Cylicospirura Species (Nematoda: Spirocercidae) and Stomach Nodules in Cougars (Puma Concolor) and Bobcats (Lynx Rufus) in Oregon

Journal of Wildlife Diseases, 47(1), 2011, pp. 140–153 # Wildlife Disease Association 2011

CYLICOSPIRURA SPECIES (NEMATODA: SPIROCERCIDAE) AND STOMACH NODULES IN COUGARS (PUMA CONCOLOR) AND BOBCATS (LYNX RUFUS) IN OREGON

Jayde A. Ferguson,1,6 Karen Woodberry,2 Colin M. Gillin,3 DeWaine H. Jackson,4 Justin L. Sanders,1 Whitney Madigan,5 Robert J. Bildfell,5 and Michael L. Kent1 1 Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis, Oregon 97331, USA 2 North Reading Veterinary Clinic, 212 Main Street, North Reading, Massachusetts 01864, USA 3 Oregon Department of Fish and Wildlife, 7118 Vandenberg Avenue, Corvallis, Oregon 97331, USA 4 Oregon Department of Fish and Wildlife, 4192 N Umpqua Highway, Roseburg, Oregon 97470, USA Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021 5 College of Veterinary Medicine, Oregon State University, 146 Magruder Hall, Corvallis, Oregon 97331, USA 6 Corresponding author (email: [email protected])

ABSTRACT: The stomachs and proximal duodena of 160 cougars (Puma concolor) and 17 bobcats (Lynx rufus), obtained throughout Oregon during 7 yr, were examined for Cylicospirura spp. and associated lesions. Prevalence in cougars was 73%, with a range in intensity of 1–562 worms. The mean diameter of nodules was 1.2 cm (SD50.5), and many extended through the submucosa to the muscularis. About 83% of cougars had nodules; most nodules contained worms, but 14% of the smaller nodules (,0.2 cm) contained porcupine (Erethizon dorsatum) quills. A mean of 12.4 worms/nodule (SD534.1) was observed, with a maximum of 340 worms/nodule. Prevalence in bobcats was 53%, with an intensity of 1–25 worms. About 65% of bobcats had nodules, which were slightly smaller than those in cougars but appeared to involve similar layers of gastrointestinal tissue. One to 25 Cylicospirura sp. were found in all but two small nodules in bobcats. Cougars killed for livestock damage or safety concerns had a significantly higher median worm intensity than did those that died of other causes. Also, the median worm intensity of older cougars was higher than that of younger lions. There were more males than females killed for livestock damage or safety concerns. The cylicospirurid from cougars was Cylicospirura subaequalis, and that of bobcats was Cylicospirura felineus. These two similar species were separated morphologically by differences in tooth and sex organ morphology. They were also differentiated by DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Worm sequences from cougars differed from those from bobcats by 11%, whereas essentially no difference was found among worms from the same host. Phylogenetic analysis showed that within the order Spirurida, both cylicospirurids were most closely related to Spirocerca lupi, based on this gene sequence. Key words: Bobcat, cougar, Cylicospirura spp., livestock damage, mtDNA sequence, phylogenetic analysis, spirurid nematodes, stomach nodules.

INTRODUCTION mation does exist from captive cougars or those euthanized for safety reasons. How- Cougars (Puma concolor)areoneofthe ever, these studies often involve small, most common large predators in Oregon with isolated reports lacking a large enough a recently estimated population of 5,100 sample size to evaluate the effect of (Oregon Department of Fish and Wildlife diseases or parasites. Several parasites have [ODFW], 2006). Each year many are been found in cougars, such as Trichinella euthanized by governmental agencies when spiralis (Kluge, 1967; Winters, 1969), they damage livestock or threaten pets and Alaria marclanae (Fischthal and Martin, people. Explanations for why cougars are 1977), Nanophyetus salmincola (Kistner et involved in these situations include habitat al., 1979), and Ollulanus tricuspis (Rickard encroachment, habituation to nonthreaten- and Foreyt, 1992). ing (nonhunting) humans, and younger Many spirurid nematodes cause nodules cougars with unestablished territories being in the gastrointestinal tract of their hosts. forced to share habitats with humans (Beier, In felines, four Cylicospirura species are 1991; Thornton and Quinn, 2009). associated with these lesions: Cylicospir- There are limited data on diseases and ura felineus, Cylicospirura subaequalis, parasites of wild cougars, but such infor- Cylicospirura advena, and Cylicospirura

140 FERGUSON ET AL.—NODULE-FORMING STOMACH WORMS IN OREGON COUGARS 141 heydoni. The latter two species have been associated with nematodes in the stomach. recorded only from felids in the South Worms were identified as Cylicospirura Pacific, whereas the former two have a sp. (Foreyt, pers. comm.). This prompted cosmopolitan distribution. Cylicospirura ODFW to conduct necropsies on all dead heydoni infects marsupials in Australia cougars, and preliminary results from 20 (Spratt et al., 1991); however, it has also cougars indicated that these parasites were been reported from feral cats (Felis catus) linked to the deaths of many of the cougars in Tasmania (Milstein and Goldsmid, killed for safety concerns (ODFW, 2006). A 1997). The only record of C. advena is question arose whether these prevalent the original species description from a infections could play a role in altering the Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021 feral cat in New Zealand (Clark, 1981). behavior of cougars in Oregon because The type species, C. subaequalis, has been there are examples of this phenomenon recorded from many hosts, such as cougar occurring in other parasitized hosts (e.g., in North America (Waid and Pence, 1988; fish; Barber and Wright, 2005). Rickard and Foreyt, 1992), domestic cat in Here, we present results of a survey of Asia (Silamban et al., 1996), lion (Panthera Cylicospirura spp. and associated nodules leo) in the Congo and leopards (Panthera in the stomach or duodenum of 160 pardus) in West Africa (Sandground, cougars and 17 bobcats from locations 1929, 1930), tiger (Panthera tigris)in around Oregon collected over 7.5 yr. Data India (Alwar and Mudaliar, 1947), jagua- were analyzed to determine which cougars rundi (Puma yagouaroundi) in South were associated with higher infections and America (Seesee et al., 1981), and even which ones were being killed for livestock the spotted hyena (Crocuta crocuta)in damage or safety concerns. Worms were Africa (Round, 1968). Cylicospirura feli- identified by morphologic comparisons neus has also been reported from many and sequence analysis of the mitochondri- felid species, including domestic and feral al DNA (mtDNA) cytochrome c oxidase cats in Australia (Gregory and Munday, subunit 1 (cox1) gene. 1976; Pavlov and Howell, 1977; O’Call- aghan et al., 2005), Africa (Junker et al., MATERIALS AND METHODS 2006), and Asia (Gill, 1972); Bengal tiger Sample collection (Panthera tigris tigris) in Indochina (Ya- maguti, 1961); and lynx (Lynx canadensis) The 160 cougar, 74 male and 86 female (Fig. 1), and the 17 bobcat specimens exam- and bobcat (Lynx rufus) in North America ined were collected between August 1999 and (Pence et al., 1978; Watson et al., 1981). February 2007. The causes of death for the Although both C. subaequalis and C. cougars were as follows: 53 (33.1%) were felineus are typically parasites of felids, killed because of livestock predation, 11 there are records of the former infecting (6.9%) were killed for public safety reasons, 77 (48.1%) were killed by hunters, four (2.5%) red fox (Vulpes vulpes) in India (Yamaguti, were found dead, seven (4.4%) were killed by 1961) and the latter infecting arctic fox vehicles, two (1.3%) were illegally killed, and (Vulpes lagopus) in Alaska (Rausch et al., six (3.7%) were killed by some other type of 1990) and red fox in Algeria (Round, 1968). accident. Cougars killed because of livestock An earlier survey of gastrointestinal and public safety concerns are referred to as problematic, whereas those in the other five helminths in cougars killed in northeast- categories were combined into a cause-of- ern Oregon in the 1970s found no death category called other. Cylicospirura spp. (Rausch et al., 1983). Regulations require any person killing a However, in 2002, two of us (D.H.J. and cougar to submit the head, female reproduc- R.J.B.) necropsied several cougars found tive organs, stomach, and proximal duodenum to ODFW. The agency analyzes both upper dead in southwestern Oregon (Rogue premolar teeth (if present) for determining Valley and Douglas County); the only sign age and also records date, method of kill, and of disease was peritonitis and nodules location where the kill occurred. Cougars are 142 JOURNAL OF WILDLIFE DISEASES, VOL. 47, NO. 1, JANUARY 2011

