In bighorn sheep (Ovis canadensis), pasteurellosis is associated with respiratory disease, death, and decreased fecundity that can limit sheep populations.1–5 Recent disease outbreaks in bighorn sheep during the winter of 2009 to 2010 in several western states and questions regarding whether domestic sheep (Ovis aries) are reservoirs for agents that cause these outbreaks are compelling reasons for the identification of Pasteurellaceae responsible for bighorn sheep deaths.6–9 Pasteurellaceae associated with disease epizootics in bighorn sheep include Mannheimia haemolytica (formerly Pasteurella haemolytica),10 Bibersteinia trehalosi (formerly Pasteurella trehalosi),11,12 and Pasteurella multocida.2,5,13–15 Isolation of pure cultures of Pasteurellaceae from multiple organs of bighorn sheep that died with evidence of respiratory disease16 as well as death of bighorn sheep following experimental inoculation with Pasteurellaceae17 suggests that these bacteria are primary pathogens of bighorn sheep. In a study18 that was conducted under controlled conditions, Pasteurellaceae were transmitted from clinically normal domestic sheep to bighorn sheep, which then developed respiratory disease. Therefore, it is necessary to identify Pasteurellaceae responsible for respiratory disease in free-ranging bighorn sheep.
Several methods of classifying Pasteurella and Mannheimia spp of Pasteurellaceae exist, such as the use of capsular serogroups, capsular antigens, and serotypes.12,19 Because many isolates from bighorn sheep cannot be characterized by use of these conventional methods, a hierarchic biovariant classification scheme based on 23 microbiological characteristics and biochemical utilization tests of isolates in culture was established.20,21 This system distinguishes a large number of Pasteurellaceae strains, which is useful for epidemiological studies. Limited data are available regarding Pasteurella or Mannheimia biovariants of bighorn sheep with respiratory disease.5,13,21–23 Consequently, the purpose of the study reported here was to identify Pasteurellaceae biovariants isolated from bighorn sheep that were clinically normal or that had evidence of respiratory disease.
Materials and Methods
Bacterial isolates cultured from antemortem oropharyngeal or nasal swab specimens obtained from free-ranging bighorn sheep submitted to the Caine Veterinary Teaching Center were included in the present study. However, isolates from a 1995–1996 respiratory disease outbreak in bighorn sheep in Hells Canyon that were previously reported13,23,24 were not included in the study reported here. Swab specimens were collected as a part of routine management and disease investigation activities, which were in compliance with institutional guidelines for research on animals for the agencies that submitted the specimens. Each swab specimen yielded zero to multiple bacterial isolates. Isolates included in the present study were from submissions that included information on the date of submission, geographic location, health classification (without clinical abnormalities [ie, healthy] or with signs of respiratory disease [ie, diseased]), sex, and age class (adult [≥ 1 year old] or lambs [< 1 year old]). Isolates were obtained from 290 bighorn sheep; 45 sheep had signs of respiratory disease, and 245 had no clinical abnormalities.
Bacterial culture and classification—Swab specimens were submitted to the Caine Veterinary Teaching Center on cold packs and inoculated by use of previously described methods22 onto selective and nonselective Columbia blood agara within 72 hours after collection. Following incubation, representatives of each colony type that were consistent with known Pasteurellaceae were propagated onto fresh Columbia blood agar for species and biovariant classification by use of a modification of a biochemical testing system developed for identification of isolates from domestic animals that was adapted for identification of isolates from wildlife.20,21 In addition, the ability of isolates to cause zones of β-hemolysis on 5% sheep blood agar was recorded.
Statistical analysis—Statistical analysis was conducted by use of statistical software,b and values of P < 0.05 were considered significant for all analyses. Histophilus somni isolates (n = 2) were not included in the analyses. Descriptive analyses were conducted, and ORs were calculated for 2 × 2 tables. The association between the host sheep's health classification (healthy or diseased) and whether an isolate was β-hemolytic was evaluated for each biovariant via χ2 analysis. Because χ2 analysis assumes independence for data, the reported significance and CIs are approximate values.
