Parasitol Res (2012) 110:1855–1862 DOI 10.1007/s00436-011-2710-z

ORIGINAL PAPER

Parasites and vector-borne pathogens of southern plains woodrats (Neotoma micropus) from southern

Roxanne A. Charles & Sonia Kjos & Angela E. Ellis & J. P. Dubey & Barbara C. Shock & Michael J. Yabsley

Received: 14 August 2011 /Accepted: 6 November 2011 /Published online: 23 November 2011 # Springer-Verlag 2011

Abstract From 2008 to 2010, southern plains woodrats reaction (PCR) testing of blood samples from 104 woodrats (Neotoma micropus) from southern Texas, were examined detected a novel Babesia sp. in one (1%) and Hepatozoon for parasites and selected pathogens. Eight helminth species sp. in 17 (16%) woodrats. Partial 18S rRNA gene sequence were recovered from 97 woodrats including, Trichuris of the Babesia was 94% similar to B. conradae. Histologic neotomae from 78 (prevalence = 80%), Ascarops sp. from examination of tissues detected intestinal coccidia in seven 42 (43%), Nematodirus neotoma from 31 (32%), Raillietina of 104 (7%), Sarcocystis neotomafelis in 26 (25%), sp. from nine (9%), Taenia taeniaeformis larvae from eight Hepatozoon sp. in 21 (20%), and Dunnifilaria meningica (8%), and an unidentified spiurid, a Scaphiostomum sp. and in four (4%) woodrats. Three woodrats (5%) were a Zonorchis sp. each from a single woodrat. Besnotia seropositive for Toxoplasma gondii. Ectoparasites recov- neotomofelis was detected in three (3%) woodrats and ered included fleas (Orchopeas sexdentatus and O. neo- microfilaria were detected in seven (7%). Polymerase chain tomae), ticks (Ixodes woodi and Ornithodoros turicata), mites (Trombicula sp. and Ornithonyssus (Bdellonyssus) bacoti) and bot flies (Cuterebra sp.). The only difference in R. A. Charles : B. C. Shock : M. J. Yabsley Southeastern Cooperative Wildlife Disease Study, prevalence related to gender was for N. neotoma (males> Department of Population Health, College of Veterinary Medicine, females, p=0.029). Prevalence of T. neotomae and all University of Georgia, intestinal parasites combined was significantly higher in 589 DW Brooks Drive, Wildlife Health Building, adults compared with juveniles (p=0.0068 and p=0.0004), Athens, GA 30602, USA respectively. Lesions or clinical signs were associated with S. Kjos Cuterebra and B. neotomofelis. Collectively, these data Marshfield Clinic Research Foundation, indicate that woodrats from southern Texas harbor several Marshfield, WI 54449, USA parasites of veterinary and/or medical importance. A. E. Ellis Athens Diagnostic Laboratory, College of Veterinary Medicine, University of Georgia, Introduction Athens, GA 30602, USA

J. P. Dubey The southern plains woodrat (Neotoma micropus), com- Department of Agriculture, Agricultural Research monly called a packrat, is a medium-sized, nocturnal Service, and Natural Resources Institute, that inhabits semiarid brush lands, low valleys and plains of Animal Parasitic Diseases Laboratory, the south-central and southwestern United States and Building 1001, Beltsville, MD 20705, USA northeastern Mexico. In Texas, N. micropus inhabits areas : dominated by thorny desert shrubs or cacti (Braun and B. C. Shock M. J. Yabsley (*) Mares 1989), and their diet consists mainly of vegetation Daniel B. Warnell School of Forestry and Natural Resources, such as succulent leaves and fruit of cacti, seeds and acorns University of Georgia, Athens, GA 30602, USA (Raun 1966). Woodrats (Neotoma spp.) are common hosts e-mail: [email protected] for ticks and fleas which are potential vectors of tularemia 1856 Parasitol Res (2012) 110:1855–1862