of male spicules, and location of the vulva in females in reference to the esophageal junction. Worm measurements were obtained using SPOT advanced digital camera and software (Diagnostic Instruments Inc., Ster- ling Heights, Michigan, USA). These characteristics were compared with the redescriptions of C. felineus and C. subaequalis by Pence et al. (1978) and Waid and Pence (1988), respectively. We examined 41 worms infecting cougars from 21 locations throughout

the state that represented different geographic Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021 areas (N, S, E, W, and central), with one to four worms from each cougar. This nonbiased FIGURE 1. Geographic locations of 160 kill sites systematic approach of subsampling was used of Oregon cougars sampled for nodular stomach because evaluating all worms was impractical. worms (Cylicospirura sp.), 1999–2007. One to two worms from every infected bobcat were examined, with 22 worms examined in considered adults at 2 yr old, based on criteria total from bobcats. previously described (Ashman et al., 1983). We divided the location of cougars broadly Molecular analysis into eastern and western Oregon samples, Single Cylicospirura spp. worms were based on the placement of sites in reference to selected from eight hosts of each species the Cascade Range, as defined by Campbell et (cougars and bobcats) from representative al. (2003). These two regions differ in climate geographic locations in the study to use for generally (and thus in habitats), with the east mtDNA (cox1) gene sequence comparison. as high desert and the west as Mediterranean. These individual worms were identified to the All bobcat samples were obtained from species level using morphologic criteria as hunters, and no further information was described above. Total DNA extraction was available for these specimens. performed using the Qiagen DNeasy Blood and Tissue kit per manufacturer’s instructions Specimen processing (QIAGEN, Valencia, California, USA) on Stomach and proximal duodenum from each worms that had been cut into smaller pieces. specimen were opened separately after thaw- The mtDNA cox1 gene from these samples ing. Contents were rinsed gently with water was amplified by polymerase chain reaction into nested sieves and sorted. Large helminths (PCR) using the nondegenerative primer set 9 9 were separated, counted, and fixed in 10% NTF (5 -TGATTGGTGGTTTTGGTAA-3 ) 9 formalin. The Cylicospirura species are red and NTR (5 -ATAAGTACGAGTATCAA- TATC-39), as previously described for other and are about 20–30 mm in length, so they members of Spirurida (Casiraghi et al., 2001). were collected by gross inspection. However, The PCR master mixes were prepared for each smaller larvae were collected from a 149-mm DNA extract in 50 ml containing 5 mlof copper screen with the aid of a hand template, 100 pmol of NTF and NTR primers, magnifying lens. The size and location of and 25 ml of Ready Mix (New England Biolabs nodules were documented before opening. Inc., Ipswich, Massachusetts, USA). Amplifi- Worms from stomach and duodenum and cations were performed on a Peltier 200 individual nodules were kept separate, count- thermocycler (MJ Research, now Bio-Rad ed, and fixed in 70% ethanol with 2% glycerin Laboratories, Hercules, California, USA) with by volume. Parasites inconsistent with Cyli- initial denaturation at 94 C for 7 min, 40 cycles cospirura spp. morphology were processed at 94 C for 1 min, 50 C for 1 min, and 72 C for and stored separately. Prevalence, abundance, 1 min, with a final extension at 72 C for 10 min. and intensity of infections are reported in The PCR products were electrophoresed in accordance with the definitions provided by a1% agarose gel mixed with SYBR Safe DNA Margolis et al. (1982) and Bush et al. (1997). gel stain (1ml/ml; Invitrogen, Carlsbad, Cali- Species identification fornia, USA) and visualized using ultraviolet transillumination. Distinct bands of around The identification of Cylicospirura spp. was 700 base pairs (bp) were excised and purified based on the examination of morphologic using the QIAquick gel extraction kit (QIAgen, characteristics, such as tooth morphology, size Hilden, Germany). The resulting amplicons FERGUSON ET AL.—NODULE-FORMING STOMACH WORMS IN OREGON COUGARS 143 were sequenced on an ABI Prism 3730 (Insightful Corporation, Seattle, Washington, Genetic Analyzer with the BigDye Terminator USA) or R version 2.7.2 (R Project for v. 3.1 Cycle Sequencing Kit (Applied Biosys- Statistical Computing, Wien, Austria). The tems, Foster City, California, USA) using the specimens were grouped into cause-of-death same primers as for the PCR. In total, quality categories labeled problematic and other as sequence data were obtained from six C. described above. The intensity of worms and felineus and five C. subaequalis specimens. abundance of nodules were compared be- Electropherograms were examined manually tween these two groups using an exact and edited using BioEdit (Ibis Therapeutics, Wilcoxon rank-sum test. This nonparametric Carlsbad, California, USA; Hall, 1999). The test was used in lieu of t-tools because the resulting sequences were entered into the assumption of normality was violated based on National Center for Biotechnology Informa- the Kolmogorov-Smirnov goodness-of-fit test. Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021 tion (Bethesda, Maryland, USA) GenBank The sex, sample site (eastern vs. western), basic local alignment search tool (BLAST; and age (youth vs. adult) were also analyzed in Altschul et al., 1997), and 15 of the closest relation to the cause-of-death categories by matches were used to create a multiple using Fisher’s exact tests. Lastly, sex, age, and sequence alignment using ClustalX (Larkin et site were analyzed in relation to intensity of al., 2007). worms and abundance of nodules. Statistical significance was set at P,0.05 and P-values Phylogenetic analysis are two-tailed. Phylogenetic analyses were performed on the multiple sequence alignment using the RESULTS neighbor-joining and maximum parsimony methods in the MEGA4 software package Almost 120 cougars were infected with (Tamura et al., 2007) with 10,000 replicates to Cylicospirura sp. (Table 1). Nodules were determine the bootstrap value as a measure of found in 83% of the cougars, with 89% of robustness. MrBayes (Ronquist and Huelsen- beck, 2003) was used to perform a Bayesian these animals infected with Cylicospirura analysis of the data set using a general time sp. The remaining 11% of cougars were reversible nucleotide model and an inverse not infected with Cylicospirura sp. but gamma substitution rate. Analyses were run had a single nodule in their stomachs. for 1,000,000 generations and sampled every 100 generations. Trees before convergence Four of these cats had porcupine (Erethi- were discarded to reconstruct the resulting zon dorsatum) quills in their nodule. At Bayesian consensus tree with posterior prob- least one nodule containing worms oc- abilities. All trees were reconstructed using curred in 73% of infected cougars, where- Strongyloides stercoralis as a putative out- as only 27% of infected cougars lacked group, because it has been used in several other phylogenetic studies involving spirurids worms in all of their nodules. The mean (e.g., Traversa et al., 2007; Iorio et al., 2009). diameter of nodules was 1.2 cm Interspecific and intraspecific pairwise dis- (range50.2–4.5 cm), and the mean depth tances between the two Cylicospirura species was 0.6 cm (range50.1–3 cm). Worms sequences and those from closely related were found in 65% of the nodules genera were calculated using MEGA4 with . the Kimura-2 parameter model including all 0.2 cm, whereas many of the very small codon positions. nodules (#0.2 cm) had worms in them, and some contained porcupine quills Histology instead. A mean of 12.4 worms/nodule Three nodules from a single cougar were (SD534.1) was observed, with a maxi- examined by histology. Previously frozen mum of 340 worms/nodule. In cougars, all nodules were fixed in 10% buffered formalin, stomach and duodenal nodules that con- processed using standard techniques, and tained worms were found near the pyloric stained either with hematoxylin and eosin or with Masson’s trichrome. sphincter, with the exception of four nodules that were about 5–10 cm distal Statistical analysis to the esophageal sphincter. Large nod- All statistical comparisons were conducted ules in cougars contained many worms with either S-PLUS version 8.0 software residing together in the same tissue cavern 144 JOURNAL OF WILDLIFE DISEASES, VOL. 47, NO. 1, JANUARY 2011

