanism. If the inhibition of growth of M. haemolytica by P. multocida occurs in vivo as well, it could explain the inconsistent isolation of M. haemolytica from the lungs of pneumonic BHS.     

Hemorrhagic disease in bighorn sheep in Arizona

Two bighorn sheep from Arizona (USA) were submitted for necropsy. One was a Rocky Mountain bighorn (Ovis canadensis canadensis) and the other was a desert bighorn (Ovis canadensis mexicana). Both had lesions consistent with those of hemorrhagic disease (HD). Epizootic hemorrhagic disease virus (EHDV) type-2 and bluetongue virus (BTV) type-17, respectively, were isolated from the sheep tissues. To our knowledge, HD caused by either EHDV or BTV infection has not been documented previously in Arizona bighorn sheep.     

Occurrence, diagnosis, and strain typing of Mycobacterium avium subspecies paratuberculosis infection in Rocky Mountain bighorn sheep (Ovis canadensis canadensis) in southwestern Alberta

The role that wildlife may play in the transmission of Mycobacterium avium subspecies paratuberculosis (Map), the causative agent of Johne's disease (JD), and the potential consequences of infection in these populations are being given increasing consideration. A yearling male Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from southwestern Alberta, Canada, was found infected with Map in August 2009. Clinical signs of emaciation and diarrhea and histologic findings of diffuse granulomatous enteritis of the distal ileum, lymphadenitis of the mesenteric lymph nodes, and lymphangitis of the ileum were similar to previously described cases of JD in bighorn sheep. Infection with Map was confirmed by bacterial isolation through fecal culture, acid-fast staining, and polymerase chain reaction (PCR) of IS900. The Map1506 gene was sequenced, and the isolate was identified as a Cattle (Type II) strain. In a follow-up herd-level survey, three of 44 fecal samples (7%) from individual bighorn sheep from the same herd as the index case were PCR-positive and identified as Type II Map strains. Twenty-five samples from a distant bighorn population were negative. Additional strain typing of the isolates from the index case and the positive fecal samples was done by sequencing three discriminatory short sequence repeat (SSR) regions. All four SSR profiles differed from one another, suggesting multiple introductions or a long-existing circulation of Map within this bighorn population. Detailed molecular analyses are essential for understanding and managing diseases at the wildlife-livestock interface.     

Restoration Potential of Bighorn Sheep in a Prairie Region

Efforts to recover Rocky Mountain bighorn sheep (Ovis canadensis canadensis) throughout western North America have had limited success with the majority of current populations remaining in small and isolated areas on a fraction of their historical range. Prairie environments with rugged topography throughout the Northern Great Plains ecoregion were historically occupied by relatively robust bighorn sheep populations. We predicted there is likely unrealized potential habitat for restoring bighorn sheep to these areas; however, relatively little attention has been devoted to identifying habitat in unoccupied prairie regions. We used global positioning system (GPS)-collar data collected from 43 female bighorn sheep in 2 populations located in the eastern Montana, USA, portion of the Northern Great Plains during 2014–2018 to estimate a population-level annual resource selection model and identify the important factors affecting bighorn sheep resource selection. We extrapolated model predictions across eastern Montana's prairie region and identified potential habitat to understand restoration potential and assist with future translocations of bighorn sheep. Resource selection of bighorn sheep was most strongly associated with terrain slope and ruggedness, tree canopy cover, and a normalized difference vegetation index metric. Within currently unoccupied areas of the historical range, the model extrapolation predicted 7,211 km² of habitat, with most owned and managed by private landowners (44%), Bureau of Land Management (33%), and the United States Fish and Wildlife Service (15%). Our results provide an empirical evaluation of landscape covariates influencing resource selection of bighorn sheep occupying prairie environments and provide a habitat model that may be generalizable to other areas in the Northern Great Plains ecoregion. We demonstrate substantial potential for restoration opportunities of bighorn sheep in the Northern Great Plains ecoregion. Broad restoration of bighorn sheep across the ecoregion would likely require strong collaboration among and between public resource managers, private landowners, and livestock producers given the heterogeneous land ownership patterns, management strategies, and domestic sheep distributions. © 2020 The Wildlife Society.