DOI: 10.7589/2014-05-130
Journal of Wildlife Diseases, 51(1), 2015, pp. 19–27 # Wildlife Disease Association 2015
BIGHORN SHEEP (OVIS CANADENSIS) SINUS TUMORS ARE ASSOCIATED WITH COINFECTIONS BY POTENTIALLY PATHOGENIC BACTERIA IN THE UPPER RESPIRATORY TRACT Karen A. Fox,1,2,5 Natalie M. Rouse,1,2 Kathryn P. Huyvaert,3 Karen A. Griffin,1 Halcyon J. Killion,4 Jessica Jennings-Gaines,4 William H. Edwards,4 Sandra L. Quackenbush,2 and Michael W. Miller1 1
Colorado Parks and Wildlife, Wildlife Health Program, 4330 W Laporte Avenue, Fort Collins, Colorado 80521, USA Department of Microbiology, Immunology, and Pathology, Colorado State University, 1619 Campus Delivery, Fort Collins, Colorado 80523, USA 3 Department of Fish, Wildlife, and Conservation Biology, Colorado State University, 1474 Campus Delivery, Fort Collins, Colorado 80523, USA 4 Wyoming Game and Fish Department, Wildlife Disease Laboratory, 1174 Snowy Range Road, Laramie, Wyoming 82070, USA 5 Corresponding author (email: [email protected]) 2
Bighorn sheep (Ovis canadensis) sinus tumors are hyperplastic to neoplastic, predominantly stromal masses of the paranasal sinuses that expand the sinus lining and obstruct the sinus cavities. Obstruction of the sinus cavities and disruption of normal sinus lining anatomy may interfere with clearance of bacterial pathogens from the upper respiratory tract. To examine this possibility, we explored whether the presence of sinus tumor features (tumor score) affected the likelihood of detecting potentially pathogenic bacteria from upper respiratory sinus lining tissues in bighorn sheep. We developed or used existing PCR assays for the detection of leukotoxigenic Pasteurellaceae and Mycoplasma ovipneumoniae in sinus lining tissues collected from 97 bighorn sheep in Colorado, US from 2009 to 2012. With the use of logistic regression analyses we found that tumor score was a good predictor of the probability of detecting potentially pathogenic bacteria in sinus lining tissues; we were more likely to detect potentially pathogenic bacteria from samples with high tumor scores. These findings add to our understanding of possible mechanisms for the maintenance and shedding of bacterial agents from the upper respiratory tracts of bighorn sheep. Key words: Bighorn sheep, leukotoxin, Mycoplasma ovipneumoniae, Ovis canadensis, Pasteurellaceae, polymerase chain reaction, sinus tumors.
ABSTRACT:
respiratory tract. In bighorn sheep, lower respiratory tract disease (bronchopneumonia) has been well described for decades as a syndrome that causes all-age die-offs (Potts 1937; Marsh 1938; Post 1962; Bear and Jones 1973; Spraker et al. 1984), as well as annually recurring lamb mortalities (Marsh 1938; Coggins 1988; Festa-Bianchet 1988; Monello 2001) in Colorado and throughout western North America. However, although bronchopneumonia is well described in bighorn sheep, the contributions of upper respiratory infections to the persistence and transmission of respiratory pathogens in bighorn sheep has not been explored. To investigate the hypothesis that bighorn sheep sinus tumors prevent optimal clearance of potentially pathogenic bacteria from the upper respiratory tract, we examined bighorn sheep sinus lining tissues that were variably affected by
INTRODUCTION
Paranasal sinus tumors are a recently described disease affecting Rocky Mountain bighorn sheep (Ovis canadensis canadensis) in Colorado, US (Fox et al. 2011; Fox 2013). Common features of these tumors include thickening of the maxillary or frontal sinus linings by hyperplastic to neoplastic, predominantly stromal tissue, and invasion of underlying bone. Although several characteristics of the disease resemble oncogenic retroviral diseases of domestic sheep and goats, the cause of bighorn sheep sinus tumors is unknown (Fox et al. 2011; Fox 2013). Regardless of etiology, thickening of the sinus lining likely alters its function, preventing normal clearance of bacteria from the sinuses and potentially leading to persistent bacterial infections in the upper 19
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TABLE 1. Gross criteria for bighorn sheep tumor score assignment to bighorn sheep (Ovis canadensis) in Colorado, USA. Tumor score
0
1
2
Criteria for inclusion
No thickening of sinus lining, and no mucus in sinus cavities, and no cystic expansions of sinus lining Sinus lining thickened, but ,5 mm, and/or mucus present in sinus cavities, and/or cystic expansions of sinus lining Sinus lining thickened to .5 mm and thickened tissue is edematous with a wet appearance and invasion or remodeling of underlying bone
features of bighorn sheep sinus tumors (tumor score). We used PCR assays to assess these sinus lining tissues for two bacterial pathogens believed to be associated with fatal bronchopneumonia in bighorn sheep, leukotoxigenic Pasteurellaceae and Mycoplasma ovipneumoniae. Pasteurellaceae bacteria have long been associated with pneumonia die-offs in bighorn sheep (Post 1962; George et al. 2008; Wehausen et al. 2011; Miller et al. 2012), and the leukotoxin A gene (lktA) has specifically been identified as the primary virulence factor associated with pathogenicity (Davies et al. 2001; Jeyaseelan et al. 2002; Dassanayake et al. 2009). Mycoplasma spp. have also long been associated with bronchopneumonia in bighorn sheep (Woolf et al. 1970; Miller et al. 2012), with M. ovipneumoniae suggested as a primary agent (Besser et al. 2008, 2012) or as a predisposing factor allowing for secondary fatal infection by leukotoxigenic Pasteurellaceae (Dassanayake et al. 2010). MATERIALS AND METHODS Cases
From February 2009 through July 2012 we examined 136 bighorn sheep heads and skulls for sinus tumors. Heads and skulls were obtained through Colorado Division of (Parks and) Wildlife health monitoring and through collaborations with Colorado taxidermists. Heads and skulls obtained through agency sources came from free-ranging or captive
animals found dead or euthanized, and submitted for necropsy. Skulls obtained through taxidermists came from hunter-harvested animals. These skulls were first processed by removal of the skull cap (with horns) for taxidermy before the rest of the skull was donated for examination. Animals ,9 mo old were not included in the study, based on the inability to assess the maxillary sinus cavity for lesions, as no molars have erupted from the maxillary sinus cavity and immature tooth material fills the sinus cavity. The most likely cause of death was recorded based on history and necropsy findings. Diagnostic criteria
For each carcass, we grossly examined the paranasal sinus cavities including at least the maxillary sinuses, and portions of the frontal sinuses that had not been removed for taxidermy. Most of the cases examined were inadequate for histopathology because of autolysis and freeze–thaw artifacts. Therefore, criteria were based on gross features only. We categorized each carcass as tumor score50, 1, or 2 based on the criteria provided in Table 1 and illustrated in Figure 1. Characteristics incorporated into the scoring scheme included thickness, consistency, and presence of cystic expansions of the sinus lining, presence of mucus in the sinus cavity, and bone invasion. Each case examined fell exclusively into one of the three categories. PCR approach
For the detection of M. ovipneumoniae, we used PCR primers (McAuliffe et al. 2003) that have been shown to amplify this organism from postmortem lung tissue samples (Besser et al. 2012). We developed novel PCR primers for the detection of leukotoxigenic Pasteurellaceae from postmortem tissues. Primers for the detection of lktA were designed by aligning sequences available through GenBank representing Pasteurellaceae including Mannheimia hemolytica, Mannheimia ruminalis, Mannheimia glucosida, and Bibersteinia trehalosi (AF314503–AF314526.2, AY425280.2, JQ423930.1, JQ423931.1, M20730.1, M24197.1, U01215.1, U01216.1, and Z26247.1) (National Center for Biotechnology Information [NCBI] 2012). We identified conserved regions between all sequences, and screened conserved regions for primer candidates with he use of Primer3 software (Rozen and Skaletsky 2000). The resulting primers, listed in Table 2 (lktA fw and lktA rv), were designed to amplify a 945– base-pair (bp) fragment of the lktA gene, and were validated by two methods.