(Francisella tularensis), plague (Yersinia pestis), Q fever were selected based on fresh tracks and rodent droppings at (Coxiella burnetti), relapsing fever (Borrelia spp.) and the base of presumed woodrat nests built among cactus Rocky Mountain spotted fever (Rickettsia rickettsi). Other (Opuntia spp.) plants. Traps were set in the afternoon and pathogenic organisms reported from woodrats include checked the following morning. Trypanosoma cruzi (causative agent of Chagas disease in humans and domestic ), Besnoitia neotomofelis, and Anesthesia and blood collection Leishmania mexicana (McHugh et al. 1990; Dubey and Yabsley 2010; Pinto et al. 2010). Captured animals were anesthetized and weighed. Briefly, Although numerous studies have looked at the ectoparasitic woodrats were anesthetized with 100 mg/kg ketamine (Fort fauna of woodrats in Texas, to date, only a few studies have Dodge Laboratories, Fort Dodge, IA) followed by blood looked at endoparasites of southern plains woodrats. Collec- collection via cardiocentesis into potassium ethylenediami- tively, in the United States and Mexico, only nine species have netetraacetic acid (K2EDTA) BD Vacutainer® tubes (Beckton been reported including: Taenia taeniaeformis, Litomosoides Dickinson, Franklin Lakes, NJ) using aseptic techniques. In carinii, Dunnifilaria meningica, Trichuris muris, L. mexicana, 2010, blood smears were made with fresh blood, air-dried, Try. cruzi, Try. neotomae, Sarcocystis neotomafelis,andB. fixed in absolute alcohol for 5 min, and stained with Geimsa neotomofelis (Packchanian 1942; Johnson 1966; Burkholder stain. All animals were euthanized by cervical dislocation et al. 1980; Gutierrez-Pena 1989; Galaviz-Silva et al. 1991; and adult and juvenile (not pups) were then necropsied and Pinto et al. 2010; Charles et al. 2011). Because higher examined for parasites. All techniques were reviewed and diversities of parasites have been reported in other species of approved by the IACUC committee at the University of woodrats in the southwestern United States, we conducted this Georgia. study to better understand the endo- and ectoparasitic fauna of southern plains woodrats from Uvalde County, Texas. Parasite collection and identification

Each woodrat was examined for ectoparasites by combing Materials and methods back the fur and collecting specimens with fine forceps. Collected ectoparasites were preserved in 100% ethanol. Trapping Bot-fly larvae were removed by gentle traction with forceps and characterized using polymerase chain reaction (PCR) A total of 104 southern plains woodrats (56 females and 48 and sequencing of two regions of the cytochrome oxidase males) were trapped during July 2008 and March and May subunit I (COI) gene as described in Table 1. Fleas, mites, 2010 at four sites in Uvalde County, Texas. Animals were and ticks were mounted on slides using saline solution and live trapped by small squirrel cage traps (Havahart, Litz, identified to species with a light microscope and published PA) and large Sherman traps (H.B. Sherman Traps, taxonomic keys (Eads 1950; Keirans and Litwak 1989; Tallahassee, FL) baited with dried apricots. Trap stations Lewis 2000; Haas et al. 2004).

Table 1 Oligonucleotide primers used in polymerase chain reaction assays

Target organisma Gene target Primer Primer sequence (5′–3′) Reference

Babesia/Hepatozoon (1°) 18S rRNA 3.1 CTCCTTCCTTTAAGTGATAAG Yabsley et al. (2005) Babesia/Hepatozoon (1°) 18S rRNA 5.1 CCTGGTTGATCCTGCCAGTAGT Yabsley et al. (2005) Babesia/Hepatozoon (2°) 18S rRNA RLBH-F GAGGTAGTGACAAGAAATAACAATA Yabsley et al. (2005) Babesia/Hepatozoon (2°) 18S rRNA RLBH-R TCTTCGATCCCCTAACTTTC Yabsley et al. (2005) Rickettsia (1°) 17 kDa antigen 17 k-3 TGTCTATCAATTCACAACTTGCC Labruna et al. (2004) Rickettsia (1°) 17 kDa antigen 17 k-5 GCTTTACAAAATTCTAAAAACCATATA Labruna et al. (2004) Rickettsia (2°) 17 kDa antigen 17Kd1 GCTCTTGCAACTTCTATGTT Labruna et al. (2004) Rickettsia (2°) 17 kDa antigen 17kD2 CATTGTTCGTCAGGTTGGCG Labruna et al. (2004) Cuterebra Cytochrome oxidase I (COI) C1-J-2183 CAACATTTATTTTGATTTTTTGG Noël et al. (2004) C1-N-2659 GCTAATCCAGTGAATAATGG C2-J-3138 AGAGCTTCACCCTTAATAGAGCAA C2-N-3661 CCACAAATTTCTGAACATTGACCA a 1°, primers used in the primary amplification; 2°, primers used in secondary amplification of a nested PCR protocol Parasitol Res (2012) 110:1855–1862 1857