Results
Six hundred seventy-five Pasteurellaceae isolates were obtained from 290 bighorn sheep from Idaho (n = 620 isolates), Oregon (45), and Wyoming (10). Isolates (n = 675) were identified from 4 species of Pasteurellaceae: H somni (2), M haemolytica (219), P multocida (33), and B trehalosi (421; Tables 1 and 2). Among the latter 3 Pasteurellaceae species, 112 subspecies, biotypes, and biovariants were identified. Bibersteinia trehalosi 2 (n = 229 [34%]) was the biovariant most frequently identified. The majority (618/675 [92%]) of isolates were obtained from adult sheep, of which 597 (97%) isolates were from sheep without apparent clinical abnormalities. In contrast, 57 isolates were obtained from lambs, of which 47 (82%) were from lambs with signs of respiratory disease. A total of 68 (10%) isolates were obtained from bighorn sheep (adults and lambs) with evidence of respiratory disease; identified isolates included H somni and 21 subspecies, biotypes, and biovariants of P multocida, B trehalosi, and M haemolytica. Biovariants, except for M haemolytica 10αB (n = 3 isolates), isolated from lambs were more often associated with signs of respiratory disease. All biovariants, except for 3 (M haemolytica 9B, B trehalosi 2BG, and B trehalosi 2CDS, which accounted for 4 isolates), isolated from diseased lambs were also isolated from apparently healthy adult sheep.
Number of Manheimia haemolytica isolates by biovariant taxonomic status identified from nasal and oropharyngeal swab specimens obtained from 290 free-ranging bighorn sheep (Ovis canadensis) in 2 age classifications (adults, ≥ 1 year old; lambs, < 1 year old) that were clinically normal (healthy) or had evidence of respiratory disease (diseased).
| M haemolytica biovariant | Adult sheep | Lambs | Total | ||
|---|---|---|---|---|---|
| Diseased | Healthy | Diseased | Healthy | ||
| 1 | 0 | 8 | 1 | 0 | 9 |
| 1α | 0 | 5 | 0 | 0 | 5 |
| 1αB | 0 | 8 | 0 | 0 | 8 |
| 1αBG | 0 | 1 | 0 | 0 | 1 |
| 1αE | 0 | 2 | 0 | 0 | 2 |
| 1E | 0 | 3 | 0 | 0 | 3 |
| 10 | 0 | 5 | 0 | 0 | 5 |
| 10α | 1 | 8 | 1 | 0 | 10 |
| 10αB | 0 | 3 | 1 | 2 | 6 |
| 10αBE | 0 | 1 | 0 | 0 | 1 |
| 10αBS | 0 | 1 | 0 | 0 | 1 |
| 10αC | 0 | 1 | 0 | 0 | 1 |