TABLE 1. Occurrence of Cylicospirura subaequalis and nodular lesions in cougars from Oregon by sex, age, geographic location, and cause of death.

Nodule abundancea Worm intensitya Infection Category prevalence (%) Range Median Mean SD Range Median Mean SD

Total 117/160 (73.1) 0–10 2.0 1.6 1.5 1–562 15.0 38.3 63.1 Female 68/86 (79.1) 0–10 2.0 2.1 1.4 1–562 14.5 37.3 75.4 Male 49/74 (66.2) 0–7 2.0 2.1 1.5 1–340 15.0 39.6 61.4 Youngb 42/48 (87.5) 0–10 2.0 1.9 1.5 1–63 5.5* 12.2 14.1 Adultb 54/85 (63.5) 0–7 2.0 2.3 1.5 1–340 26.0* 59.8 70.3 Eastern 43/71 (60.6) 0–7 2.0 2.2 1.3 1–340 13.5 31.7 51.5 Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021 Western 74/89 (83.1) 0–10 2.0 2.0 1.7 1–562 21.0 49.5 92.5 Problematicc 43/64 (67.2) 0–7 2.0 1.9 1.2 1–562 23.0* 62.9 104.0 Otherc 74/96 (77.1) 0–10 2.0 2.2 1.6 1–155 11.0* 24.0 30.0 a Data from infected cats only. b Cougars considered adults at 24 mo (Ashman et al., 1983); age was not determined for 27 cougars. c Problematic cougars were those killed for damage control or safety concerns. * Statistically significant difference (P,0.05) between the medians within comparative groups (the mean was not tested).

(Fig. 2A, B). In one instance, an intact 65% of bobcats had nodules, and 82% of nodule from a cougar extended to the these were infected with Cylicospirura sp. serosa and was filled with a suppurative The mean diameter of nodules in bobcats exudate. was 1.1 cm (range50.1–2.5 cm), and the Prevalence in bobcats was 53%, with mean depth was 0.4 cm (range50.1– intensity ranging from 1 to 25 worms 0.8 cm), which was slightly smaller than found in all but two small nodules. About those in cougars were and appeared to

FIGURE 2. Cylicospirura spp. from cougars and bobcats. (A) Dissected nodule showing numerous C. subaequalis nematodes. Bar51 cm. (B) Intact nodule from cougar stomach. Bar51 cm. (C) Dissected mouth region of C. felineus showing trifid teeth. Bar525 mm. (D) Anterior region of C. subaequalis with bifid teeth (arrow). Bar525 mm. FERGUSON ET AL.—NODULE-FORMING STOMACH WORMS IN OREGON COUGARS 145

trabeculae of woven bone that lack osteoid. The densely packed mesenchymal cells between the collagenous septae had a high nucleus:cytoplasm ratio with poorly defined cell boundaries. Nuclear details were obscured by freezing and autolysis but one or more nucleoli were typically visible. Comparing cougar sexes, no statistical

