Alaria Infection in Companion Animals in North Central Oklahoma from 2006 Through 2015 and Detection in Wildlife

Prevalence of Alaria infection in companion animals in north central Oklahoma from 2006 through 2015 and detection in wildlife

Eileen M. Johnson dvm, phd OBJECTIVE To determine the prevalence of Alaria infection in cats and dogs in north Yoko Nagamori dvm central Oklahoma over various periods and investigate whether wild animal Rebecca A. Duncan-Decocq ms species in this region were also infected. Patrick N. Whitley DESIGN Akhilesh Ramachandran bvsc&ah, phd Combined cross-sectional study and case series. Mason V. Reichard phd SAMPLE From the Department of Veterinary Pathobiology, Results of parasitological testing of fecal samples from 5,417 client-owned Center for Veterinary Health Sciences, Oklahoma dogs and 1,246 client-owned cats (2006 through 2014); fecal samples from State University, Stillwater, OK 74078 (Johnson, Naga- 837 shelter or rescue dogs and 331 shelter or rescue cats (2013 and 2014) mori, Duncan-Decocq, Ramachandran, Reichard); and and 268 feral cats (2015); tongue or jowl samples from cadavers of 43 wild the USDA APHIS Wildlife Service, 2800 N Lincoln Blvd, Oklahoma City, OK 73105 (Whitley). pigs, 3 opossums, and 1 raccoon; and intestinal tract segments from cadavers of 48 cats and 5 coyotes. Address correspondence to Dr. Johnson (eileen.johnson@ okstate.edu). PROCEDURES Various parasite recovery techniques were performed to detect various Alaria stages in samples. Recovered adult trematodes and mesocercariae were used for PCR assay and sequencing of the 28S rRNA gene. RESULTS Prevalence of Alaria infection was significantly higher in feral cats (9.0%) than in shelter or rescue cats (0.6%) and client-owned cats (1.4%) and in shelter or rescue dogs (1.8%) than in client-owned dogs (0.2%). Mesocer- cariae were recovered from tissue samples from 11 (26%) wild pigs and 1 opossum. Amplicon sequences from adult trematodes and mesocercariae were 100% identical to each other and 99% homologous to GenBank sequences of Alaria alata and Alaria mustelae. CONCLUSIONS AND CLINICAL RELEVANCE Prevalence of Alaria infection in the study area has increased in dogs and cats since 1990, when infections were rare. Prevalence in wild pigs was similar to that in Eurasia, where A alata is considered an emerging zoonotic parasite. (J Am Vet Med Assoc 2017;250:881–886)

iplostomatid trematodes in the genus Alaria Alaria arisaemoides, Alaria intermedia, Alaria Dhave a worldwide distribution. Domestic and taxidae, and Alaria marcianae.6 wild carnivores are the definitive hosts.1–6 Adult trem- In reported cases of human alariosis in North atodes in the small intestine pass eggs in the feces, America, Alaria infection was acquired through and the eggs embryonate and hatch in fresh water. consumption of improperly cooked game meat.9,10 Newly hatched miracidiae penetrate susceptible spe- The only Alaria sp recorded to date in Europe is cies of fresh water snails (eg, Helistoma, Planorbis, A alata.1,6,13,14 Although not definitively associated Lymnea, and Anisus spp) that support asexual devel- with human disease, A alata has been experimen- opment of miracidiae into sporocysts and furcocer- tally transmitted to a rhesus monkey.11,13 Over the cous cercariae.1,2,6,7 Cercariae penetrate the skin of past decade during mandatory testing of wild pigs tadpoles or frogs and become mesocercariae.1,2,8 Vari- (Sus scrofa scrofa) for Trichinella spp in the Euro- ous species of birds, reptiles, and mammals (includ- pean Union, A alata mesocercariae have been dis- ing humans) that ingest the infected frogs can serve covered in wild pig tissues, primarily in muscle fas- as collector (paratenic) hosts.1,2,6,9–13 cia and adipose tissue.1,6,14–19 Carnivores become infected when they ingest in- In Europe, alariosis had been classified as a low-risk fected frogs or paratenic hosts or through transmam- zoonosis.1,6,13,14 However, with expansion of wild pig pop- mary transmission.1–6 The mesocercariae migrate ulations, an increase in the prevalence of mesocercariae to the lungs and encyst as metacercariae that are in wild pigs, an increase in human consumption of coughed up and swallowed. Recognized Alaria spp meat from wild pigs, changes in cooking methods, include Alaria alata, Alaria mustelae, Alaria canis, and recognition of severe and fatal disease in hu-