During necropsy, the viscera of all woodrats were set of DNA extractions, and one water control for each set of grossly examined for the presence of parasites such as primary and secondary PCR reactions. Amplicons were Taenia and Besnoitia cysts. The entire length of gastroin- visualized by trans-illumination of an ethidium bromide- testinal tract and some organs (pancreas, liver, and spleen) stained 1.5% agarose gel. were removed from the abdominal cavity, dissected under a dissecting scope, and closely examined for helminths. Statistical analyses Contents were filtered through a 100 μm sieve (W.S. Tyler Incorporated, Mentor, OH) for concentration of parasites. Parasite prevalence, intensity and range were calculated as All parasites were stored in 100% ethanol and examined defined by Bush et al. (1997). Fisher’s exact test was used under a light or dissecting microscope for identification. to test for differences in parasite prevalence (by species and Large nematodes were cleared with a 70% ethanol/30% collectively) among age classes and gender. A two-way phenol solution. analysis of variance (ANOVA) implemented by SAS, was used to determine if gastrointestinal nematode intensity Histopathology varied according to age and/or gender.

Tissue samples including brain, lung, liver, heart, kidney, spleen, lymph nodes, quadriceps, gonads and sections of Results the gastrointestinal tract were preserved in 10% buffered formalin for histopathological examination. Small sections A total of nine helminth species were recovered from of formalin-fixed tissues were embedded in paraffin, 90 of 97 woodrats (54 females/43 males and 79 adults/ sectioned at 5 μm and stained with hematoxylin and eosin 18 juveniles) including four species of nematodes (H&E) for light microscopic examination. (Trichuris neotomae, Nematodirus neotoma, Ascarops sp. and a single unidentified female spiurid), two species Serologic testing for Toxoplasma gondii of cestodes (Raillietina sp. and Taenia taeniaeformis) and two species of trematodes (Scaphiostomum sp., a pancreatic Sera samples from 66 woodrats were tested for antibodies fluke, and Zonorchis sp., a liver fluke) (Table 2). to T. gondii by the modified agglutination test (MAT) as Additionally, microfilaria of an unknown filarial nema- described by Dubey and Desmonts (1987). tode species (likely either Litosomoides carinii or Dunni- filaria meningica) was found in the blood. The T. Molecular detection of parasites taeniaformis cysticerci were found encysted in the liver and the adult stage of a Raillietina sp. was found in the Several PCR assays were used to test woodrats for lumen of the small intestine. The three nematodes were all hemoparasites and vector-borne bacteria. DNA was found in the gastrointestinal tract, Ascarops sp. in the extracted from 100 μl of whole blood using the DNeasy stomach, N. neotomae in the small intestine, and T. blood and tissue kit (Qiagen, Inc., Valencia, CA) according neotomae in the large intestine (Table 2). The only to the manufacturer’s protocol. The extracted DNA was helminth that was associatedwithgrosslesionswasthe used as a template to test for species of Babesia, Ascarops sp., which were surrounded by areas of Hepatozoon, and Rickettsia as described in Table 1. Briefly, thickened gastric mucosa (~1 cm diameter) that housed for primary amplification, 5 μl DNA was added to 20 μlof multiple worms, up to 17 in one case. Seven woodrats a master mix containing 11 μl of molecular grade biological (three females/four males and two adults/five juveniles) water (MGBW), 2.5 μl of 25 mM MgCl2,5μl of GoTaq were negative for all helminths. Flexi Clear Buffer (Promega, Madison, WI), 0.25 μlof No difference in prevalence of whipworms was noted for 20 mM dNTP, 0.5 μl of each primer (40 μM), 0.25 μlof gender (p=1.000) but adults had a significantly higher GoTaq Flexi (Promega). For each secondary reaction (if prevalence compared with juveniles (p=0.0068). In con- needed), 1 μl of primary product was used as a template in trast, prevalence of N. neotomae was significantly higher in a 25-μl reaction containing similar PCR components with males (19 of 43) compared to females (12 of 54) (p= the exception of an additional 4 μl of water and different 0.0285), but no difference was noted for age (p=0.165). primers. Overall, gender was not associated with differences in Stringent protocols and controls were utilized in all PCR prevalence of intestinal parasites (p=0.460) but age was, reactions to prevent and detect contamination. Separate with more adults 77 of 79 (98%) being infested than dedicated laboratory areas were used for DNA extraction, juveniles (12 of 18 [67%]; p=0.0004). Based on ANOVA, primary amplification, secondary amplification, and prod- the interaction of age and gender on parasite intensity for all uct analysis. A negative water control was included in each gastrointestinal nematodes was not significant (p>0.05). 1858 Parasitol Res (2012) 110:1855–1862