difference was found in the median worm Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021 intensity or nodule abundance (P50.72 and P50.88, respectively, exact Wilcoxon rank-sum test; Table 1). Also, there were no geographic differences in median worm intensity and nodule abundance FIGURE 3. Cylicospirura subaequalis nodule (P50.26 and P50.17, respectively, exact from cougar stomach. Hematoxylin and eosin. (A) Wilcoxon rank-sum test); infections were Low magnification of nodule showing cross-sections of worms in lumen (right side of photograph). the same east and west of the Cascade Bar5500 mm. (B) Higher magnification demonstrat- Range (Table 1). Median worm intensity ing inner aspect of capsular wall, with alternating was greater in adult than young cougars bands of tightly packed mesenchymal cells and (P,0.01, exact Wilcoxon rank-sum test; collagenous trabeculae. Bar525 mm. Table 1), with adult cougars having about 16.5 more worms than young cougars had involve similar layers of the gastrointesti- (95% confidence interval [CI]58–37 nal tissue. worms). There was no difference in Histology of cougar nodules showed median nodule abundance between adult that they tended to be confined to the and young cougars (P50.20, exact Wil- submucosal layer but, in one instance, coxon rank-sum test; Table 1). elicited mild serosal inflammation through There were no significant differences a compressed tunica muscularis. The when comparing the two categoric causes nodule lumen usually communicated with of death with cougar age and geographic the gastric lumen via a fistula or a small location (P50.20 and P50.11, respective- ulcer through the mucosal layer. The core ly, Fisher’s exact test). However, there of the nodule included one or more worms were significantly more male (n524) than plus necrotic (sometimes partially miner- female (n519) cougars that were classified alized) cellular debris with an outer zone as problematic (P,0.01, Fisher’s exact of macrophages, neutrophils, plasma cells, test). and a few multinucleate giant cells. The Cougars that were killed because of wall consisted of regularly alternating safety and livestock concerns (problematic lamina of broad collagenous trabeculae cougars) had more than a two-and-a-half separated by cords of densely packed, times greater mean intensity of infection pleomorphic mesenchymal cells with min- than did those that died from other causes imal stroma (Fig. 3). At the periphery, the (Table 1), and the median worm intensity fibers in these collagenous septa were of these cougars was significantly greater oriented in a parallel pattern, and there than those grouped as other (P50.03, were fewer pleomorphic mesenchymal exact Wilcoxon rank-sum test; Table 1). cells. Along the inner aspect of the The problematic cougars had a median capsule, the fibers were interwoven worm intensity of 8.0 worms more than around evenly spaced stellate cells, result- the cougars killed for other reasons (95% ing in an appearance that resembles CI51–21 worms). However, there was no 146 JOURNAL OF WILDLIFE DISEASES, VOL. 47, NO. 1, JANUARY 2011 significant difference in median nodule in parentheses): the long spicule ranged abundance between the two death cate- from 1.9 to 3.5, with a mean of 2.9 (0.53); gories (P50.31, exact Wilcoxon rank-sum the short spicule was 0.2–0.6, with a mean test). of 0.4 (0.07). Those of C. felineus (n533) Infection prevalence in bobcats was were as follows: the long spicule was 1.0– 53% (9 of 17 bobcats), which was about 2.1, with a mean of 1.4 (0.4), and the short two-thirds the prevalence of cougar infec- spicule was 0.4–0.9, with a mean of 0.5 tions. Worm intensity in bobcats ranged (0.2). Also, although not a definitive from 1 to 25 worms/cat, with a mean of characteristic, C. subaequalis males typi-

10.9 worms/cat (SD57.3), which was cally have four papillae on the tail, Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021 about three-and-a-half times less than that whereas those of C. felineus have five of cougars. The biggest difference be- (Waid and Pence, 1988). tween infections of bobcats and cougars The length and width measurements for was the maximum intensity, with a cougar C. subaequalis (n566), respectively, were harboring approximately 22 times more 6.2–29.3, with a mean of 19.4 (5.7), and worms than a bobcat. The abundance of 0.2–0.6 with a mean of 0.4 (0.10). The nodules in bobcats ranged from 0 to 3 length and width measurements for C. nodules/cat, with a mean of 1 nodule/cat felineus (n533), respectively, were 12.5– (SD50.94). As with cougars, most nodules 25.4, with a mean of 18.1 (4.0), and 0.3–0.9 in bobcats contained worms, with only two with a mean of 0.6 (0.2). The female worms of 11 bobcats (18%) containing nodules of both species were always much larger without worms. In contrast to cougars, all than the male worms. The measurements nodules containing worms were located in of C. felineus and C. subaequalis were the stomach near the esophageal sphinc- consistent with that of previously published ter. Also, nodules in bobcats had multiple, redescriptions by Pence et al. (1978) and small, tube-like spaces, and each con- Waid and Pence (1988), respectively. tained two worms. Histology was not The single worms occupying one nodule conducted on nodules from bobcats. each, which were found near the esoph- Regarding worm identification, repre- ageal sphincter in four cougars, were of sentative samples from infected cougars too poor integrity to identify based on were identified as C. subaequalis, whereas morphology. However, these samples samples from all infected bobcats were were used for sequence analysis and two identified as C. felineus. This was based on of the four samples provided sequences the differences in distinct tooth morphol- identified as C. subaequalis. Lastly, there ogy between the two species, C. subae- were two cougars that contained either 1 qualis having bifid teeth and C. felineus or 15 Physaloptera sp. (spirurid) inside a trifid teeth (Fig. 2C, D). Morphologic nodule with C. subaequalis worms. Simi- differences in genitalia were also used larly, there was one bobcat that contained for species differentiation. In females of C. a single Physaloptera sp. inside a nodule subaequalis, the vulva is posterior to the with C. felineus worms. These worms were esophagointestinal junction, whereas in C. identified to the genus level based on felineus it is anterior. In males of C. observation of the prominent expansion of subaequalis, the spicules are more un- their cuticular sheath on the anterior ends. equal in length than are those of C. Sequence data from the mtDNA cox1 felineus, with the large spicule of C. gene were obtained from six C. felineus subaequalis approximately twice as long (GenBank accession number GQ342967) as that of C. felineus. The following and five C. subaequalis (GenBank acces- measurements are all in millimeters. The sion number GQ342968) specimens. The measurements of male spicules of C. contiguous consensus sequences con- subaequalis were as follows (n566; SD structed were both 689 bp. Pairwise FERGUSON ET AL.—NODULE-FORMING STOMACH WORMS IN OREGON COUGARS 147 Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021