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mans, A alata is now considered an emerging threat feral cats as part of the teaching hospital’s neuter-and- to human health.1,6,14–20 release program, Operation Catnip, in 2015 were also Surveillance of Trichinella spp in the United States included in the prospective portion of the study. is not mandatory and does not include wild pig popu- Forty-six cadavers of shelter cats, 1 stray cat, and lations.21 Control has been achieved in domesticated 1 client-owned cat submitted to the Oklahoma Animal pigs (Sus scrofa domesticus) through enforcement of Disease Diagnostic Laboratory for disposal between laws that prevent feeding of raw garbage and through March and July 2015 and 5 cadavers of coyotes that had improvements in biosecurity and hygiene in confine- recently been hit by cars and submitted to the same ment-type swine production units. Serologic testing laboratory between September 10, 2014, and March 5, (ELISA) is used in swine production herds to deter- 2015, were included in the study. Also included were mine Trichinella status. No information is available on tongues or jowl tissues collected from 43 wild pigs the role of wild pigs as a source of human infection through the cooperation of the USDA APHIS Wildlife with Alaria mesocercariae in the United States. Service during routine hunting and disease surveil- Efforts to control expanding wild pig populations lance in 6 Oklahoma counties (Jefferson, Love, Osage, in the United States through regulated hunting and Pawnee, Pittsburg, and Tillman) between February 23 trapping have been unsuccessful.22,23 This population and May 12, 2015, as well as tongue and jowl tissues expansion is believed to be due to periodic release of from 1 raccoon and 3 opossums that had been recently wild pigs from Eurasia into different areas of the Unit- hit by cars. Tissues were shipped on ice overnight to ed States for hunting and the escape of domesticated the Oklahoma Animal Disease Diagnostic Laboratory pigs that subsequently interbreed with these released for evaluation. wild pigs.22,23 Adaptability of wild pigs is related to their omnivorous feeding habits, high reproductive potential, Parasitological evaluation intelligence, and reclusive nature. They are considered of fecal samples large game and are used for hunting purposes and hu- Historical laboratory results for fecal samples man consumption in many parts of the world.20,24 from owned cats and dogs were reviewed retro- Studies1,6,13,18,25 in Europe have revealed a high spectively for findings of Alaria ova. All other fecal prevalence of A alata infection in red fox and wolf samples were prospectively evaluated specifically populations, which are also believed to be expanding. for the study. For all fecal samples, including those In North America, most reports4,5,26–32 of Alaria infec- represented in the retrospective portion of the study, tions in domesticated and wild carnivores pertain to a centrifugal fecal flotation technique with Sheath- Canada, the northern United States, and Florida. In a er sucrose or 33% zinc sulfate solution was used to study33 of endoparasitism in client-owned dogs con- evaluate samples for evidence of parasites.34 Slides ducted in north central Oklahoma from 1981 through prepared in this manner were examined at 100X and 1990, no Alaria ova were detected in 12,515 fecal sam- 400X magnification with a binocular microscope. ples. The purpose of the study reported here was to obtain an updated estimate of the prevalence of Alaria Parasitological evaluation of cadavers infection in various dog and cat populations and to de- Cadavers were processed and examined for gastro- termine whether infection existed in wildlife reservoir intestinal parasites by use of the following technique. hosts, primarily wild pigs, in north central Oklahoma. Intestinal tracts were removed. Segments (stomach, small intestine, and colon) were