FOX ET AL.—BIGHORN SHEEP SINUS TUMORS AND BACTERIAL COINFECTIONS
21
FIGURE 1. Paranasal sinus cavities of Colorado, USA bighorn sheep (Ovis canadensis) illustrating assignment of tumor scores reflecting severity of sinus tumors. (A) Case with tumor score50, demonstrating normal, paper-thin sinus lining. (B) Tumor score51. Note thickened maxillary sinus lining ,5 mm thick. (C) Tumor score52. Note thickened maxillary sinus lining .5 mm thick, wet/edematous appearance to the tissue, and pool of mucus within the sinus cavity. (D) Case demonstrating cystic expansions of the maxillary sinus. This lesion was sometimes found in association with a distinct sinus tumor (tumor score52), but was also present in some cases in the absence of a distinct sinus tumor (tumor score51). (E) Unilateral tumor in frontal sinus (left side of image) of a yearling male bighorn sheep (tumor score52). (F) Unilateral sinus tumor filling frontal sinus (on right side of image) of an adult female bighorn sheep (tumor score52). lktA PCR validation
To confirm the specificity of our lktA primers, we screened archived DNA extracts from 16 bacterial isolates of Pasteurellaceae obtained from Colorado bighorn sheep. Each isolate was previously shown to be leukotoxin positive by PCR (Davies et al. 2001) and the
full-length lktA gene sequences were previously determined (Miller et al. 2013). Isolates were representative of multiple Pasteurellaceae species including B. trehalosi (n55), M. hemolytica (n53), M. ruminalis (n54), and M. glucosida (n54), based on 16S rRNA gene sequences (Miller et al. 2013).
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TABLE 2. Primers and reaction conditions for PCR assays assessing presence of Mycoplasma ovipneumoniae and leukotoxigenic Pasteurellaceae in bighorn sheep (Ovis canadensis), Colorado, USA. Cycling conditions Primers
Expected product (bp)
Leukotoxin A (designed for this study) lktA F: 59-GCCCGTTATCTTGCGAATTT-39 lktA R: 59-TACCACCAAATAAGCGGTCA-39 Leukotoxin A (Davies et al. 2001) lktA9: 59-TCAAGAAGAGCTGGCAAC-39 lktA7: 59-AGTGAGGGCAACTAAACC-39 M. ovipneumoniae (McAuliffe et al. 2003) LMF1: 59-TGAACGGAATATGTTAGCTT-39 LMR1: 59-GACTTCATCCTGCACTCTGT-39
Reaction (mL)
1 cycle (uC3min)
945
25a
95310 7233
,2,850
50b
9532 7235
419
25a
9435 7237
a
22.5 mL Platinum PCR SuperMix (Invitrogen), 100 ng genomic DNA, 0.4 mM each primer.
b
23 mL Promega Supermix (M713B), 50–100-ng genomic DNA, 0.4 mM each primer.
To confirm the sensitivity of our lktA primers in postmortem tissue, we analyzed fresh (,24 h postmortem) lung tissues from 13 bighorn sheep lambs that died with or without pneumonia during the summer of 2013. For each lung sample, a location-matched sample of lung was also cultured with the use of standard microbiologic methods (Quinn 1994) followed by removal of all bacterial growth from the primary culture plate with 15 mL of phosphate-buffered saline to wash colonies from the culture media. We extracted DNA from the plate washes with the use of a commercially available extraction kit (E.Z.N.A. Tissue DNA kit, Omega Bio-Tek, Norcross, Georgia, USA) per manufacturer’s instructions, and screened for lktA with our primers, as well as primers to amplify the full-length gene (Davies et al. 2001). Finally, to confirm the sensitivity of our primers in autolysed tissue, we allowed lung tissues to decompose for 1 wk at outdoor temperature (range 1.7– 29.4 C) and repeated the PCR assay with DNA extracted directly from the autolysed tissue. PCR methods for sinus lining tissues
Fresh tissue from the sinus lining was collected and yielded amplifiable DNA, as assessed by glyceraldehyde 3 phosphate dehydrogenase (GAPDH) PCR, from 97 of 136 bighorn sheep postmortem cases. The 39 cases that did not have amplifiable DNA were omitted from the study, as negative results for these cases could reflect degradation of DNA versus absence of bacteria. Each of the 97 adequate samples was assessed by PCR assays for lktA and M. ovipneumoniae. PCR products obtained with our lktA primers were sequenced and DNA extracts found to be
35 cycles (uC3s)
95330 60330 72360 95330 58330 72360 94330 55330 72330
positive with our lktA primers were screened for lktA with the use of available primers to amplify the full-length lktA gene (Davies et al. 2001). DNA extraction was performed with a commercially available extraction kit (QIAmp DNA mini kit, QIAGEN, Valencia, California, USA) per manufacturer’s instructions. PCR primers and cycling conditions are given in Table 2. Statistical methods
Three sets of logistic regression models were developed to explore the association between tumor score and the probability of detecting bacterial pathogens in the upper respiratory tracts of bighorn sheep. For each analysis two competing models were considered, one with a tumor score effect, and one without (i.e., a null model or model of no effect). This was performed for each of three outcome variables: 1) detection of M. ovipneumoniae, 2) detection of lktA, and 3) detection of one or both of the above. Akaike’s Information Criterion values, corrected for small sample sizes (AICc; Burnham and Anderson 2010), were calculated and the models were ranked by the AICc differences (DAICc) relative to the best model in the set of two. Akaike weights (wi), a measure of the evidence for each model, and evidence ratios, the ratio of the weights of the models being compared, were also calculated and used for inference. RESULTS Cases
Fresh tissue from the sinus lining yielded amplifiable DNA, as assessed by GAPDH PCR, from 97 of 136 bighorn
FOX ET AL.—BIGHORN SHEEP SINUS TUMORS AND BACTERIAL COINFECTIONS