FIGURE 4. Phylogenetic relationships of Cylicospirura species with related nematodes. Reconstruction computed using MrBayes software with a general time-reversible model and an inverse gamma substitution rate. Values shown at nodes represent posterior probability values based on 1,000,000 generations. The tree was rooted using Strongyloides stercoralis as a putative outgroup. distance between the two sequences from were not robust. Although they showed the different species was 11.4%.In high bootstrap values for the clade formed contrast, the maximum difference within by the Cylicospirura spp. and the cluster- species (i.e., worms from the same host ing with Spirocerca lupi, the values for species) was ,0.5%. deeper branches of the tree were low. The The multiple sequence alignment of 18 tree produced using Bayesian analysis spirurid taxa consisted of 600 nucleotides (Fig. 4) was the most robust at all levels with 375 sites conserved among all 18. based on posterior probabilities and again Overall mean distance was 0.15. Phyloge- showed that the Cylicospirura spp. form a netic reconstructions using all three meth- clade and group closely with S. lupi. ods produced trees with similar topology; however, phylogenetic trees reconstructed DISCUSSION using the neighbor-joining and maximum parsimonymethodsbasedonthese- The main aims of this study were to quence of the cox1 gene (data not shown) identify parasites associated with stomach 148 JOURNAL OF WILDLIFE DISEASES, VOL. 47, NO. 1, JANUARY 2011 nodules in cougars and bobcats in Oregon Subsequently, authors such as Round and to map the distribution of infections. (1968) recognized that and included the Using this large data set, we evaluated the proper name in their checklists of hel- correlation between nodule-forming minth parasites of African felids. Similarly, worms and problematic cougars. These previous reports of C. subaequalis infect- parasites were confidently identified as C. ing lynx in Canada (Threlfall, 1969; Van subaequalis in cougars and C. felineus in Zyll De Jong, 1966) and bobcat in Texas bobcats by morphologic and molecular (Little et al., 1971) have been regarded as comparisons. These two species have been dubious by Pence et al. (1978), based on found previously in wild felids in North their identification of specimens as C. Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021 America. Cylicospirura felineus infects felineus in bobcats and lynx from the same bobcat and lynx (Pence et al., 1978) but locations. not cougars. Cylicospirura subaequalis Further complicating the views of host was first found in cougars in Texas (Waid specificity is that C. felineus has been listed and Pence, 1988) and later in Washington as a rare parasite of Arctic foxes, with a (Rickard and Foreyt, 1992), but a survey prevalence of ,0.2% in .1,500 samples in of cougars in neighboring northeastern Alaska (Rausch et al., 1990). Round (1968) Oregon in the 1970s did not detect any also lists this parasite infecting red fox in cylicospirurids (Rausch et al., 1983). Algeria. Another species, Cylicospirura However, that study did not include visual skrjabini, also infects foxes from Russia inspection of the gastroduodenal mucosa. (Kozlov, et al., 1964) and displays similar The first infection in Oregon was found by trifid tooth morphology as C. felineus, one of us (D.H.J.) in 2002. based on the drawings of Baylis (1927), Some early studies considered C. feli- but with slightly more pointed ends. neus and C. subaequalis to be conspecific Rausch et al. (1990) speculated that C. (Chabaud, 1959; Skrjabin, 1991), but skrjabini, which typically infects foxes, may others have maintained them as separate also occur in Alaska because of its close species (Sandground, 1932; Yamaguti, proximity to Russia; thus this parasite 1961; Pence et al., 1978, Waid and Pence, could have been misidentified as the 1988), based on the distinctive tooth commonly felid-specific C. felineus. Eval- morphology of C. felineus (trifid teeth) uation of these worms using molecular and C. subaequalis (bifid teeth). Our methods would likely help to resolve these results support the views of the latter issues. authors because we found differences in The taxonomic and phylogenetic rela- both morphology and gene (cox1)se- tionships within the order Spirurida have quence. Here, we demonstrated an 11% been unclear. This order is split into two difference in the mtDNA cox1 gene suborders, Camallanina and Spirurina, sequence between C. felineus and C. which are divided into two and 10 subaequalis, which is consistent with that superfamilies, respectively, based on mor- found between other species of spirurids phology and life-cycle characteristics (Traversa et al., 2007; Iorio et al., 2009). (Chabaud, 1975). However, with the Some accounts of Cylicospirura spp. advent of molecular biology and sequence infecting felids might be erroneous. For analyses, other taxonomic arrangements example, the early record by Seurat (1913) have been proposed (e.g., Traversa et al., of C. subaequalis infecting the African 2007; Iorio et al., 2009). Based on wild cat (Felis silvestris lybica) in Algeria available sequences in GenBank, the two is clearly C. felineus,basedonhis species in our study were closely related, description and illustration of trifid teeth. and their closest relative appears to be This was likely due to C. subaequalis being Spirocerca lupi. Both genera are placed in the only known species at the time. the family Spirocercidae (Chabaud, 1975). FERGUSON ET AL.—NODULE-FORMING STOMACH WORMS IN OREGON COUGARS 149

Both C. felineus and C. subaequalis are vores; however, they may also prey on cosmopolitan parasites, with records of black-tailed deer (Odocoileus hemionus either species infecting felids or canids hemionus) and mountain beaver (Aplo- from six of the seven continents (Junker et dontia rufa; Toweill and Anthony, 1988). al., 2006); Antarctica lacks suitable hosts. Cougars in Oregon prey on (in descending Prevalence of C. felineus in bobcats may frequency) deer (Odocoileus spp.), elk vary. Pence et al. (1978) reported the (Cervus elaphus), porcupine, snowshoe prevalence in bobcats from Texas and in hare, and deer mice (Peromyscus spp.; lynx from Alberta, Canada, to be about Maser and Rohweder, 1983; Toweill and