separated and placed Materials and Methods into individual buckets. Each segment was opened into a bucket and washed with running tap water. Washed Animals and samples intestinal contents were allowed to sit undisturbed for This study involved a retrospective and a prospec- 15 to 30 minutes, and the supernatant was carefully tive portion. For the retrospective portion, all results decanted. This procedure was repeated until the su- of parasitological examination of fecal samples from pernatant fluid appeared clear. Portions of sediment client-owned dogs and cats submitted to the Boren from each segment were transferred to clear glass pie Veterinary Medical Hospital of Oklahoma State Uni- plates, and grossly detectable parasites were removed versity or the Oklahoma Animal Disease Diagnostic and stored in jars containing 70% ethanol at 4°C. The Laboratory from 2006 through 2014 were included. sediment was then examined in 95 X 15-mm disposable In the prospective portion approved by the Insti- polystyrene Petri dishes with a dissecting microscope tutional Animal Care and Use Committee of Oklahoma (0.8X to 5.6X objective lenses) until all of the sediment State University, fecal samples were collected from from each segment had been examined and all para- shelter or rescue dogs and cats for 2 years (2013 and sites recovered. Trematode parasites from the intestinal 2014) through a newly implemented spay-neuter pro- tracts were identified by use of a taxonomic key.35 gram of the Boren Veterinary Medical Hospital. Dur- ing this 2-year period, additional fecal samples from Parasitological evaluation rescue cats were provided by 2 private feline practi- of tissue samples tioners located within 50 miles of the study area of Individual tissue samples were evaluated with Stillwater, Okla. Fecal samples collected from trapped the mesocercariae migration technique.36 Samples

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were weighed and cut into 2.5-cm3 cubes, then greater than the prevalence in both shelter or rescue placed in an electric meat grinder. The ground tissue cats and client-owned cats. The prevalence of Alaria (47.5 to 525.0 g) was wrapped in 2 layers of cheese- infection was also significantly P( < 0.001) greater in cloth, placed in disposable champagne glasses, and shelter or rescue dogs than in client-owned dogs. submerged in warm tap water for 90 minutes. Fluid Presence of Alaria spp in north central Okla- from the stem ends of the glasses was transferred into homa was further confirmed when the cadavers of a Petri dish by use of a disposable 5-mL pipet and ex- a client-owned cat (May 2014) and a stray cat (July amined with a dissecting microscope (0.8X to 5.6X 2014) were processed for adult parasite recovery. objective lenses) for motile mesocercariae. Samples Several 1.5- to 1.7-mm-long trematodes with distinct, in which motile mesocercariae were not identified spoon-shaped forebodies that contained oral and ven- were allowed to sit overnight and reexamined. Equip- tral suckers, a bulbous tribocytic organ, and 2 anteri- ment was washed with soap and water between each or spike-like pseudosuckers were recovered from the sample to prevent contamination. small intestinal contents (Figure 2). The flukes had cylindrical hind bodies that contained the reproduc- Molecular evaluation of Alaria spp tive organs. Typical Alaria ova were also identified Total DNA extractiona and amplification of the via zinc sulfate centrifugal flotation of fecal samples partial 28S rRNA gene were conducted on 2 of 11 collected from these cats. Five of 5 cadavers of coy- isolates of Alaria mesocercariae recovered from tis- otes that had been hit by cars from September 2014 sue samples from wild pigs, Alaria mesocercariae through May 2015 were also found to harbor numer- recovered from tissue samples from 1 opossum, and adult Alaria trematodes recovered from ca- ous adult Alaria trematodes in the small intestines. davers of 1 coyote, 1 stray cat, and 1 client-owned No adult trematodes were found in the intestinal cat as described elsewhere.12,37 Three to 5 para- tracts of the cadavers of 46 urban shelter cats submit- site stages/sample were used. The forward primer ted for disposal. 