sheep postmortem cases. Of those 97 cases, 39 came from full carcasses, and 58 were heads or skulls only. Cases included male (n552) and female (n545), with ages ranging from 9 mo to .10 yr. Five of the 97 cases were desert bighorn sheep (Ovis canadensis nelsoni), and the remainder were Rocky Mountain bighorn sheep. Causes of death included hunter-killed (n539), struck by vehicle (n518), other trauma (n58), capture mortality (n55), predation (n51), cull due to ill thrift (n52), cull due to disease in population (n53), cull due to interaction with domestic animals (n51), death from respiratory disease (n54), death from other disease (n52), and unknown history (n514). Tumor Score Results
Based on diagnostic criteria provided in Table 1, of the 97 cases included in the study, 50 were classified as tumor score50, 33 were classified as tumor score51, and 14 were classified as tumor score52. Although histologic criteria were not included in category determination, suspected tumor cases (tumor score52) that were suitable for histologic examination (n512) did demonstrate features of stromal hyperplasia with myxedema (n510), myxofibroma (n52), bone invasion by stromal components (n512), surface epithelial hyperplasia (n510), adenomatous hyperplasia of submucosal glands (n51), lymphoplasmacytic sinusitis (n510), and submucosal mucus-filled cysts (n53) as previously described (Fox et al. 2011). Two cases also demonstrated bone production. Features of malignant neoplasia were not identified in any of the cases examined histologically, despite the previous description of index tumor cases with adenocarcinoma (Fox et al. 2011). PCR validation
We confirmed the specificity of our lktA primers with the use of DNA extracted from Pasteurellaceae isolates with known lktA and 16S rRNA sequences. We used
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our lktA primers to amplify and sequence an approximately 945-bp product from each of the 16 DNA samples. For all 16 isolates, the 945-bp fragment sequence exactly matched the corresponding sequence from the previously determined full-length lktA sequence. As noted by Miller et al. (2013), species determination based on lktA sequence alignments (NCBI 2012) often differed from species determination based on 16S rRNA sequence alignments. We confirmed the sensitivity of our lktA primers by using lung tissue samples from bighorn sheep lambs that died of pneumonia and had been examined by culture and plate wash PCR. For 12 of the 13 bighorn sheep lamb cases, PCR results from our lktA primers and DNA extracted directly from lung tissue matched the results acquired with the use of plate wash DNA and full-length lktA gene primers; eight of 12 cases were positive for lktA. For one of the 13 cases, a pure culture of hemolytic B. trehalosi was isolated from a sample of pneumonic lung, and plate wash DNA was negative for lktA with primers used for the fulllength lktA gene. The same sample was positive for lktA with our newly designed primers and DNA extracted from fresh tissue. Histologically, a location-matched sample of lung from this case demonstrated lesions consistent with the presence of leukotoxin, including features such as nuclear streaming (oat cells), suggesting that our positive result for lktA was not false. Autolysis had no affect on the sensitivity of the assay. Sinus lining PCR results
Twenty-two sinus lining samples were positive by one or both PCR assays for M. ovipneumoniae and lktA. Seven were positive by both assays, six were positive for M. ovipneumoniae only, and nine were positive for lktA only. Of the 16 cases that were PCR positive with our lktA primers, 13 yielded a single lktA sequence read, and three yielded mixed sequence reads or were unamplifiable by sequencing reac-
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TABLE 3. PCR results for bacterial agents in the upper respiratory tracts of bighorn sheep (Ovis canadensis) from Colorado, USA, with varying degrees of involvement by bighorn sheep sinus tumors.
Bacteria status
Tumor score50
Tumor score51
Tumor score52
Bacteria negative Bacteria positive Mycoplasma Leukotoxin A
47 3 1 3
22 11 7 8
6 8 5 5
tion for other, unknown reasons. Of the 13 samples that yielded a single lktA sequence read, six aligned most closely with M. hemolytica, six aligned most closely with B. trehalosi, and one aligned most closely with M. glucosida. Of the 16 cases positive for lktA with our primers used to amplify a 945-bp fragment of the gene, only one was positive with primers used to amplify the full-length (,2,850-bp) gene, consistent with our expectation the smaller product would allow for higher sensitivity when analyzing autolysed tissues with variable DNA quality. Statistical results
We found positive PCR results for M. ovipneumoniae in 1/50 (2%) of tumor score50, 7/33 (21%) of tumor score51, and 5/14 (36%) of tumor score52 cases. Similarly, we found positive PCR results for lktA in 3/50 (6%) of tumor score50, 8/33 (24%) of tumor score51, and 5/14 (36%) of tumor score52 cases (Table 3). Based on logistic regression analyses, models including tumor score were clearly more likely than models without an effect of tumor score based on DAICc values (Table 4). Evidence ratios for each of the three analyses indicated that the tumor score models were 152.4 (M. ovipneumoniae), 13.2 (lktA), and 2,725.8 (one or both types of bacteria) times more likely than the respective null models (Table 4). These data strongly support an effect of tumor score on the probability of detecting these bacteria in the upper respiratory tract.