70%, whereas prevalence in bobcats from Maser, 1985). Some mice feed heavily on Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021 Nebraska and West Virginia were only insects, and thus they would be a likely about 12% (Watson et al., 1981) and 10% paratenic host for Cylicospirura spp. in (Tiekotter, 1985), respectively. This spe- cougars and bobcats. cies occurs at a very low prevalence (4%) Most of the stomach nodules in bobcats in domestic and feral cats in Australia and cougars in Oregon were infected with (Pavlov and Howell, 1977). In our study, Cylicospirura spp., and thus we conclude C. felineus was detected in 50% of bobcats that they were the cause of the lesions. All from Oregon. For C. subaequalis, cougars but 15 cougars contained nodules associ- from Texas are infected at a prevalence of ated with C. subaequalis, and four of these 50% (Waid and Pence, 1988), which was worm-free nodules contained porcupine lower than the prevalence detected in our quills. Two cougars and one bobcat had study. The mean intensities of C. felineus nodules that were coinfected with a have been documented to be consistently Physaloptera sp., which was likely a less than that of C. subaequalis.For secondary invader. A similar scenario has example, the mean intensity of C. felineus been noted with nodules formed by C. in bobcats was 8 worms/cat in Nebraska felineus in North American bobcats that (Tiekotter, 1985), 8–10 worms/cat in Texas also contained Cyathospirura chevreuxi (Stone and Pence, 1978; Pence and Eason, (Pence et al., 1978). These authors stated 1980), and 3 worms/cat in West Virginia that C. felineus was always in nodules, and (Watson et al., 1981). These intensities C. chevreuxi primarily occupied the lu- were much lower than the mean intensity men, suggesting that this parasite raided of C. subaequalis in Texas cougars (53.6 lesions produced by C. felineus. Previous worms/cat; Waid and Pence, 1988). Our studies have implicated the Cylicospirura findings are in agreement with these species as the cause of gastrointestinal accounts because the mean intensity of nodules in felids. For example, the pres- C. subaequalis in cougars was about three- ence of C. subaequalis inside nodules has and-a-half times higher than that of C. been recorded in cougars (Waid and felineus in bobcats. Pence, 1988; Rickard and Foreyt, 1992) The life cycles of Cylicospirura species and a jungle cat (Felis chaus; Pillai, 1981), are unknown, but based on descriptions of and C. felineus has been found inside other members in the Spirocercidae fam- nodules of bobcats (Watson et al., 1981). ily (e.g., Spirocerca lupi), they probably Other studies have further described this involve insect intermediate hosts or a relationship by evaluating the histopathol- paratenic vertebrate host. Stone and ogy of these lesions associated with C. Pence (1978) proposed that spirurids, felineus in bobcats (Tiekotter, 1985; Pence such as C. felineus, may be transmitted et al., 1978) and in a domestic cat (Junker via paratenic hosts because bobcats are et al., 2006), and with C. subaequalis in a strictly carnivorous. Oregon bobcats target domestic cat (Rahmen and Hegde, 1971), smaller species, such as snowshoe hare and C. advena in a feral cat (Clark, 1981). (Lepus americanus), rodents, and insecti- Our findings were consistent with previ- 150 JOURNAL OF WILDLIFE DISEASES, VOL. 47, NO. 1, JANUARY 2011 ous reports in terms of the overall we did not detect significantly more microscopic organization of the nodules. problem cougars from the more populated Unlike Spirocerca lupi, nodules associated western part of the state than the eastern with Cylicospirura thus far have not been side. This finding is not in accordance with shown to progress to neoplasms. the habitat-encroachment hypothesis; It is not possible to determine the clinical however, we may not have been able to effect of the nodules, but in one instance, a detect such a pattern because of the nodule from a cougar that extended to the coarse scale used in our study. serosa might have progressed to perfora- In conclusion, we have demonstrated tion and peritonitis. Occasional large nod- with molecular and morphologic methods Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021 ules partially occluded the pyloric sphinc- that C. felineus and C. subaequalis are ter, which may have compromised separate species, as has been noted by digestion. Thus, these gastric parasites have previous authors (Sandground, 1932; Ya- the potential to render their hosts less fit to maguti, 1961; Pence et al., 1978, Waid and hunt wild prey. Few cases have attempted Pence, 1988). Although we found a to evaluate the role of disease on cougars statistical correlation with increased worm attacking humans. In a review of cougar intensity in problematic cougars, a plausi- attacks during a 100-yr period, Beier (1991) ble scenario is that the presence of large found only two reports of diseased cougars numbers of these parasites causes minor attacking people: one cougar had rabies debilitation in the cat but is not directly and the other had cataracts. Indeed, our responsible for the poor health and analysis showed that cougars that were associated problem behavior. Alternative- killed because of their interactions with ly, cougars near farms or in urban areas livestock or humans (problematic cougars) may consume more infected intermediate had a significantly higher mean worm or paratenic hosts, or perhaps debilitated intensity compared with those killed for cougars prefer prey items that serve as other reasons. these hosts. Cougar age and sex were both significant factors in our analysis that could be ACKNOWLEDGMENTS potentially confounding variables. In gen- J.A.F would like to graciously thank the eral, more young cougars are killed for funding agency, ODFW, for the employment problem reasons than those taken during opportunity to conduct research unrelated to the hunting season in Oregon (Trainer and his thesis. We acknowledge R. Green (ODFW) for coordinating transference of samples and Golly, 1992), presumably because of accompanied information and A. Kent for inadequate territory. However, we did assistance with sample necropsies. not find younger cougars to be more frequently problematic. Perhaps this is LITERATURE CITED because adult cougars were associated ALTSCHUL, S. F., W. GISH,W.MILLER,E.W.MYERS, with higher worm burdens, which may AND D. J. LIPMAN. 1990. Basic local alignment be debilitating, and thus contributing search tool. Journal of Molecular Biology 215: more to the pool of problematic cougars 403–410. while diluting the association seen with ALWAR, V. S., AND S. V. MUDALIAR. 1947. A check list of parasites (class Nematoda) in the Department younger cougars. The increased burden of Parasitology, Madras Veterinary College observed in older cougars could be Laboratory. Indian Veterinary Journal 24: 77–94. attributable to new infections continuously ASHMAN, D., G. C. CHRISTENSEN,M.L.HESS,G.K. being obtained throughout time. Our TSUKAMOTO, AND M. S. WICKERSHAM. 1983. The findings that more male cougars were mountain lion in Nevada. Nevada Division of Wildlife, Reno, Nevada, 91 pp. associated with being problematic than BARBER, I., AND H. A. WRIGHT. 2005. Effects of were females could be related to differ- parasites on fish behaviour: Interactions with ences in behavior and territoriality. Lastly, host physiology. Fish Physiology 24: 109–149. FERGUSON ET AL.—NODULE-FORMING STOMACH WORMS IN OREGON COUGARS 151