| 10αE | 0 | 2 | 0 | 0 | 2 |
| 10αβ | 0 | 1 | 0 | 0 | 1 |
| 10B | 0 | 3 | 0 | 0 | 3 |
| 10BES | 0 | 1 | 0 | 0 | 1 |
| 10βB | 0 | 1 | 0 | 0 | 1 |
| 10E | 0 | 3 | 0 | 0 | 3 |
| 11 | 0 | 5 | 0 | 0 | 5 |
| 11α | 0 | 2 | 0 | 0 | 2 |
| 11αβG | 0 | 1 | 0 | 0 | 1 |
| 11αGX | 0 | 1 | 0 | 0 | 1 |
| 11αβ | 0 | 1 | 0 | 0 | 1 |
| 16α | 0 | 1 | 0 | 0 | 1 |
| 16αB | 0 | 4 | 0 | 0 | 4 |
| 16αE | 1 | 0 | 0 | 0 | 1 |
| 16B | 0 | 1 | 0 | 0 | 1 |
| 3 | 0 | 8 | 4 | 1 | 13 |
| 3α | 0 | 7 | 0 | 0 | 7 |
| 3αB | 0 | 3 | 0 | 0 | 3 |
| 3αBE | 0 | 3 | 0 | 0 | 3 |
| 3αBEX | 0 | 1 | 0 | 0 | 1 |
| 3αC | 0 | 2 | 0 | 0 | 2 |
| 3αCD | 0 | 1 | 0 | 0 | 1 |
| 3αE | 0 | 2 | 0 | 0 | 2 |
| 3αES | 0 | 1 | 0 | 0 | 1 |
| 3αG | 0 | 1 | 0 | 0 | 1 |
| 3B | 0 | 1 | 0 | 0 | 1 |
| 3BCX | 0 | 1 | 0 | 0 | 1 |
| 3BE | 1 | 1 | 0 | 0 | 2 |
| 3BEX | 0 | 2 | 0 | 0 | 2 |
| 3BX | 0 | 1 | 0 | 0 | 1 |
| 3CDE | 0 | 3 | 0 | 0 | 3 |
| 3E | 0 | 3 | 0 | 0 | 3 |
| 5 | 0 | 18 | 0 | 0 | 18 |
| 5αB | 0 | 1 | 0 | 0 | 1 |
| 5B | 0 | 1 | 0 | 0 | 1 |
| 6 | 0 | 2 | 0 | 0 | 2 |
| 6α | 0 | 1 | 0 | 0 | 1 |
| 6R | 0 | 1 | 0 | 0 | 1 |
| 6RX | 0 | 1 | 0 | 0 | 1 |
| 7 | 0 | 1 | 1 | 0 | 2 |
| 7B | 0 | 1 | 0 | 0 | 1 |
| 7BX | 0 | 1 | 0 | 0 | 1 |
| 8 | 0 | 5 | 0 | 0 | 5 |
| 8β | 0 | 2 | 0 | 0 | 2 |
| 9αβB | 0 | 3 | 1 | 0 | 4 |
| 9αBR | 0 | 3 | 0 | 0 | 3 |
| 9αBRX | 0 | 1 | 0 | 0 | 1 |
| 9αβ | 0 | 2 | 1 | 0 | 3 |
| 9αβR | 0 | 9 | 0 | 0 | 9 |
| 9αR | 0 | 1 | 0 | 0 | 1 |
| 9B | 0 | 0 | 1 | 0 | 1 |
| 9βR | 0 | 1 | 0 | 0 | 1 |
| U | 0 | 1 | 0 | 0 | 1 |
| Uα | 0 | 2 | 0 | 0 | 2 |
| UβBEX | 0 | 3 | 1 | 0 | 4 |
| UαB | 0 | 2 | 0 | 0 | 2 |
| UαβBC | 0 | 2 | 0 | 0 | 2 |
| Uβ | 1 | 2 | 2 | 0 | 5 |
| UαβBERX | 0 | 1 | 0 | 0 | 1 |
| UαβE | 0 | 1 | 0 | 0 | 1 |
| UαER | 0 | 2 | 0 | 0 | 2 |
| Uαβ | 0 | 7 | 0 | 0 | 7 |
| UαR | 0 | 1 | 0 | 0 | 1 |
| UβBX | 0 | 2 | 0 | 0 | 2 |
| UβB | 0 | 1 | 0 | 0 | 1 |
| UαβB | 0 | 2 | 0 | 0 | 2 |
| Total | 4 | 198 | 14 | 3 | 219 |
Isolates were obtained from bighorn sheep in Idaho, Oregon, and Wyoming.
Number of isolates of Histophilus somni, Pasteurella multocida (by subspecies and biotype), and Bibersteinia trehalosi (by biovariant taxonomic status) identified from nasal and oropharyngeal swab specimens obtained from free-ranging bighorn sheep.