5′-CTTAGCTGCGGGTTCCTGCT-3′ and reverse prim- Mesocercariae were detected in tissues from 11 of er 5′-CGGCACATAAGCAAATACCTCG-3′ were used 43 (26%) wild pigs and 1 of 3 opossums (Figure 3). for PCR amplification.12 Amplicons were sent for se- These 11 wild pigs originated from 4 of 6 Oklahoma quencingb and alignment.c counties (Jefferson, Osage, Pawnee, and Tillman). The opossums and raccoon originated from Payne Statistical analysis County. Distinct bands (approx 250 bp in length) were Prevalence of Alaria spp infection among vari- detected via agarose gel electrophoresis of PCR prod- ous animal groups was compared by means of the χ2 ucts from 3 samples of mesocercariae collected from test.38 Values of P ≤ 0.05 were considered significant.d the cadavers of 2 wild pigs and 1 opossum and adult flukes collected from the cadavers of 2 cats and 1 coy- Results ote. Amplicon sequencing and subsequent alignment revealed 100% sequence identity among the 6 animals, Over the 9-year period, 9 of 5,417 (0.2%) fecal samples from client-owned dogs and 17 of 1,246 (1.4%) fecal samples from client-owned cats contained Alaria ova that measured 98 to 134 µm X 62 to 68 µm (Figure 1). Prevalence varied from year to year and ranged from 0% for both dogs (0/285) and cats (0/59) in 2006 to 0.3% (2/705) for dogs in 2013 and 2.3% (3/131) in cats in 2010. In 2013, 12 of 503 (2.4%) fecal samples from shelter or rescue dogs and 1 of 84 (1.2%) fecal samples from shelter or rescue cats contained Alaria ova. For 2014, these numbers were 3 of 334 (0.9%) and 1 of 247 (0.4%), respectively. Overall prevalence of Alaria infection in shelter or rescue dogs and cats for both years was 1.8% (15/837) and 0.6% (2/331), respectively. Twenty-four of 268 (9.0%) fecal samples collected in 2015 from trapped feral cats were posi- tive for Alaria ova. The prevalence of Alaria infection Figure 1—Photomicrograph of unstained Alaria ova recovered via zinc sulfate in feral cats was significantly P( < 0.001) centrifugal flotation from the fecal sample of a dog. Bar = 20 mm.

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mammary infections in dogs and cats have been reported, free-roaming animals rely on predation and scavenging for food, and feral animals travel and hunt near accessible water sources, which is an important component in the epidemiology of Alaria infection because of the required snail and frog intermediate hosts.3–5,8,13,25 Alariosis in definitive hosts such Figure 2—Photomicrograph of unstained adult Alaria trematodes recovered as dogs, cats, and wild carnivores is from the intestinal tract of a coyote. Bar = 200 mm. considered an infection of low patho- genicity, with clinical signs that are mild and nonspecific.1,6,32 However, these definitive hosts act as reservoirs of the adult parasite and contaminate the environment with ova. Human infections with Alaria mesocercariae, acquired through in- gestion of infected intermediate or paratenic hosts, can have serious se- quelae and fatal outcomes.9,10 Vague and varied clinical presentations in humans and lack of noninvasive diagnostic tests make diagnosis of human alariosis difficult. Published reports1,6,9,10,13 of alariosis in humans often include a history of consumption of improperly cooked game meat. The discovery of A alata mesocercariae in wild pigs from Europe over the past decade during mandatory testing for Trichinella lar- vae suggests that wild pig products, Figure 3—Photomicrograph of unstained Alaria mesocercariae recovered from particularly those consumed raw or jowl tissue from a wild pig. Bar = 100 µm. undercooked, could be a potential source of alariosis for humans.1,6,14–19 Identification of optimal methods for with 99% homology to GenBank sequences for A alata detection of infected carcasses and establishing risk and A mustelae. factors for human exposure have led to several stud- ies17,36,37,39–41 on the effects of tissue digestion solutions, sodium chloride and ethanol concentrations, Discussion simulated gastric solutions, curing processes, and In the present study, an increase in the preva- temperatures on the viability and