Table 2 Helminth parasites of 97 southern plains woodrats Parasite No. positive (%) No. collected Mean intensity Range (Neotoma micropus) from Uvalde County, Texas Nematoda Trichuris neotomae 78 (80.4) 448A, 55Ia 6.4 1–53 Ascarops sp. 42 (43) 100A, 82I 4.3 1–17 Nematodirus neotoma 31 (32) 637 20.5 1–134 Filarial nematodes 7 (7) n.a. n.a. n.a. Dunnifilaria meningica 4 (4) n.a. n.a. n.a. Unidentified female spiurid 1 (1) 1 1.0 1 Cestoda Raillietina sp. 9 (9) 24 2.7 1–13 T. taeniformis cysts 8 (8) 19 2.4 1–8 Trematoda Scaphiostomum sp. 1 (1) 1 1 0–1 Zonorchis sp. 1 (1) 3 3 0–3 aA adults, I immature

A total of 181 ectoparasites (118 fleas, nine ticks, 39 negative in 2010 (Table 4). Partial 18S rRNA gene sequence mites and 15 bots) were collected from 42 of the 104 (40%) of the Babesia species was most similar (434 of 460 bp, woodrats (Table 3). Bot fly larvae were found predomi- 94.4% similarity) to B. conradae (AF158702), a parasite of nantly on the chest and neck regions of infested woodrats domestic dogs in California. Other similar Babesia species but one (first instar) larva was found in the nasal cavity. A included B. lengau (94.1%) from cheetahs (Acinonyx single woodrat (juvenile female, weight 118 g) that had four jubatus) (GQ411405–GQ411417), B. vesperuginis (92.4%) larvae (1.5–2 cm in length) under the chin and on the chest from a Pipistrellus sp. bat from England (AJ871610), and B. region was found severely emaciated, lethargic, and duncani (92%) from humans in California (AY027815). nonresponsive a few inches outside one of our traps. We Hepatozoon sp. was commonly detected in blood samples of were able to collect the woodrat by hand after which it died woodrats by PCR (Table 4); no gamonts were observed in while we performed an external examination. Sequence of a blood smears. All woodrat blood samples were PCR fragment of the COI gene (411 bp) from a single bot fly negative for Rickettsia. Three of 66 (5%) woodrats were was 87.9% similar to C. grisea (AY507182) and 86.8% positive for antibodies to T. gondii (titers of 1:25, 1:25, and similar to C. fontinella (AY507188). Sequences of another 1:400). Additionally, T. gondii was isolated from digested region of the COI gene (544 bp) from two bot-fly larvae brain, heart and tongue tissue from one seronegative wood- from two different animals were identical and 90.4% rat, but not from any of the three seropositive woodrats (data similar to C. fontinella (AY507197) and 89.9% similar to not shown; Dubey et al. 2011). C. grisea (AY507222). Several parasites were detected during histologic exam- Based on PCR testing and sequencing, a novel Babesia ination of tissues. A meningeal worm (Dunnifilaria was detected in one of 42 (2%) woodrats in 2008; all were meningica), associated with mild lymphocytic and eosino-