BAYLIS, H. A. 1927. Some new parasitic nematodes (cox1) gene analysis. Parasitology Research 104: from Australia. Annals and Magazine of Natural 979–984. History, Series 9 20: 214–225. JUNKER, K., J. H. VORSTER, AND J. BOOMKER. 2006. BEIER, P. 1991. Cougar attacks on humans in the First record of Cylicospirura (Cylicospirura) United States and Canada. Wildlife Society felineus (Chandler, 1925) Sandground, 1933 Bulletin 19: 403–412. (Nematoda: Spirocercidae) from a domestic cat BUSH, A. O., K. D. LAFFERTY,J.M.LOTZ, AND A. W. in South Africa. Onderstepoort Journal of SHOSTAK. 1997. Parasitology meets ecology on its Veterinary Research 73: 257–262. own terms: Margolis et al. revisited. Journal of KISTNER, T. P., D. WYSE, AND J. A. SCHMITZ. 1978. Parasitology 83: 575–583. Pathogenicity attributed to massive infection of CAMPBELL, S., D. AZUMA, AND D. WEYERMANN. 2003. Nanophyetus salmincola in a cougar. Journal of Forests of eastern Oregon: An overview. U.S. Wildlife Diseases 15: 419–420. Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021 Department of Agriculture, Forest Service, KLUGE, J. P. 1967. Trichinosis and sarcosporidiosis in Pacific Northwest Research Station, General puma. Wildlife Disease Association Bulletin 3: Technical Report PNW-GTR-578, Portland, 110–111. Oregon, 31 pp. KOZLOV, D. P., N. I. OVSYOKOVA, AND Z. P. RADKEVICH. CASIRAGHI, M., T. J. C. ANDERSON,C.BANDI,C. 1964. Cylicospirura skrjabini n. sp. (Spirurata) BAZZOCCHI, AND C. GENCHI. 2001. A phylogenetic from foxes. Trudy Gel’mintologicheskoi Labor- analysis of filarial nematodes: Comparison with atorii Academiya Nauk SSSR 14: 105–108. [In the phylogeny of Wolbachia endosymbionts. Russian.] Parasitology 122: 93–103. LARKIN, M. A., G. BLACKSHIELDS,N.P.BROWN,R. CHENNA,P.A.MCGETTIGAN,H.MCWILLIAM,F. CHABAUD, A. G. 1959. Sur la systematique des nematodes proches de Spirocerca lupi (Rud., VALENTIN,I.M.WALLACE,A.WILM,R.LOPEZ,J. D. THOMPSON,T.J.GIBSON, AND D. G. HIGGINS. 1809). Parassitologia 1: 129–135. [In French.] 2007. Clustal W and Clustal X version 2.0. ———. 1975. Keys to genera of the order Spirurida, Bioinformatics 23: 2947–2948. part 2: Spiruroidea, Habronematoidea and LITTLE, J. W., J. P. SMITH,F.F.KNOWLTON, AND R. R. Acuarioidea. In CIH (Commonwealth Institute BELL. 1971. Incidence and geographic distribu- of Helminthology) keys to the nematode para- tion of some nematodes in Texas bobcats. Texas sites of vertebrates, R. C. Anderson, A. G. Journal of Science 22: 302–314. Chabaud and S. Willmott (eds.). Commonwealth MARGOLIS, L., G. W. ESCH,J.C.HOLMES,A.M. Agricultural Bureaux, Farnham Royal, UK, KURIS, AND G. A. SCHAD. 1982. The use of pp. 29–58. ecological terms in parasitology (report of an ad CLARK, W. C. 1981. Cylicospirura advena n. sp. hoc committee of the American Society of (Nematoda: Spirocercidae) a stomach parasite Parasitologists). Journal of Parasitology 68: from a cat in New Zealand, with observations on 131–133. related species. Systematic Parasitology 3: 185– MASER, C., AND R. S. ROHWEDER. 1983. Winter food 191. habits of cougars from northeastern Oregon. FISCHTHAL, J. H., AND R. L. MARTIN. 1977. Alaria Great Basin Naturalist 43: 425–428. (alaria) marclanae (LaRue 1917) Walton 1950 MILSTEIN, T. E., AND J. M. GOLDSMID. 1997. Parasites (Trematoda: Diplostomatidae) from a mountain of feral cats from southern Tasmania and their lion, Felis concolor acrocodia Goldman, from potential significance. Australian Veterinary Paraguay. Journal of Parasitology 63: 202. Journal 75: 218–219. GILL, H. S. 1972. Incidence of gastro-intestinal O’CALLAGHAN, M., J. REDDIN, AND D. LEHMANN. 2005. helminths in cats (Felis catus) in Delhi. Journal Helminth and protozoan parasites of feral cats of Communicable Diseases 4: 109–111. from Kangaroo Island. Transactions of the Royal GREGORY, G. G., AND B. L. MUNDAY. 1976. Internal Society of South Australia 129: 81–83. parasites of feral cats from the Tasmanian OREGON DEPARTMENT OF FISH AND WILDLIFE. 2006. Midlands and King Island. Australian Veterinary Oregon cougar management plan. www.azgfd.gov/ Journal 52: 317–320. pdfs/w_c/bhsheep/OregonCougarManagement HALL, T. A. 1999. BioEdit: A user-friendly biological Plan2006.pdf. Accessed October 2009. sequence alignment editor and analysis program PAVLOV, P. M., AND M. J. HOWELL. 1977. Helminth for Windows 95/98/NT. Nucleic Acids Sympo- parasites of Canberra cats. Australian Veterinary sium Series 41: 95–98. Journal 53: 599–600. IORIO, R., J. SLAPETA,D.OTRANTO,B.PAOLETTI,A. PENCE, D. B., AND S. EASON. 1980. Comparison of the GIANGASPERO, AND T. DONATO. 2009. Phylogenet- helminth faunas of two sympatric top carnivores ic relationships of Habronema microstoma and from the rolling plains of Texas. Journal of Habronema muscae (Spirurida: Habronemati- Parasitology 66: 115–120. dae) within the order Spirurida inferred using ———, H. P. SAMOIL, AND J. E. STONE. 1978. mitochondrial cytochrome c oxidase subunit 1 Spirocercoid stomach worms (Nematoda: Spir- 152 JOURNAL OF WILDLIFE DISEASES, VOL. 47, NO. 1, JANUARY 2011

ocercidae) from wild felids in North America. carnivores of the Chaco Boreal, Paraguay. In Canadian Journal of Zoology 56: 1032–1042. Proceedings of the worldwide furbearer confer- PILLAI, K. M., C. PYTHAL, AND R. K. SUNDARAM. 1981. ence, J. A. Chapman and D. Pursley (eds.). A note on the occurrence of Cylicospirura International Fur Trade Federation, Frostburg, subaequalis (Molin, 1860) (Spiruridae: Nemato- Maryland Collection II, pp. 846–858. da) in a jungle kitten (Felis chaus) in Kerala. SEURAT, L. G. 1913. Sur deux spiropteres du chat Kerala Journal of Veterinary Science 12: 155– gante (Felis ocreata). Comptes Rendus des 156. Se´ances de la Socie´te´ de Biologie et de ses RAHMAN, S. A., K. S. HEDGE,S.MOHIYUDEEN,S.J. Filiale (Paris, France) 74: 676–679.. [In French.] SESHADRI,P.M.RAHAMATULLA, AND D. RAJASE- SILAMBAN, S., K. M. RAMACHANDRAN,T.L.DEVI, AND KHARAN. 1971. Study of Cylicospirura subaequa- K. M. PILLAI. 1996. Histopathology of Cylicos-