DISCUSSION
To date, no bighorn sheep mortalities have been attributed to sinus tumors. However, by changing the anatomy of the upper respiratory tract, these tumors may contribute to morbidity and mortality at the herd level by impairing clearance of bacterial infections in the upper respiratory tract. To examine this possibility further, we used PCR assays to screen bighorn sheep sinus lining tissues variably affected by sinus tumor features for the presence of potentially pathogenic bacteria including lktA-positive Pasteurellaceae and M. ovipneumoniae. The probability of detecting one or both of these agents in the sinus lining tissue was strongly associated with tumor score. Based on our results it is reasonable to suspect that any cause for sinus lining thickening, including sinus tumors or sinusitis of viral or bacterial origin, could lead to increased maintenance of these bacterial pathogens in the upper respiratory tract. This finding supports the notion that bighorn sheep respiratory disease is the product of complex interactions between pathogens. Our results have implications for diagnostics, surveillance, and management of bighorn sheep respiratory disease. From a diagnostic standpoint, we demonstrated the use of culture-independent methods to assess bacterial infections in sinus lining tissues. Because bacterial cultures were not possible for the sample set available for this study, we used PCR to detect both M. ovipneumoniae and leukotoxigenic Pasteurellaceae with DNA extracted directly from postmortem tissue. This involved development of PCR primers to detect a relatively small (945-bp) fragment of the lktA gene with the use of primers within conserved regions across all species of leukotoxigenic Pasteurellaceae. We demonstrated markedly increased sensitivity of this assay as compared to results that used primers to amplify the fulllength lktA gene, when applied to postmortem tissues. Although not all postmor-
FOX ET AL.—BIGHORN SHEEP SINUS TUMORS AND BACTERIAL COINFECTIONS
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TABLE 4. Model sets and rankings of pairs of models exploring the importance of tumor score on the probability of detecting a) Mycoplasma ovipneumoniae, b) leukotoxigenic Pasteurellaeae, or c) one or both types of bacteria in the upper respiratory tracts of bighorn sheep (Ovis canadensis) in Colorado, USA. Modela
a) No effect Score effect b) No effect Score effect c) No effect Score effect a
K
22 LL
AICc
DAICc
wi
1 3 1 3 1 3
76.428 62.159 86.87 77.501 103.865 83.828
78.470 68.417 88.912 83.759 105.907 90.086
10.053 0 5.153 0 15.821 0
0.007 0.993 0.071 0.929 0.0004 0.9996
ER
152.399 13.15 2,725.75
The number of parameters (K), 22 log-likelihood (22 LL), and small sample size-corrected Akaike information criterion valuces (AICc) are presented. Models are ranked by their AICc differences (DAICc) relative to the best model in the set of two. The evidence ratio (ER) is a ratio of the weights of the two models of interest. In model set a) the model with a tumor score effect is ,152 times more likely than the model without a tumor score effect.
tem tissues were suitable for PCR (we obtained amplifiable DNA from only 97 of 136 samples), our methods provided useful comparative data for this diagnostically challenging (autolysed, frozen, and thawed), yet valuable sample set. From a surveillance standpoint this study shows that bacterial agents, including both leukotoxigenic Pasteurellaceae and M. ovipneumoniae, can be present in the upper respiratory tract, where their detection seems more likely related to shedding than detection in the oropharynx. Currently, nasal swabs are the preferred sample for detecting M. ovipneumoniae, but oropharyngeal swabs are the sample of choice for Pasteurellaceae detection. Historically, the detection of Pasteurellaceae from nasal swabs has been difficult. However, based on our results, leukotoxigenic Pasteurellaceae can be present, and are perhaps more biologically relevant, in the upper respiratory tract. For the sinus lining tissues examined here, the majority of the lktA sequences we detected aligned most closely with M. hemolytica and B. trehalosi, the two Pasteurellaceae species that are most consistently associated with recurring respiratory disease in bighorn sheep herds in Colorado (Miller et al. 2013). Finally, a management implication for this study is the suggestion that shedding of bacteria from the upper respiratory tract may be variable between individuals within a herd, based on features such as
the presence of sinus tumors. This leads to the hypothesis that, within a single herd, there may be a few individuals shedding many more infectious bacteria from the upper respiratory tract than others (supershedders; Chase-Topping et al. 2008). If these individuals could be identified, then they could be selectively culled to improve management of respiratory disease within the herd. How to identify such supershedders practically remains undetermined, although detecting pneumonia- or sinusitis-causing agents from the upper respiratory tract may be approaches worth exploring. To this end, an antemortem assay for the detection of bighorn sheep sinus tumors is in development parallel to investigation of the specific etiologic agent of this disease (Fox 2013). ACKNOWLEDGMENTS
This project was funded by Colorado Division of (Parks and) Wildlife, a grant from the Boone and Crockett Club, a grant from the Colorado State University College of Veterinary Medicine and Biomedical Sciences College Research Council, and contributions from the Colorado sheep and goat auction and raffle fund. We thank all of the hunters and taxidermists who provided specimens for examination and sampling especially Barry Smith, Greg Brumfield, and Gary Bohochik. We are grateful for the many agency personnel who went to great lengths to collect carcasses from the field, including Mark Vieira, Shane Craig, Kim Woodruff, and Jamin Grigg. Ivy LeVan and Tracy Dubovos provided assistance