Table 3 Ectoparasite infestation a of 104 southern plains woodrats, Ectoparasites No. collected No. infested (%) Mean intensity Range Neotoma micropus, from Uvalde County, Texas Fleas Orchopeas sexdentatus 33♂,73♀ 40 (39) 2.7 1–13 O. neotomae 7♂ 6 (10) 1.2 1–2 Ticks Ixodes woodi 3A♀, 5 N 5 (5) 1.6 1–2 Ornithodoros turicata 1A 1 (1) 1.0 1 Mites Trombicula sp. 3 L 2 (2) 1.5 1–2 Ornithonyssus (=Bdellonyssus) bacoti 36A 6 (6) 2.0 1–29 Bot flies Cuterebra sp. 15 L 7 (12) 2.1 1–4 aL larva, N nymphs, A adults Parasitol Res (2012) 110:1855–1862 1859

Table 4 Bacterial and protozoan parasites of 104 southern plains Organism Diagnostic method No. infected (%) woodrats (Neotoma micropus) from Uvalde County, Texas Rickettsia spp. Polymerase chain reaction 0 (0) Babesia sp. Polymerase chain reaction 1 (1) Hepatozoon sp. Polymerase chain reaction 17 (16) Histology 21 (20) Besnoitia neotomofelis Gross examination and histology 3 (3) Sarcocystis neotomafelis Histology 26 (25) Coccidian oocysts Histology 7 (7) Toxoplasma gondii Serology 3 (5) philic meningitis, was observed in the meningeal and Texas, are hosts to several parasites and pathogens, and submeningeal spaces of four (4%) woodrats. Sarcocysts of although most were not associated with significant gross or S. neotomafelis were observed in 26 of 104 (25%) woodrats histological lesions, some may cause disease in humans, with more sarcocysts being found in the quadriceps muscle woodrats, and other animals. (26/26; 100%) compared with the myocardium (4/26; All ectoparasites recovered from the woodrats in this 15%). S. neotomafelis cysts ranged from a few (3–4) to study have been previously recorded from this host with the numerous (>50) per tissue sample. A few cysts were exception of O. neotomae (Johnson 1966). This flea species accompanied with a mild multifocal myositis. Hepatazoon feeds primarily on Neotoma spp., especially the Mexican cysts were observed in the liver of 21 (20%) woodrats woodrat (N. mexicana). The range of N. mexicana overlaps compared to 17 (16%) that were positive with PCR of with that of N. micropus in , far western Texas, blood samples. Only six woodrats were positive for and parts of Mexico but not in our study area. Similar to Hepatozoon sp. by PCR and histology. Two encysted our findings, Stark (1958) found coinfestations of with O. flukes surrounded by fibrous capsules were found in the neotomae and O. sexdentatus on the same host (Stark liver of one woodrat. Based on histology, Besnoitia 1958). Both flea species are involved in the host–flea neotomofelis was detected in the tissues of two woodrats complex in the spread of Yersinia pestis in western United (2%). A third woodrat, which was moribund at capture, had States (Anderson and Williams 1997). numerous grossly visible cysts present in the facial skin and Flies of the genus Cuterebra areverycommonin throughout the musculature and subcutaneous layer of the most temperate and tropical regions in the western body (Charles et al. 2011). Developmental stages (micro- hemisphere (Sabrosky 1986). Larvae of these flies infest and macrogametocytes and oocysts) of coccidia were found the subcutis of lagomorphs and (including Neo- in the small and large intestine of seven (7%) woodrats. toma spp.). Although bots are relatively large compared to their hosts, they rarely cause mortality. Young animals are more prone to injury or increased susceptibility to Discussion predation if larval burdens are high. In this study, two infested woodrats exhibited good body condition, but one Southern plains woodrats are known to be hosts for a suite juvenile woodrat that was infested with four bots ~2 cm of parasites, some of which are of medical and veterinary long, had poor body condition, and was likely moribund importance (Packchanian 1942; Johnson 1966). Consider- due to Cuterebra infestation. Sequences from our woodrat able work has been done to characterize the ectoparasitic samples were most similar to C. fontinella and C. grisea fauna of southern plains woodrats, but to date, there is a (only two Cuterebra spp. in Genbank), which are both paucity of reported