tenacity of A ala- lence of Alaria infection was detected in dogs and ta mesocercariae. In a 2% sodium chloride solution, cats in north central Oklahoma since last measured mesocercariae were viable for up to 21 days but died in 1990.33 This increase was particularly high in free- within 24 hours in a 3% sodium chloride solution.39 roaming cats. The presence of Alaria spp (adult trem- Ethanol solutions from 8% to 70% were detrimental atodes) in the study area was further confirmed in to mesocercariae viability in < 1 minute. Mesocer- the cadavers of 1 client-owned cat, 1 stray cat, and 5 cariae survived in simulated gastric solutions for 120 coyotes and in tissue samples (mesocercariae) from 1 minutes. In raw, infected, homemade wild pig prod- opossum and 11 wild pigs. Recovery of Alaria ova in ucts prepared by curing, fermentation, cold smok- client-owned dogs and cats of the present study was ing, and drying, the greatest danger of human expo- unexpected because the parasite was not previously sure came from consumption of raw cured products detected in the earlier study33 and was not considered (particularly certain types of sausage) within 24 endemic to the area. However, the prevalence of Alar- hours after starting the curing process.40 Mesocer- ia infection in these client-owned animals was gener- cariae survived in meat products for 21 days at 2° ally low. A higher prevalence in shelter or rescue dogs to –7°C and for 5 days at –18°C.17 In Ringer solution, and cats, feral cats, and coyotes in an endemic area temperatures > 60°C inactivated mesocercariae, as might be expected owing to various factors. Trans- did 90 seconds in a microwave oven.41

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In the United States, the prevalence of infection Alaria alata (Goeze 1792) in natural conditions in France: with Alaria mesocercariae in wild pigs is unknown. molecular evidence. Vet Parasitol 2012;190:151–158. 8. Patrelle C, Portier C, Jouet D, et al. Prevalence and intensity In the present study conducted in north central Okla- of Alaria alata (Goeze, 1792) in water frogs and brown frogs homa, 26% of wild pigs tested were infected with in natural conditions. Parasitol Res 2015;114:4405–4412. Alaria mesocercariae, a prevalence similar to that in 9. Fernandes BJ, Cooper JD, Cullen JB, et al. Systemic infec- parts of Europe.1,6,14–19 Improperly cooked meat from tion with Alaria americana (Trematoda). Can Med Assoc J infected wild pigs can be a potential source of human 1976;115:1111–1114. 10. Otranto D, Eberhard ML. Zoonotic helminths affecting the alariosis in the United States. human eye. Parasit Vectors 2011;4:1–21. Factors that could account for emergence of Alar- 11. Odening K. The “Duncker muscle fluke” can be transmitted ia spp in north central Oklahoma over the past 25 experimentally to monkeys (in Germany). Monatsh Vet Med years likely include expansion of wild pig and coyote 1961;16:395–399. 12. Rentería-Solis ZM, Hamedy A, Michler F, et al. Alaria alata populations in the area; several years of drought with mesocercariae in raccoons (Procyon lotor) in Germany. Par- concentration of temporary water sources and subse- asitol Res 2013;112:3595–3600. quent concentration of snails, frogs, wild paratenic 13. Portier J, Vallée I, Lacour SA, et al. Increasing circulation of hosts, and wild definitive hosts around limited water Alaria alata mesocercaria in wild boar populations of the sources; continuing expansion of suburban develop- Rhine valley, France, 2007–2011. Vet Parasitol 2014;199:153– 159. ments into wildlife habitats; or a combinations of all 14. Riehn K, Große K, Hamedy A, et al. Detection of Alaria these factors.1,6,8,13,27 These factors would increase spp. mesocercariae in game meat in Germany. In: Paulsen exposure risk for companion animals, particularly P, Bauer A, Vodnansky M, et al, eds. Game meat hygiene in free-roaming dogs and cats in rural settings. Wild focus. Wageningen, The Netherlands: Wageningen Academic Publishers, 2011;119–125. pigs, as paratenic hosts of Alaria spp and large game 15. Paulsena P, Ehebruster J, Irschik I, et al. Findings of Alaria used for human consumption, increase the risk for alata metacercariae in wild boars (Sus scrofa) in Eastern human exposure.13 Austria. Eur J Wildl Res 2012;58:991–995. 