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with necropsy procedures, and Connie Brewster and Joel Rovnak provided valuable laboratory assistance. LITERATURE CITED Bear GD, Jones GW. 1973. History and distribution of bighorn sheep in Colorado. Colorado Division of Wildlife, Denver, Colorado, 232 pp. Besser TE, Cassirer EF, Potter KA, VanderSchalie J, Fischer A, Knowles DP, Herndon DR, Rurangirwa FR, Weiser GC, Srikumaran S. 2008. Association of Mycoplasma ovipneumoniae infection with population-limiting respiratory disease in free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis). J Clin Microbiol 46:423–430. Besser TE, Highland MA, Baker K, Cassirer EF, Anderson NJ, Ramsey JM, Mansfield K, Bruning DL, Wolff P, Smith JB, et al. 2012. Causes of pneumonia epizootics among bighorn sheep, western United States, 2008–2010. Emerg Infect Dis 18:406–414. Burnham KP, Anderson DR. 2010. Model selection and multimodel inference: A practical information-theoretic approach. 2nd Ed. Springer, New York, New York, 488 pp. Chase-Topping M, Gally D, Low C, Matthews L, Woolhouse M. 2008. Super-shedding and the link between human infection and livestock carriage of Escherichia coli O157. Nat Rev Microbiol 6:904–912. Coggins V. 1988. The Lostine Rocky Mountain bighorn sheep die-off and domestic sheep. In: Proceedings: 6th Biennial Symposium of the Northern Wild Sheep and Goat Council, Northern Wild Sheep and Goat Council, 11–15 April, Banff, Alberta, Canada, pp. 57–64. Dassanayake RP, Shanthalingam S, Herndon CN, Lawrence PK, Cassirer EF, Potter KA, Foreyt WJ, Clinkenbeard KD, Srikumaran S. 2009. Mannheimia haemolytica serotype A1 exhibits differential pathogenicity in two related species, Ovis canadensis and Ovis aries. Vet Microbiol 133:366–371. Dassanayake RP, Shanthalingam S, Herndon CN, Subramaniam R, Lawrence PK, Bavananthasivam J, Cassirer EF, Haldorson GJ, Foreyt WJ, Rurangirwa FR, et al. 2010. Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia. Vet Microbiol 145:354–359. Davies RL, Whittam TS, Selander RK. 2001. Sequence diversity and molecular evolution of the leukotoxin (lktA) gene in bovine and ovine strains of Mannheimia (Pasteurella) haemolytica. J Bacteriol 183:1394–1404. Festa-Bianchet M. 1988. A pneumonia epizootic in bighorn sheep, with comments on preventive management. In: Proceedings: 6th Biennial
Symposium of the Northern Wild Sheep and Goat Council, Northern Wild Sheep and Goat Council, 11–15 April, Banff, Alberta, Canada, pp. 66–76. Fox KA. 2013. Sinus tumors of Rocky Mountain bighorn sheep: Investigation of an infectious etiology. PhD Thesis, Pathology, Colorado State Univerisity, Fort Collins, Colorado, 152 pp. Fox KA, Wootton SK, Quackenbush SL, Wolfe LL, Levan IK, Miller MW, Spraker TR. 2011. Paranasal sinus masses of Rocky Mountain bighorn sheep (Ovis canadensis canadensis). Vet Pathol 48:706–712. George JL, Martin DJ, Lukacs PM, Miller MW. 2008. Epidemic pasteurellosis in a bighorn sheep population coinciding with the appearance of a domestic sheep. J Wildl Dis 44:388–403. Jeyaseelan S, Sreevatsan S, Maheswaran SK. 2002. Role of Mannheimia haemolytica leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis. Anim Health Res Rev 3:69–82. Marsh H. 1938. Pneumonia in Rocky Mountain bighorn sheep. J Mammal 19:214–219. McAuliffe L, Hatchell FM, Ayling RD, King AI, Nicholas RA. 2003. Detection of Mycoplasma ovipneumoniae in Pasteurella-vaccinated sheep flocks with respiratory disease in England. Vet Rec 153:687–688. Miller DS, Hoberg E, Weiser G, Aune K, Atkinson M, Kimberling C. 2012. A review of hypothesized determinants associated with bighorn sheep (Ovis canadensis) die-offs. Vet Med Int 2012;2012:796527. Miller MW, Hause BM, Killion HJ, Fox KA, Edwards WH, Wolfe LL. 2013. Phylogenetic and epidemiologic relationships among Pasteurellaceae from Colorado bighorn sheep herds. J Wildl Dis 49:653–660. Monello R, Murray DL, Cassirer EF. 2001. Ecological correlates of pneumonia epizootics in bighorn sheep herds. Can J Zool–Rev Can Zool 79:1423–1432. National Center for Biotechnology Information (NCBI). 2012. GenBank, www.ncbi.nlm.nih. gov/genbank. Accessed August 2012. Post G. 1962. Pasteurellosis of Rocky Mountain bighorn sheep (Ovis canadensis canadensis). Wildl Dis 23:1–14. Potts MK. 1937. Hemorrhagic septicemia in the bighorn of Rocky Mountain National Park. J Mammal 18:105–106. Quinn PJ, Carter ME, Markey B, Carter GR. 1994. Clinical veterinary microbiology. Wolfe Publishing, London, UK, 648 pp. Rozen S, Skaletsky H. 2000. Primer3 on the www for general users and for biologist programmers. Methods Mol Biol 132:365–386. Spraker TR, Hibler CP, Schoonveld GG, Adney WS. 1984. Pathologic changes and microorganisms
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Journal of Wildlife Diseases, 51(1), 2015, pp. 19–27 # Wildlife Disease Association 2015
BIGHORN SHEEP (OVIS CANADENSIS) SINUS TUMORS ARE ASSOCIATED WITH COINFECTIONS BY POTENTIALLY PATHOGENIC BACTERIA IN THE UPPER RESPIRATORY TRACT Karen A. Fox,1,2,5 Natalie M. Rouse,1,2 Kathryn P. Huyvaert,3 Karen A. Griffin,1 Halcyon J. Killion,4 Jessica Jennings-Gaines,4 William H. Edwards,4 Sandra L. Quackenbush,2 and Michael W. Miller1 1
Colorado Parks and Wildlife, Wildlife Health Program, 4330 W Laporte Avenue, Fort Collins, Colorado 80521, USA Department of Microbiology, Immunology, and Pathology, Colorado State University, 1619 Campus Delivery, Fort Collins, Colorado 80523, USA 3 Department of Fish, Wildlife, and Conservation Biology, Colorado State University, 1474 Campus Delivery, Fort Collins, Colorado 80523, USA 4 Wyoming Game and Fish Department, Wildlife Disease Laboratory, 1174 Snowy Range Road, Laramie, Wyoming 82070, USA 5 Corresponding author (email: [email protected]) 2
Bighorn sheep (Ovis canadensis) sinus tumors are hyperplastic to neoplastic, predominantly stromal masses of the paranasal sinuses that expand the sinus lining and obstruct the sinus cavities. Obstruction of the sinus cavities and disruption of normal sinus lining anatomy may interfere with clearance of bacterial pathogens from the upper respiratory tract. To examine this possibility, we explored whether the presence of sinus tumor features (tumor score) affected the likelihood of detecting potentially pathogenic bacteria from upper respiratory sinus lining tissues in bighorn sheep. We developed or used existing PCR assays for the detection of leukotoxigenic Pasteurellaceae and Mycoplasma ovipneumoniae in sinus lining tissues collected from 97 bighorn sheep in Colorado, US from 2009 to 2012. With the use of logistic regression analyses we found that tumor score was a good predictor of the probability of detecting potentially pathogenic bacteria in sinus lining tissues; we were more likely to detect potentially pathogenic bacteria from samples with high tumor scores. These findings add to our understanding of possible mechanisms for the maintenance and shedding of bacterial agents from the upper respiratory tracts of bighorn sheep. Key words: Bighorn sheep, leukotoxin, Mycoplasma ovipneumoniae, Ovis canadensis, Pasteurellaceae, polymerase chain reaction, sinus tumors.