information on the diversity and parasites of mice in the genus (Noeletal. prevalence of parasites and vector-borne pathogens in this 2004). Although bot flies are common in woodrats, rodent species. In this current study, we report eight new unfortunately there are no sequences available for the parasite–host associations including one flea (Orchopeas many Cuterebra spp. reported from Neotoma spp. for neotomae), two trematodes (Scaphiostomum sp. and comparison with the sequences we obtained (Baird 1979, Zonorchis sp.), one stomach nematode (Ascarops sp.), an 1997; Wilson et al. 1997). intestinal nematode (N. neotoma), and three protozoan Several of the helminths detected in this study are parasites (T. gondii, Babesia sp., and coccidia). Addition- common parasites of Neotoma spp. including Trichuris ally, we report S. neotomafelis infection in N. micropus for spp., N. neotomae, Raillietina sp., and T. taeniaeformis. the first time in the United States. Collectively, these data Prevalence of Trichuris spp. in woodrats is typically very indicate that southern plains woodrats in Uvalde Co., high; a previous study on N. micropus reported T. muris in 1860 Parasitol Res (2012) 110:1855–1862 all four woodrats examined (Johnson 1966). T. muris (likely A novel Babesia sp. was detected in a single woodrat from now considered to be T. neotomae) has also been reported our 2008 collection. Unfortunately, no blood smears were from the eastern woodrat (N. floridana) from Oklahoma made in 2008, so no morphologic data are available. Based and T. neotomae has been reported from the dusky-footed on partial 18S rRNA gene sequence, this woodrat Babesia woodrat (N. fuscipes) in California (Chandler 1945; Boren was most similar to B. conradae, a canine species from et al. 1993). Although this is the first report of N. neotoma California (Kjemtrup et al. 2006). This represents the first (=N. tortuosus) (Hoberg et al. 1988)inN. micropus, it has report of a Babesia species in a Neotoma spp.; however, a been reported from numerous other woodrat species different piroplasm, Theileria youngi, has been reported from including N. fuscipes, the bushy-tailed woodrat (N. cinerea), N. fuscipes from California (Kjemtrup et al. 2001). Possible the desert woodrat (N. lepida), N. mexicana,andN. floridana vectors include I. woodi, a common tick found in the current (Hall 1916; Tucker 1942;MillerandSchmidt1982). Neither study and previous studies (Eads et al. 1952;Johnson1966) of these helminths has been associated with disease in and the less commonly found ticks, Amblyomma inornatum, woodrats. Dermacentor variabilis,andD. parumapertus (Eads et al. Woodrats were hosts to two different species of 1952; Eads and Hightower 1956;Johnson1966). tapeworms: T. taeniaeformis, which were found as larvae Based on histology and PCR testing of blood, Hepatozoon in the liver, and a Raillietina sp. that was found as adults infections were common in this woodrat species. Recently, in the small intestine. T. taeniaeformis has been reported genetic characterization of Hepatozoon sp. from southern from southern plains woodrats previously with 26% of 88 plains woodrats found it was related to a Hepatozoon sp. being positive (Johnson 1966). Strobilocerci of T. from N. fuscipes in California as well as other rodent taeniaeformis are commonly found in the liver of wood- Hepatozoon (Allen et al. 2011). Similar to a study of rats, rabbits, squirrels,muskrats,bats,voles,othersmall Hepatazoon in cotton rats (Sigmodon hispidus), meronts rodents and occasionally humans, and the adult form in were present in the liver of our naturally infected woodrats felids, which are definitive hosts (Johnson 1966;Theis (Johnson et al. 2007). Interestingly, Hepatozoon from N. and Schwab 1992; Fichet-Calvet et al. 2003). Rarely does fuscipes and N. micropus were genetically similar to this parasite cause any significant lesions or disease in Hepatozoon detected in snakes and other rodents (Allen et intermediate hosts; however, high numbers can cause al. 2011). Genetic characterization of Hepatozoon from infertility and hepatic neoplasia in some rodents (Lin