| Species | Subspecies, biotype, or biovariant | Adult sheep | Lambs | Total | ||
|---|---|---|---|---|---|---|
| Diseased | Healthy | Diseased | Healthy | |||
| H somni P multocida | — | 0 | 0 | 2 | 0 | 2 |
| Mult A | 0 | 2 | 1 | 0 | 3 | |
| Mult B | 0 | 6 | 1 | 0 | 7 | |
| Gallicida | 0 | 4 | 0 | 0 | 4 | |
| Stomatis | 0 | 2 | 0 | 0 | 2 | |
| Testudinis | 0 | 1 | 0 | 0 | 1 | |
| U11 | 0 | 1 | 0 | 0 | 1 | |
| U16 | 0 | 1 | 0 | 0 | 1 | |
| U2 | 0 | 3 | 0 | 0 | 3 | |
| U23 | 0 | 1 | 0 | 0 | 1 | |
| U6 | 0 | 5 | 2 | 0 | 7 | |
| U8 | 0 | 3 | 0 | 0 | 3 | |
| B trehalosi | 2 | 13 | 194 | 15 | 7 | 229 |
| 2B | 2 | 109 | 5 | 0 | 116 | |
| 2αB | 0 | 1 | 0 | 0 | 1 | |
| 2BE | 0 | 1 | 0 | 0 | 1 | |
| 2BG | 0 | 0 | 1 | 0 | 1 | |
| 2BS | 0 | 13 | 0 | 0 | 13 | |
| 2C | 0 | 4 | 0 | 0 | 4 | |
| 2CDS | 0 | 0 | 2 | 0 | 2 | |
| 2CD | 0 | 1 | 0 | 0 | 1 | |
| 2CS | 0 | 1 | 0 | 0 | 1 | |
| 2E | 0 | 6 | 0 | 0 | 6 | |
| 2EDG | 0 | 1 | 0 | 0 | 1 | |
| 2GS | 0 | 1 | 0 | 0 | 1 | |
| 2S | 0 | 14 | 0 | 0 | 14 | |
| 4 | 1 | 5 | 4 | 0 | 10 | |
| 4B | 1 | 6 | 0 | 0 | 7 | |
| 4βBS | 0 | 2 | 0 | 0 | 2 | |
| 4BS | 0 | 3 | 0 | 0 | 3 | |
| 4CDE | 0 | 1 | 0 | 0 | 1 | |
| 4CDS | 0 | 4 | 0 | 0 | 4 | |
| 4DGS | 0 | 1 | 0 | 0 | 1 | |
| 4DS | 0 | 1 | 0 | 0 | 1 | |
| 4S | 0 | 1 | 0 | 0 | 1 | |
| Total | — | 17 | 399 | 33 | 7 | 456 |
— = Not applicable.
See Table 1 for remainder of key.
The species most commonly identified among the 675 isolates were B trehalosi (421/675 [62%]), M haemolytica (219/675 [32%]), and P multocida (33/675 [5%]). Pasteurella multocida was the species with the greatest proportion (4/33 [12%]) of isolates obtained from diseased sheep; however, the proportion of isolates obtained from diseased sheep did not differ significantly (P = 0.60) among these 3 bacterial species.
The most common biovariants identified among the isolates were B trehalosi 2 (229/675 [34%]), B trehalosi 2B (116/675 [17%]), M haemolytica 5 (18/675 [3%]), and B trehalosi 2S (14/675 [2%]). Bibersteinia trehalosi 2 was the biovariant with the greatest proportion (28/229 [12%]) of isolates obtained from diseased sheep. Comparisons of the proportions of isolates obtained from diseased sheep among biovariants were not conducted because only 2 biovariants (B trehalosi 2 and B trehalosi 2B, which comprised 51% of the data) provided > 5 isolates from both diseased and healthy sheep.
For isolates obtained from adult bighorn sheep, a β-hemolytic isolate was 2.59 (95% CI, 1.10 to 6.07) times as likely to have been obtained from a diseased sheep as from a healthy sheep. Conversely, for isolates obtained from lambs, a β-hemolytic isolate was only 0.74 (95% CI, 0.19 to 2.81) times as likely to have been obtained from a diseased lamb as from a healthy lamb.
Discussion
Findings from the present study provided preliminary baseline data of Pasteurellaceae strains in bighorn sheep with and without evidence of respiratory disease. The majority (47/57 [82%]) of isolates from lambs were obtained from animals with signs of respiratory disease, whereas relatively few (21/618 [3%]) isolates from adult sheep were obtained from animals with signs of respiratory disease. Although most of the isolates in the present study were obtained from clinically normal adult bighorn sheep, isolates from diseased sheep were identified as H somni and 21 subspecies, biotypes, and biovariants of M haemolytica, P multocida, and B trehalosi. Biovariants of P multocida subspecies similar to those identified in the study reported here have been isolated from bighorn sheep with respiratory disease.5,7,13,21,23,24 Biovariants previously isolated from bighorn sheep with respiratory disease that were not associated with diseased bighorn sheep in the present study included Pasturella multocida gallicida,13 M haemolytica 1G, and M haemolytica 1αG.5 It is unknown whether the findings reported here can be extrapolated to bighorn sheep in a larger geographic area.