ABSTRACT:
respiratory tract. In bighorn sheep, lower respiratory tract disease (bronchopneumonia) has been well described for decades as a syndrome that causes all-age die-offs (Potts 1937; Marsh 1938; Post 1962; Bear and Jones 1973; Spraker et al. 1984), as well as annually recurring lamb mortalities (Marsh 1938; Coggins 1988; Festa-Bianchet 1988; Monello 2001) in Colorado and throughout western North America. However, although bronchopneumonia is well described in bighorn sheep, the contributions of upper respiratory infections to the persistence and transmission of respiratory pathogens in bighorn sheep has not been explored. To investigate the hypothesis that bighorn sheep sinus tumors prevent optimal clearance of potentially pathogenic bacteria from the upper respiratory tract, we examined bighorn sheep sinus lining tissues that were variably affected by
INTRODUCTION
Paranasal sinus tumors are a recently described disease affecting Rocky Mountain bighorn sheep (Ovis canadensis canadensis) in Colorado, US (Fox et al. 2011; Fox 2013). Common features of these tumors include thickening of the maxillary or frontal sinus linings by hyperplastic to neoplastic, predominantly stromal tissue, and invasion of underlying bone. Although several characteristics of the disease resemble oncogenic retroviral diseases of domestic sheep and goats, the cause of bighorn sheep sinus tumors is unknown (Fox et al. 2011; Fox 2013). Regardless of etiology, thickening of the sinus lining likely alters its function, preventing normal clearance of bacteria from the sinuses and potentially leading to persistent bacterial infections in the upper 19
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TABLE 1. Gross criteria for bighorn sheep tumor score assignment to bighorn sheep (Ovis canadensis) in Colorado, USA. Tumor score
0
1
2
Criteria for inclusion
No thickening of sinus lining, and no mucus in sinus cavities, and no cystic expansions of sinus lining Sinus lining thickened, but ,5 mm, and/or mucus present in sinus cavities, and/or cystic expansions of sinus lining Sinus lining thickened to .5 mm and thickened tissue is edematous with a wet appearance and invasion or remodeling of underlying bone
features of bighorn sheep sinus tumors (tumor score). We used PCR assays to assess these sinus lining tissues for two bacterial pathogens believed to be associated with fatal bronchopneumonia in bighorn sheep, leukotoxigenic Pasteurellaceae and Mycoplasma ovipneumoniae. Pasteurellaceae bacteria have long been associated with pneumonia die-offs in bighorn sheep (Post 1962; George et al. 2008; Wehausen et al. 2011; Miller et al. 2012), and the leukotoxin A gene (lktA) has specifically been identified as the primary virulence factor associated with pathogenicity (Davies et al. 2001; Jeyaseelan et al. 2002; Dassanayake et al. 2009). Mycoplasma spp. have also long been associated with bronchopneumonia in bighorn sheep (Woolf et al. 1970; Miller et al. 2012), with M. ovipneumoniae suggested as a primary agent (Besser et al. 2008, 2012) or as a predisposing factor allowing for secondary fatal infection by leukotoxigenic Pasteurellaceae (Dassanayake et al. 2010). MATERIALS AND METHODS Cases
From February 2009 through July 2012 we examined 136 bighorn sheep heads and skulls for sinus tumors. Heads and skulls were obtained through Colorado Division of (Parks and) Wildlife health monitoring and through collaborations with Colorado taxidermists. Heads and skulls obtained through agency sources came from free-ranging or captive
animals found dead or euthanized, and submitted for necropsy. Skulls obtained through taxidermists came from hunter-harvested animals. These skulls were first processed by removal of the skull cap (with horns) for taxidermy before the rest of the skull was donated for examination. Animals ,9 mo old were not included in the study, based on the inability to assess the maxillary sinus cavity for lesions, as no molars have erupted from the maxillary sinus cavity and immature tooth material fills the sinus cavity. The most likely cause of death was recorded based on history and necropsy findings. Diagnostic criteria
For each carcass, we grossly examined the paranasal sinus cavities including at least the maxillary sinuses, and portions of the frontal sinuses that had not been removed for taxidermy. Most of the cases examined were inadequate for histopathology because of autolysis and freeze–thaw artifacts. Therefore, criteria were based on gross features only. We categorized each carcass as tumor score50, 1, or 2 based on the criteria provided in Table 1 and illustrated in Figure 1. Characteristics incorporated into the scoring scheme included thickness, consistency, and presence of cystic expansions of the sinus lining, presence of mucus in the sinus cavity, and bone invasion. Each case examined fell exclusively into one of the three categories. PCR approach
For the detection of M. ovipneumoniae, we used PCR primers (McAuliffe et al. 2003) that have been shown to amplify this organism from postmortem lung tissue samples (Besser et al. 2012). We developed novel PCR primers for the detection of leukotoxigenic Pasteurellaceae from postmortem tissues. Primers for the detection of lktA were designed by aligning sequences available through GenBank representing Pasteurellaceae including Mannheimia hemolytica, Mannheimia ruminalis, Mannheimia glucosida, and Bibersteinia trehalosi (AF314503–AF314526.2, AY425280.2, JQ423930.1, JQ423931.1, M20730.1, M24197.1, U01215.1, U01216.1, and Z26247.1) (National Center for Biotechnology Information [NCBI] 2012). We identified conserved regions between all sequences, and screened conserved regions for primer candidates with he use of Primer3 software (Rozen and Skaletsky 2000). The resulting primers, listed in Table 2 (lktA fw and lktA rv), were designed to amplify a 945– base-pair (bp) fragment of the lktA gene, and were validated by two methods.
FOX ET AL.—BIGHORN SHEEP SINUS TUMORS AND BACTERIAL COINFECTIONS
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FIGURE 1. Paranasal sinus cavities of Colorado, USA bighorn sheep (Ovis canadensis) illustrating assignment of tumor scores reflecting severity of sinus tumors. (A) Case with tumor score50, demonstrating normal, paper-thin sinus lining. (B) Tumor score51. Note thickened maxillary sinus lining ,5 mm thick. (C) Tumor score52. Note thickened maxillary sinus lining .5 mm thick, wet/edematous appearance to the tissue, and pool of mucus within the sinus cavity. (D) Case demonstrating cystic expansions of the maxillary sinus. This lesion was sometimes found in association with a distinct sinus tumor (tumor score52), but was also present in some cases in the absence of a distinct sinus tumor (tumor score51). (E) Unilateral tumor in frontal sinus (left side of image) of a yearling male bighorn sheep (tumor score52). (F) Unilateral sinus tumor filling frontal sinus (on right side of image) of an adult female bighorn sheep (tumor score52). lktA PCR validation
To confirm the specificity of our lktA primers, we screened archived DNA extracts from 16 bacterial isolates of Pasteurellaceae obtained from Colorado bighorn sheep. Each isolate was previously shown to be leukotoxin positive by PCR (Davies et al. 2001) and the
full-length lktA gene sequences were previously determined (Miller et al. 2013). Isolates were representative of multiple Pasteurellaceae species including B. trehalosi (n55), M. hemolytica (n53), M. ruminalis (n54), and M. glucosida (n54), based on 16S rRNA gene sequences (Miller et al. 2013).
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TABLE 2. Primers and reaction conditions for PCR assays assessing presence of Mycoplasma ovipneumoniae and leukotoxigenic Pasteurellaceae in bighorn sheep (Ovis canadensis), Colorado, USA. Cycling conditions Primers
Expected product (bp)
Leukotoxin A (designed for this study) lktA F: 59-GCCCGTTATCTTGCGAATTT-39 lktA R: 59-TACCACCAAATAAGCGGTCA-39 Leukotoxin A (Davies et al. 2001) lktA9: 59-TCAAGAAGAGCTGGCAAC-39 lktA7: 59-AGTGAGGGCAACTAAACC-39 M. ovipneumoniae (McAuliffe et al. 2003) LMF1: 59-TGAACGGAATATGTTAGCTT-39 LMR1: 59-GACTTCATCCTGCACTCTGT-39
Reaction (mL)
1 cycle (uC3min)
945
25a
95310 7233
,2,850
50b
9532 7235
419
25a
9435 7237
a
22.5 mL Platinum PCR SuperMix (Invitrogen), 100 ng genomic DNA, 0.4 mM each primer.
b
23 mL Promega Supermix (M713B), 50–100-ng genomic DNA, 0.4 mM each primer.