et snakes, as potential hosts, in southern Texas may clarify al. 1990; Irizarry-Rovira et al. 2007). Although Raillietina the natural history of this understudied group of parasites. spp. are uncommon in woodrats, they have been Three tissue cyst-forming coccidians, S. neotomafelis, B. reported in N. cinerea, N. fuscipes,andN. lepida neotomofelis, and T. gondii, were detected in woodrats in (Linsdale and Tevis 1951; Grundmann 1958; Miller and the current study and interestingly, all are suspected to Schmidt 1982). utilize felids as definitive hosts. Although S. neotomafelis Prior to this study, no trematode species have been was common in the woodrats in the current study, this reported from Neotoma spp. We detected at least two parasite had previously only been reported in N. micropus species, Scaphiostomum sp. and Zonorchis sp., and a from Nuevo Leon, Mexico (Galaviz-Silva et al. 1991). possible third species was found encysted in the liver of Only sections of heart and quadriceps were examined in one woodrat that is not believed to be Zonorchis (typically this present study and cysts were more prevalent in found in the gall bladder, bile ducts or small intestine quadriceps muscles whereas in a previous study, cysts were proximal to the bile duct opening) (McIntosh 1939; Santos more common in the masseter muscles and only rarely et al. 2010). Scaphiostomum spp. are usually found in the found in quadriceps muscles (Galaviz-Silva et al. 1991). pancreatic duct of its definitive rodent hosts, including the Currently, the only suspected definitive host for S. neo- white-ankled mouse (Peromyscus pectoralis) in Texas tomafelis is the domestic cat; however, the only study that (Santos et al. 2010). The primary intermediate host is the has attempted to experimentally determine the life cycle terrestrial flamed disc snail, Anguispira alternata, and depicts an oocyst passed in cat feces that is morphologically secondary intermediate terrestrial snail hosts include Neo- more similar to Isospora than Sarcocystis (Galaviz-Silva et helix (=Triodopsis) albolaris and Haplotrema concavum al. 1991). However, we believe that felids could be the (Schell 1985). Zonorchis sp. has also been reported from definitive host because a recent study found a Sarcocystis- the white-ankled mice in Texas, but this parasite utilizes the infected woodrat coinfected with two other felid- terrestrial snail Polygyra texasiana as an intermediate host transmitted protozoans, Besnoitia neotomofelis and T. (Schell 1985). The low prevalence of Scaphiostomum sp. gondii (Charles et al. 2011). Cysts of B. neotomofelis were and Zonorchis sp. is likely due to a low density of detected in low numbers in two woodrats which had no appropriate intermediate hosts or because these snails are lesions associated with infection; however, the finding of a rarely consumed by N. micropus. single woodrat that was moribund due to Besnoitia Parasitol Res (2012) 110:1855–1862 1861 infection indicates that B. neotomofelis can be a cause of Anderson SH, Williams ES (1997) Plague in a complex of white- clinical disease (Charles et al. 2011). Although Besnoitia tailed prairie dogs and associated small in Wyoming. J Wildl Dis 33:720–732 spp. are typically considered nonpathogenic, B. jellisoni,a Baird CR (1979) Incidence of infection and host specificity of parasite of deer mice (Peromyscus maniculatus)and Cuterebra tenebrosa in bushy-tailed wood rats (Neotoma kangaroo rats (Dipodomys ordii and D. microps) can cause cinerea) from central Washington. J Parasitol 65:639–644 disease if parasite numbers become very high (Ernst et al. Baird CR (1997) Bionomics of Cuterebra austeni (Diptera: Cuterebridae) and its association with Neotoma albigula 1968; Chobotar et al. 1970). Similarly, T. gondii infection (Rodentia: ) in the southwestern United States. J was rare in woodrats; only three had low antibody titers and Med Entomol 34:690–695 a single seronegative woodrat had viable T. gondii isolated Boren JC, Lochmiller RL, Boggs JF, Leslie DM Jr (1993) Gastroin- from tissues (data not shown) (Dubey et al. 2011). This testinal helminths of eastern