Serogroup classification schemes for Pasteurellaceae may represent group lineages with various amounts of pathogenicity. Therefore, the refined scale of the biovariant classification scheme may be most useful for the identification of lineages with the highest pathogenicity. In the present study, Pasteurellaceae isolates were identified most frequently as B trehalosi, followed by M haemolytica, and most of the biovariants were identified within these 2 species. Even though no significant association was detected between health status (diseased or healthy) of sheep and any particular subspecies, biotype, or biovariant identified in the present study, the proportion of isolates obtained from diseased sheep was greater for B trehalosi 4 (5/10 [50%]) and M haemolytica 3 (4/13 [31%]) than that for B trehalosi 2 (28/229 [12%]), which was the biovariant most frequently identified. This finding suggested that the most prevalent biovariants may not be the most pathogenic, although it is difficult to confirm this without baseline information for the populations (ie, the denominator) from which these samples were collected. Additional information would be required to identify the biovariants that have the greatest impact on natural populations because the effect of a biovariant within a population is determined by many factors, including, but not limited to, the pathogenicity and transmission efficiency of the biovariant and the risk of sheep being exposed to the biovariant. In the present study, there was a large range of prevalences for identified biovariants; B trehalosi 2 and B trehalosi 2B accounted for 345 of 675 (51%) isolates, whereas none of the other 111 biovariants identified accounted for > 3% of the isolates.
In the present study, most biovariants were isolated from healthy adult bighorn sheep, with the exception of M haemolytica 16αE (only 1 isolate obtained from a diseased adult sheep). This finding is similar to pasteurellosis in domestic animals,25 in which Pasteurellaceae are part of the normal respiratory tract flora and are only associated with disease when favored by adverse combinations of host, agent, and environmental factors. However, all the biovariants (except for M haemolytica 10αB) obtained from lambs were more frequently isolated from diseased animals. Also, with the exception of 3 biovariants (M haemolytica 9B, B trehalosi 2BG, and B trehalosi 2CDS), each of the other 15 biovariants isolated from diseased lambs was also isolated from apparently healthy adult sheep. Whether this was because a high percentage of the specimens obtained from lambs were from diseased animals or was attributable to an increased susceptibility of lambs to disease caused by these biovariants requires further research.
Results of other studies5,26 indicate an association between β-hemolytic M haemolytica and pneumonia in bighorn sheep. Similarly, for adult bighorn sheep in the present study, β-hemolytic isolates were almost 2.6 times as likely to be obtained from diseased as from healthy sheep. Therefore, the ability of an isolate to cause β-hemolysis on blood agar may be an indication of an isolate's virulence in adult bighorn sheep, much as it is for Streptococcus spp in other animal species.27 However, for isolates obtained from lambs, β-hemolytic isolates were not associated with disease (OR, 0.74; 95% CI, 0.19 to 2.81); thus, β-hemolysis may not be an accurate indicator of an isolate's pathogenicity in bighorn lambs.
In the present study, the presence of disease was determined on the basis of clinical signs or necropsy results recorded by multiple submitters of specimens who likely used various assessment criteria. Precise definitions of pathogenicity and virulence are not possible until pathological criteria are defined and consistently applied and associations between specific Pasteurellaceae biovariants and respiratory disease in bighorn sheep are established. The present study represented the first step in resolving the challenges that exist for making these determinations. Additional data are needed to determine whether the biovariants most frequently identified from diseased animals represent particularly pathogenic strains or are a reflection of the most common biovariants of the respiratory tract flora of the general population of bighorn sheep. If the latter scenario is true, the diversity of isolates associated with diseased sheep in the present study was similar to the diversity of isolates associated with bovine respiratory disease (ie, shipping fever) complex in cattle, in which many Pasteurellaceae that are part of the normal bovine respiratory tract flora25,28 cause disease only sporadically as secondary, opportunistic infections. On the basis of both epidemiological29 and microbial pathogenesis models,30 a host's ability to mount an immune response to a microbe and the extent to which that immune response affects a microbe's pathogenicity and virulence are critical components for understanding the causes of and potential responses to respiratory disease outbreaks in bighorn sheep.
ABBREVIATION
| CI | Confidence interval |
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