To confirm the sensitivity of our lktA primers in postmortem tissue, we analyzed fresh (,24 h postmortem) lung tissues from 13 bighorn sheep lambs that died with or without pneumonia during the summer of 2013. For each lung sample, a location-matched sample of lung was also cultured with the use of standard microbiologic methods (Quinn 1994) followed by removal of all bacterial growth from the primary culture plate with 15 mL of phosphate-buffered saline to wash colonies from the culture media. We extracted DNA from the plate washes with the use of a commercially available extraction kit (E.Z.N.A. Tissue DNA kit, Omega Bio-Tek, Norcross, Georgia, USA) per manufacturer’s instructions, and screened for lktA with our primers, as well as primers to amplify the full-length gene (Davies et al. 2001). Finally, to confirm the sensitivity of our primers in autolysed tissue, we allowed lung tissues to decompose for 1 wk at outdoor temperature (range 1.7– 29.4 C) and repeated the PCR assay with DNA extracted directly from the autolysed tissue. PCR methods for sinus lining tissues
Fresh tissue from the sinus lining was collected and yielded amplifiable DNA, as assessed by glyceraldehyde 3 phosphate dehydrogenase (GAPDH) PCR, from 97 of 136 bighorn sheep postmortem cases. The 39 cases that did not have amplifiable DNA were omitted from the study, as negative results for these cases could reflect degradation of DNA versus absence of bacteria. Each of the 97 adequate samples was assessed by PCR assays for lktA and M. ovipneumoniae. PCR products obtained with our lktA primers were sequenced and DNA extracts found to be
35 cycles (uC3s)
95330 60330 72360 95330 58330 72360 94330 55330 72330
positive with our lktA primers were screened for lktA with the use of available primers to amplify the full-length lktA gene (Davies et al. 2001). DNA extraction was performed with a commercially available extraction kit (QIAmp DNA mini kit, QIAGEN, Valencia, California, USA) per manufacturer’s instructions. PCR primers and cycling conditions are given in Table 2. Statistical methods
Three sets of logistic regression models were developed to explore the association between tumor score and the probability of detecting bacterial pathogens in the upper respiratory tracts of bighorn sheep. For each analysis two competing models were considered, one with a tumor score effect, and one without (i.e., a null model or model of no effect). This was performed for each of three outcome variables: 1) detection of M. ovipneumoniae, 2) detection of lktA, and 3) detection of one or both of the above. Akaike’s Information Criterion values, corrected for small sample sizes (AICc; Burnham and Anderson 2010), were calculated and the models were ranked by the AICc differences (DAICc) relative to the best model in the set of two. Akaike weights (wi), a measure of the evidence for each model, and evidence ratios, the ratio of the weights of the models being compared, were also calculated and used for inference. RESULTS Cases
Fresh tissue from the sinus lining yielded amplifiable DNA, as assessed by GAPDH PCR, from 97 of 136 bighorn
FOX ET AL.—BIGHORN SHEEP SINUS TUMORS AND BACTERIAL COINFECTIONS
sheep postmortem cases. Of those 97 cases, 39 came from full carcasses, and 58 were heads or skulls only. Cases included male (n552) and female (n545), with ages ranging from 9 mo to .10 yr. Five of the 97 cases were desert bighorn sheep (Ovis canadensis nelsoni), and the remainder were Rocky Mountain bighorn sheep. Causes of death included hunter-killed (n539), struck by vehicle (n518), other trauma (n58), capture mortality (n55), predation (n51), cull due to ill thrift (n52), cull due to disease in population (n53), cull due to interaction with domestic animals (n51), death from respiratory disease (n54), death from other disease (n52), and unknown history (n514). Tumor Score Results
Based on diagnostic criteria provided in Table 1, of the 97 cases included in the study, 50 were classified as tumor score50, 33 were classified as tumor score51, and 14 were classified as tumor score52. Although histologic criteria were not included in category determination, suspected tumor cases (tumor score52) that were suitable for histologic examination (n512) did demonstrate features of stromal hyperplasia with myxedema (n510), myxofibroma (n52), bone invasion by stromal components (n512), surface epithelial hyperplasia (n510), adenomatous hyperplasia of submucosal glands (n51), lymphoplasmacytic sinusitis (n510), and submucosal mucus-filled cysts (n53) as previously described (Fox et al. 2011). Two cases also demonstrated bone production. Features of malignant neoplasia were not identified in any of the cases examined histologically, despite the previous description of index tumor cases with adenocarcinoma (Fox et al. 2011). PCR validation
We confirmed the specificity of our lktA primers with the use of DNA extracted from Pasteurellaceae isolates with known lktA and 16S rRNA sequences. We used
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our lktA primers to amplify and sequence an approximately 945-bp product from each of the 16 DNA samples. For all 16 isolates, the 945-bp fragment sequence exactly matched the corresponding sequence from the previously determined full-length lktA sequence. As noted by Miller et al. (2013), species determination based on lktA sequence alignments (NCBI 2012) often differed from species determination based on 16S rRNA sequence alignments. We confirmed the sensitivity of our lktA primers by using lung tissue samples from bighorn sheep lambs that died of pneumonia and had been examined by culture and plate wash PCR. For 12 of the 13 bighorn sheep lamb cases, PCR results from our lktA primers and DNA extracted directly from lung tissue matched the results acquired with the use of plate wash DNA and full-length lktA gene primers; eight of 12 cases were positive for lktA. For one of the 13 cases, a pure culture of hemolytic B. trehalosi was isolated from a sample of pneumonic lung, and plate wash DNA was negative for lktA with primers used for the fulllength lktA gene. The same sample was positive for lktA with our newly designed primers and DNA extracted from fresh tissue. Histologically, a location-matched sample of lung from this case demonstrated lesions consistent with the presence of leukotoxin, including features such as nuclear streaming (oat cells), suggesting that our positive result for lktA was not false. Autolysis had no affect on the sensitivity of the assay. Sinus lining PCR results
Twenty-two sinus lining samples were positive by one or both PCR assays for M. ovipneumoniae and lktA. Seven were positive by both assays, six were positive for M. ovipneumoniae only, and nine were positive for lktA only. Of the 16 cases that were PCR positive with our lktA primers, 13 yielded a single lktA sequence read, and three yielded mixed sequence reads or were unamplifiable by sequencing reac-
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TABLE 3. PCR results for bacterial agents in the upper respiratory tracts of bighorn sheep (Ovis canadensis) from Colorado, USA, with varying degrees of involvement by bighorn sheep sinus tumors.