16. Paulsen P, Forejtek P, Hutarova Z, et al. Alaria alata mesocercariae in wild boar (Sus scrofa, Linnaeus, 1758) in south re- Acknowledgments gions of the Czech Republic. Vet Parasitol 2013;197:384–387. The authors report that there were no conflicts of interest. 17. Portier J, Jouet D, Ferte H, et al. New data in France on the Presented as a poster at the 60th Annual Meeting of the trematode Alaria alata (Goeze, 1792) obtained during American Association of Veterinary Parasitology, Boston, July Trichinella inspections. Parasite 2011;18:271–275. 2015, and at the 25th International Conference of the World Asso- 18. Esite Z, Deksae G, Bagrade G. Overview of Alaria alata dis- ciation for the Advancement of Veterinary Parasitology, Liverpool, tribution in different host animals in Latvia, in Proceedings. England, August 2015. Anim Health Food Hyg Conf 2012;36. The authors thank Brooke Horinek and Larinda Holland for 19. Riehn K, Lalkovski N, Hamedy A, et al. First detection of conducting the molecular analyses; Travis Roberts for providing Alaria alata mesocercariae in wild boars (Sus scrofa, Lin- wild pig tissues from Pawnee County, Okla; and Dr. Anne Barrett naeus, 1758) from Bulgaria. J Helminthol 2014;88:247–249. for editorial assistance. 20. Duscher GG, Leschnik M, Fuehrer HP, et al. Wildlife reservoirs for vector-borne canine, feline and zoonotic infections in Austria. Int J Parasitol Parasites Wildl 2014;4:88–96. Footnotes 21. Gamble HR. Trichinae. Available at: www.aphis.usda.gov/vs/ trichinae/docs/fact_sheet.htm. Accessed Jan 28, 2015. a. DNeasy Blood and Tissue Kit, Qiagen, Germantown, Md. 22. USDA National Agricultural Library. Wild boar. Available at: b. Eurofins Genomics, Eurofins MWG Operon LLC, Huntsville, Ala. www.invasivespeciesinfo.gov/animals/wildboar.shtml. Ac- c. Clustal W2 EMBL−EBI, Wellcome Trust Genome Campus, cessed Sep 29, 2015. Hinxton, Cambridgeshire, England. 23. Stevens RL. Current status and distribution. In: The feral hog d. SigmaPlot, Systat Software Inc, San Jose, Calif. in Oklahoma: Ardmore, Oklahoma. Ardmore, Okla: The Samuel Roberts Noble Foundation, 2010;1–19. References 24. Meng XJ, Lindsay DS, Sriranganathan N. Wild boars as sources for infectious diseases in livestock and humans. Parasite 1. Möhl K, Große K, Hamedy A, et al. Biology of Alaria spp. and 2011;18:271–275. human exposition risk to Alaria mesocercariae—a review. 25. Széll Z, Tolnai Z, Sreter T. Environmental determinants of the Parasitol Res 2009;105:1–15. spatial distribution of Alaria alata in Hungary. Vet Parasitol 2. Pearson JC. Studies on the life cycles and morphology of the 2013;198:116–121. larval stages of Alaria arisaemoides Augustine and Uribe, 26. Blagburn BL, Schenker R, Gagne F, et al. Prevalence of intes- 1927 and Alaria canis Larue and Fallis, 1936 (Trematoda: tinal parasites in companion animals in Ontario and Quebec, Diplostomatidae). Can J Zool 1956;34:295–387. Canada during the winter months. Vet Ther 2008;9:169–175. 3. Shoop WL, Corkum KC. Maternal transmission by Alaria 27. Hiestand SJ, Nielsen CK, Jimenez FA. Epizootic and zoonotic marcianae (Trematoda) and the concept of amphiparaten- helminths of the bobcat (Lynx rufus) in Illinois and a com- esis. J Parasitol 1987;73:110–115. parison of its helminth component communities across the 4. Foster GW, Kinsella JM, Sheppard BJ, et al. Transmammary American midwest. Parasite 2014;21:4. infection of free-ranging Florida panther neonates by Alar- 28. Foster GW, Main MB, Kinsella JM, et al. Parasitic helminths ia marcianae (Trematoda: Diplostomatidae). J Parasitol and arthropods of coyotes (Canis latrans) from Florida, 2009;95:238–239. USA. Comp Parasitol 2003;70:162–166. 5. Pence DB, Knowlton FF, Windberg LA. Transmission of An- 29. Rausch R, Williamson FS. Studies on the helminth fauna of cylostoma caninum and Alaria marcianae in coyotes (Ca- Alaska. XXXIV. The parasites of wolves, Canis lupus. J Para- nis latrans). J Wildl Dis 1988;24:560–563. sitol 1959;45:395–403. 