woodrat populations in central Oklahoma. Southwest Nat 38:146–149 woodrat T. gondii isolate was genetically characterized as a Braun JK, Mares MA (1989) Neotoma micropus. Mamm Species type 12 lineage (Dubey et al. 2011). The only previous 330:1–9 study on woodrats failed to find any antibodies to T. gondii Burkholder JE, Allison TC, Kelly VP (1980) Trypanosoma cruzi in N. fuscipes in California (Dabritz et al. 2008). (Chagas') (Protozoa, Kinetoplastida) in invertebrate, reservoir, and human hosts of the Lower Rio-Grande valley of Texas. J Parasitol 66:305–311 Bush AO, Lafferty KD, Lotz JM, Shostak AW (1997) Pasitology Conclusions meets ecology on its own terms: Margolis et al., revisited. J Parasitol 83:575–583 Chandler AC (1945) Trichuris species from California rodents. J Our results provide evidence that southern plains woodrats in Parasitol 31:284–286 this locale have a high diversity of parasites and at least one of Charles RA, Ellis AE, Dubey JP, Barnes JC, Yabsley MJ (2011) the parasites detected, T. gondii, is zoonotic. A concurrent Besnoitiosis in a southern plains woodrat (Neotoma micropus) study also detected a high prevalence of Trypanosoma cruzi, from Uvalde, Texas. J Parasitol 97:838–841 Chobotar B, Anderson LC, Ernst JV, Hammond DM (1970) another important zoonosis (Charles, unpublished data). Of Pathogenicity of Besnoitia jellisoni in naturally infected kangaroo interest is the absence of gross lesions consistent with rats (Dipodomys ordii) in Northwestern Utah. J Parasitol 56:195– Leishmania mexicana on any of our woodrats (data not 197 shown) as this parasite has been reported in woodrats, Dabritz HA, Miller MA, Gardner IA, Packham AE, Atwill ER, Conrad PA (2008) Risk factors for Toxoplasma gondii infection including N. micropus, in Texas (McHugh et al. 1990;Grogl in wild rodents from central coastal California and a review of T. et al. 1991;McHughetal.2003). Additionally, several of the gondii prevalence in rodents. J Parasitol 94:675–683 parasites detected (i.e., B. neotomofelis, T. gondii, and Dubey JP, Desmonts G (1987) Serological responses of equids fed Cuterebra) can cause disease in woodrats. Additionally, the Toxoplasma gondii oocysts. Equine Vet J 19:337–339 Dubey JP, Velmurugan G, Rajendran C, Yabsley MJ, Thomas NJ, detection of a novel Babesia, which has unknown medical or Beckmen KB, Sinnett D, Ruid D, Hart J, Fair PA, McFee WE, veterinary importance, highlights the need for further work Shearn-Bochsler V, Kwok OCH, Ferreira LR, Choudhary S, Faria on parasites or vector-borne pathogens of these common, EB, Zhou H, Felix TA, Su C (2011) Genetic characterization of often peridomestic, rodents. Toxoplasma gondii in wildlife from North America revealed widespread and high prevalence of the fourth clonal type. Int J Parasitol 41:1139–1147 Acknowledgements The authors thank J. Edwards, E. Blizzard, and Dubey JP, Yabsley MJ (2010) Besnoitia neotomofelis n. sp. (Protozoa: D. Roellig for field assistance and L. Saunders, A. Page, and J. Huang Apicomplexa) from the southern plains woodrat (Neotoma for laboratory assistance. We also thank several landowners who micropus). Parasitology 137:1731–1747 generously allowed us to sample animals on their properties. This Eads RB (1950) The fleas of Texas. Texas State Health Department, study was primarily supported by the National Institutes of Health Austin, p 85 (R15 AI067304-02) which was focused on understanding the ecology Eads RB, Hightower BG (1956) The ticks of Texas, with notes on of Trypanosoma cruzi in these woodrats. Additional support was their medical significance. Tex J Sci 8:7–24 provided by the Southeastern Cooperative Wildlife Disease Study Eads RB, Menzies GC, Miles VI (1952) Acarina taken during west (SCWDS) (http://scwds.org/), through continued sponsorship of Texas plague studies. Proc Entomol Soc Wash 54:250–253 SCWDS member state and federal agencies. RAC was supported by Ernst JV, Chobotar G, Oaks EC, Hammond DM (1968) Besnoitia the Fulbright (LASPAU) program. jellisoni (Sporozoa: Toxoplasmea) in rodents from Utah and California. 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