Bacteria status
Tumor score50
Tumor score51
Tumor score52
Bacteria negative Bacteria positive Mycoplasma Leukotoxin A
47 3 1 3
22 11 7 8
6 8 5 5
tion for other, unknown reasons. Of the 13 samples that yielded a single lktA sequence read, six aligned most closely with M. hemolytica, six aligned most closely with B. trehalosi, and one aligned most closely with M. glucosida. Of the 16 cases positive for lktA with our primers used to amplify a 945-bp fragment of the gene, only one was positive with primers used to amplify the full-length (,2,850-bp) gene, consistent with our expectation the smaller product would allow for higher sensitivity when analyzing autolysed tissues with variable DNA quality. Statistical results
We found positive PCR results for M. ovipneumoniae in 1/50 (2%) of tumor score50, 7/33 (21%) of tumor score51, and 5/14 (36%) of tumor score52 cases. Similarly, we found positive PCR results for lktA in 3/50 (6%) of tumor score50, 8/33 (24%) of tumor score51, and 5/14 (36%) of tumor score52 cases (Table 3). Based on logistic regression analyses, models including tumor score were clearly more likely than models without an effect of tumor score based on DAICc values (Table 4). Evidence ratios for each of the three analyses indicated that the tumor score models were 152.4 (M. ovipneumoniae), 13.2 (lktA), and 2,725.8 (one or both types of bacteria) times more likely than the respective null models (Table 4). These data strongly support an effect of tumor score on the probability of detecting these bacteria in the upper respiratory tract.
DISCUSSION
To date, no bighorn sheep mortalities have been attributed to sinus tumors. However, by changing the anatomy of the upper respiratory tract, these tumors may contribute to morbidity and mortality at the herd level by impairing clearance of bacterial infections in the upper respiratory tract. To examine this possibility further, we used PCR assays to screen bighorn sheep sinus lining tissues variably affected by sinus tumor features for the presence of potentially pathogenic bacteria including lktA-positive Pasteurellaceae and M. ovipneumoniae. The probability of detecting one or both of these agents in the sinus lining tissue was strongly associated with tumor score. Based on our results it is reasonable to suspect that any cause for sinus lining thickening, including sinus tumors or sinusitis of viral or bacterial origin, could lead to increased maintenance of these bacterial pathogens in the upper respiratory tract. This finding supports the notion that bighorn sheep respiratory disease is the product of complex interactions between pathogens. Our results have implications for diagnostics, surveillance, and management of bighorn sheep respiratory disease. From a diagnostic standpoint, we demonstrated the use of culture-independent methods to assess bacterial infections in sinus lining tissues. Because bacterial cultures were not possible for the sample set available for this study, we used PCR to detect both M. ovipneumoniae and leukotoxigenic Pasteurellaceae with DNA extracted directly from postmortem tissue. This involved development of PCR primers to detect a relatively small (945-bp) fragment of the lktA gene with the use of primers within conserved regions across all species of leukotoxigenic Pasteurellaceae. We demonstrated markedly increased sensitivity of this assay as compared to results that used primers to amplify the fulllength lktA gene, when applied to postmortem tissues. Although not all postmor-
FOX ET AL.—BIGHORN SHEEP SINUS TUMORS AND BACTERIAL COINFECTIONS
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TABLE 4. Model sets and rankings of pairs of models exploring the importance of tumor score on the probability of detecting a) Mycoplasma ovipneumoniae, b) leukotoxigenic Pasteurellaeae, or c) one or both types of bacteria in the upper respiratory tracts of bighorn sheep (Ovis canadensis) in Colorado, USA. Modela
a) No effect Score effect b) No effect Score effect c) No effect Score effect a
K
22 LL
AICc
DAICc
wi
1 3 1 3 1 3
76.428 62.159 86.87 77.501 103.865 83.828
78.470 68.417 88.912 83.759 105.907 90.086
10.053 0 5.153 0 15.821 0
0.007 0.993 0.071 0.929 0.0004 0.9996
ER
152.399 13.15 2,725.75
The number of parameters (K), 22 log-likelihood (22 LL), and small sample size-corrected Akaike information criterion valuces (AICc) are presented. Models are ranked by their AICc differences (DAICc) relative to the best model in the set of two. The evidence ratio (ER) is a ratio of the weights of the two models of interest. In model set a) the model with a tumor score effect is ,152 times more likely than the model without a tumor score effect.
tem tissues were suitable for PCR (we obtained amplifiable DNA from only 97 of 136 samples), our methods provided useful comparative data for this diagnostically challenging (autolysed, frozen, and thawed), yet valuable sample set. From a surveillance standpoint this study shows that bacterial agents, including both leukotoxigenic Pasteurellaceae and M. ovipneumoniae, can be present in the upper respiratory tract, where their detection seems more likely related to shedding than detection in the oropharynx. Currently, nasal swabs are the preferred sample for detecting M. ovipneumoniae, but oropharyngeal swabs are the sample of choice for Pasteurellaceae detection. Historically, the detection of Pasteurellaceae from nasal swabs has been difficult. However, based on our results, leukotoxigenic Pasteurellaceae can be present, and are perhaps more biologically relevant, in the upper respiratory tract. For the sinus lining tissues examined here, the majority of the lktA sequences we detected aligned most closely with M. hemolytica and B. trehalosi, the two Pasteurellaceae species that are most consistently associated with recurring respiratory disease in bighorn sheep herds in Colorado (Miller et al. 2013). Finally, a management implication for this study is the suggestion that shedding of bacteria from the upper respiratory tract may be variable between individuals within a herd, based on features such as
the presence of sinus tumors. This leads to the hypothesis that, within a single herd, there may be a few individuals shedding many more infectious bacteria from the upper respiratory tract than others (supershedders; Chase-Topping et al. 2008). If these individuals could be identified, then they could be selectively culled to improve management of respiratory disease within the herd. How to identify such supershedders practically remains undetermined, although detecting pneumonia- or sinusitis-causing agents from the upper respiratory tract may be approaches worth exploring. To this end, an antemortem assay for the detection of bighorn sheep sinus tumors is in development parallel to investigation of the specific etiologic agent of this disease (Fox 2013). ACKNOWLEDGMENTS
This project was funded by Colorado Division of (Parks and) Wildlife, a grant from the Boone and Crockett Club, a grant from the Colorado State University College of Veterinary Medicine and Biomedical Sciences College Research Council, and contributions from the Colorado sheep and goat auction and raffle fund. We thank all of the hunters and taxidermists who provided specimens for examination and sampling especially Barry Smith, Greg Brumfield, and Gary Bohochik. We are grateful for the many agency personnel who went to great lengths to collect carcasses from the field, including Mark Vieira, Shane Craig, Kim Woodruff, and Jamin Grigg. Ivy LeVan and Tracy Dubovos provided assistance
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