6. Wasiluk A. Alaria alata infection—threatening yet rarely de- 30. Samuel WM, Ramalingam S, Carbyn LN. Helminths in coy- tected trematodiasis. 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31. Unruh DHA, King JE, Eaton RDP, et al. Parasites of dogs from ples using the Alaria spp. mesocercariae migration (AMT). Indian settlements in northwestern Canada: a survey with Parasitol Res 2013;112:2653–2660. public health implications. Can J Comp Med 1973;37:25–32. 37. Riehn K, Hamedy A, Alter T, et al. Development of a PCR 32. Lucio-Forster A, Bowman DD. Prevalence of fecal-borne parasites approach for differentiation of Alaria spp. mesocercariae. detected by centrifugal flotation in feline samples from two shel- Parasitol Res 2011;108:1327–1332. ters in upstate New York. J Feline Med Surg 2011;13:300–303. 38. Sokal RR, Rohlf FJ. 1997. The chi-square distribution. In: Biom- 33. Jordan HE, Mullens ST, Stebbins ME. Endoparasitism in dogs: etry. 3rd ed. New York: WH Freeman and Co, 1997;152–154. 21,584 cases (1981–1990). J Am Vet Med Assoc 1993;203:547–549. 39. González-Fuentes H, Riehn K, Koethe M, et al. Effects of in vi- 34. Zajac AM, Conboy GA, eds. Fecal examination for the diag- tro conditions on the survival of Alaria alata mesocercariae. nosis of parasitism. In: Veterinary clinical parasitology. 7th Parasitol Res 2014;113:2983–2989. ed. Ames, Iowa: Blackwell Publishing, 2006;3–8. 40. González-Fuentes H, Hamedy A, von Borell E, et al. Tenacity 35. Schell SC. Key to families and genera of subclass digenea. of Alaria alata mesocercariae in homemade German meat In: Handbook of trematodes of North America. Moscow: products. Int J Food Microbiol 2014;176:9–14. University Press of Idaho, 1985;78–85, 90–94. 41. González-Fuentes H, Hamedy A, Koethe M, et al. Effect of 36. Riehn K, Hamedy A, Saffaf J, et al. First interlaboratory test temperature on the survival of Alaria alata mesocercariae. for the detection of Alaria spp. mesocercariae in meat sam- Parasitol Res 2015;114:1179–1187.

From this month’s AJVR

Assessment of contrast-enhanced ultrasonography of the hepatic vein for detection of hemodynamic changes associated with experimentally induced portal hypertension in dogs Keitaro Morishita et al

OBJECTIVE To assess the use of contrast-enhanced ultrasonography (CEUS) of the hepatic vein for the detection of hemodynamic changes associated with experimentally induced portal hypertension in dogs. April 2017 ANIMALS 6 healthy Beagles. See the midmonth PROCEDURES A prospective study was conducted. A catheter was surgically placed in the portal vein of each dog. issues of JAVMA Hypertension was induced by intraportal injection of microspheres (10 to 15 mg/kg) at 5-day intervals for the expanded via the catheter. Microsphere injections were continued until multiple acquired portosystemic shunts were created. Portal vein pressure (PVP) was measured through the catheter. Contrast-enhanced ultra- table of contents sonography was performed before and after establishment of hypertension. Time-intensity curves were for the AJVR generated from the region of interest in the hepatic vein. Perfusion variables measured for statistical analysis were hepatic vein arrival time, time to peak, time to peak phase (TTPP), and washout ratio. The or log on to correlation between CEUS variables and PVP was assessed by use of simple regression analysis. avmajournals.avma.org RESULTS Time to peak and TTPP were significantly less after induction of portal hypertension. Simple re- for access gression analysis revealed a significant negative correlation between TTPP and PVP. to all the abstracts. CONCLUSIONS AND CLINICAL RELEVANCE CEUS was useful for detecting hemodynamic changes associated with experimentally induced portal hypertension in dogs, which was characterized by a rapid increase in the intensity of the hepatic vein. Furthermore, TTPP, a time-dependent variable, provided useful complementary information for predicting portal hypertension. IMPACT FOR HUMAN MEDICINE Because the method described here induced presinusoidal portal hypertension, these results can be applied to idiopathic portal hypertension in humans. (Am J Vet Res 2017;78:465–471)

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