lis (Molin, 1860) and the histopathology of the pirura subaequalis (Molin, 1860) induced nod- Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021 nodule in the stomach of a cat. Indian Veterinary ule in the stomach of a cat. Journal of Veterinary Journal 48: 683–687. Animal Sciences 27: 68–69. RAUSCH, R. L., F. H. FAY, AND F. S. L. WILLIAMSON. SKRJABIN, K. I. 1991. Key to parasitic nematodes, 1990. The ecology of Echinococcus multilocu- Vol. 1: Spirurata and Filariata. E.J. Brill, Leiden, laris (Cestoda, Taeniidae) on St. Lawrence New York, New York, 497 pp. Island, Alaska, 2: Helminth populations in the SPRATT, D. M., IBEVERIDGE, AND E. L. WALTER. 1991. definitive host. Annales de Parasitologie Hu- A catalogue of Australasian monotremes and maine et Comparee 65: 131–140. marsupials and their recorded helminth para- ———, C. MASER, AND E. P. HOBERG. 1983. sites. Records of the South Australian Museum Gastrointestinal helminths of the cougar, Felis Monograph Series 1: 1–105. concolor L., in northeastern Oregon. Journal of STONE, J. E., AND D. B. PENCE. 1978. Ecology of Wildlife Diseases 19: 14–19. helminth parasitism in the bobcat from west RICKARD, L. G., AND W. J. FOREYT. 1992. Gastroin- Texas. Journal of Parasitology 64: 295–302. testinal parasites of cougars (Felis concolor)in TAMURA, K., J. DUDLEY,M.NEI, AND S. KUMAR. 2007. Washington and the first report of Ollulanus MEGA4: Molecular evolutionary genetics anal- tricuspis in a sylvatic felid from North America. ysis (MEGA) software version 4.0. Molecular Journal of Wildlife Diseases 28: 130–133. Biology and Evolution 24: 1596–1599. RONQUIST, F., AND J. P. HUELSENBECK. 2003. MrBayes THORTON, C., AND M. S. QUINN. 2009. Coexisting with 3: Bayesian phylogenetic inference under mixed cougars: Public perceptions, attitudes, and models. Bioinformatics 19: 1572–1574. awareness of cougars on the urban-rural fringe ROUND, M. C. 1968. Check list of the helminth parasites of Calgary, Alberta, Canada. Human-Wildlife of African mammals of the orders Carnivora, Conflicts 3: 282–295. Tubulidentata, Proboscidea, Hyracoidea, Artiodac- THRELFALL, W. 1969. Further records of helminths tyla and Perissodactyla. Commonwealth Agricul- from Newfoundland mammals. Canadian Jour- tural Bureau Technical Communication 38 of the nal of Zoology 47: 197–201. Commonwealth Bureau of Helminthology, St. TIEKOTTER, K. L. 1985. Helminth species diversity and Albans, Hertfordshire, UK, 252 pp. biology in the bobcat, Lynx rufus (Schreber), from SANDGROUND, J. H. 1929. A new liver fluke from a Nebraska. Journal of Parasitology 71: 227–234. monkey and new parasitic roundworms from TOWEILL, D. E., AND R. G. ANTHONY. 1988. Annual various African animals. Proceedings of the U.S. diet of bobcats in Oregon’s Cascade Range. National Museum 75: 1–11. Northwest Science 62: 99–103. ———. 1930. Report on the Harvard African ———, AND C. MASER. 1985. Food of cougars in the expedition upon the African Republic of Liberia Cascade Range of Oregon. Great Basin Natu- and the Belgian Congo, part III: Medical and ralist 45 (1): 77–80. biological investigations—Notes and descrip- TRAINER, C. E., AND N. E. GOLLY. 1992. Cougar age tions of some parasitic helminths collected by and reproduction: Pittman-Robertson Federal the expedition. Contributions of the Department Aid Project W-87-R, progress report, subproject of Tropical Medicine and the Institute of 281, job 3. Oregon Department of Fish and Tropical Biology and Medicine (Harvard Uni- Wildlife, Corvallis, Oregon, 14 pp. versity) 5: 462–486. TRAVERSA, D., F. COSTANZO,R.IORIO,I.AROCH, AND ———. 1932. Report on the nematode parasites E. LAVY. 2007. Mitochondrial cytochrome c collected by the Kelley-Roosevelts expedition to oxidase subunit 1 (cox1) gene sequence of Indo-China with descriptions of several new Spirocerca lupi (Nematoda, Spirurida): Avenues species, part 1: Parasites of birds, part 2— for potential implications. Veterinary Parasitolo- Parasites of mammals. Zeitschrift fu¨ r Parasiten- gy 146: 263–270. kunde (Berlin, Germany) 5: 542–583. VAN ZYLL DE JONG, C. G. 1966. Parasites of the SEESEE, F. M., D. E. WORLEY, AND R. L. MARTIN. Canada lynx, Felis (Lynx) canadensis (Kerr). 1981. A survey of nematode parasites from Canadian Journal of Zoology 44: 499–509. FERGUSON ET AL.—NODULE-FORMING STOMACH WORMS IN OREGON COUGARS 153

WAID, D. D., AND D. B. PENCE. 1988. Helminths of WINTERS, J. B. 1969. Trichiniasis in Montana moun- mountain lions (Felis concolor) from southwest- tain lions. Wildlife Disease Association Bulletin ern Texas with a redescription of Cylicospirura 5: 400. subaequalis (Molin, 1860) Vevers, 1922. Cana- YAMAGUTI, S. 1961. Systema helminthum, Vol. III: dian Journal of Zoology 66: 2110–2117. The nematodes of vertebrates, parts I and II. WATSON, T. G., V. F. NETTLES, AND W. R. DAVIDSON. Interscience Publishers, New York, New York, 1981. Endoparasites and selected infectious 1261 pp. agents in Bobcats (Felis rufus) from West Virginia and Georgia. Journal of Wildlife Dis- Submitted for publication 10 December 2009. eases 17: 547–554. Accepted 3 August 2010. Downloaded from http://meridian.allenpress.com/jwd/article-pdf/47/1/140/2238941/0090-3558-47_1_140.pdf by guest on 28 September 2021