Efficacy of Fenbendazole and Milbemycin Oxime for Treating Baboons (Papio Cynocephalus Anubis) Infected with Trichuris Trichiura

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Efficacy of Fenbendazole and Milbemycin Oxime for Treating Baboons (Papio cynocephalus anubis) Infected with Trichuris trichiura

Mason V Reichard,1,* Roman F Wolf,2 David W Carey,2 Jennifer Jane Garrett,3 and Heather A Briscoe1

We evaluated the efficacy of fenbendazole (FBZ) and milbemycin oxime (MO) in the treatment of baboons Papio( cynocephalus anubis) with naturally acquired Trichuris trichiura infection by comparing fecal egg count reduction (FECR) tests. We assigned 7 baboons, each singly housed and confirmed infected withT. trichiura, to treatment groups of FBZ (n 3) or MO (n 3), or as a control (n 1). All (100%) baboons that received FBZ stopped shedding T. trichiura eggs within 6 d of treatment, and fecal egg counts remained negative at 65 d after treatment. Although the number of T. trichiura eggs shed per gram of feces from 2 (67%) baboons decreased significantly after the second treatment with MO, this regimen never totally eliminated eggs of T. trichiura. The results of our study indicate that FBZ was more effective for treating baboons with T. trichiura than was MO.

Abbreviations: FBZ, fenbendazole; MO, milbemycin oxime; FECR, fecal egg count reduction; EPG, eggs per gram

Members of the genus Trichuris are nematode parasites that infect the intestinal tract of a wide variety of mammals. Trichu- ris spp are commonly referred to as ‘whipworms’ because of their long, filamentous bodies that thicken distally, resembling a bullwhip.16,20 Trichuris trichiura, the species that infects man, is also found in wild and captive baboons (Papio spp),6,7,11-14 which are increasingly in demand as models for biomedical research. Clinical trichuriasis in nonhuman primates is similar to that seen in man, and signs include profuse watery diar- rhea, lethargy, abdominal pain, weight loss, inappetance, and dehydration.8,12,16 Trichuris spp chiefly inhabit the large bowel and cecum, threading through mucosal layers as immature larvae but becoming stationary as adults.8 Consequently, pathogenesis commonly involves anaphylactoid-type reactions and ischemic necrosis, both limited to tissue where adult worms reside in situ and direct injury occurs. Infection of T. trichiura in mammals is diagnosed through microscopic observation of whipworm Figure 1. A characteristic double-plugged egg of Trichuris trichiura. eggs in host feces. Eggs of T. trichiura measure 49.5 to 65.0 μm Magnification,t 400. t 22.5 to 30.0 μm and are distinctive barrel- or lemon-shaped ellipses with polar plugs at each end to facilitate emerging larvae 6 d of treatment. In contrast, 2 treatments with MO reduced the (Figure 1).20 Because of a direct fecal–oral route of transmission numbers of T. trichiura eggs but never completely eliminated and the ability to persist within the environment, treatment and them from the feces of infected baboons. control of T. trichiura is requisite to maintaining healthy baboon colonies, providing quality animals for research, and reducing -ATERIALS¬AND¬-ETHODS the threat of zoonotic transmission. %XPERIMENTAL¬DESIGN Seven adult olive baboons (Papio cyno- Standard treatment of whipworm infection in animals in- cephalus anubis), naturally infected with T. trichiura but without cludes either fenbendazole (FBZ) or milbemycin oxime (MO) signs of clinical trichuriasis, were utilized for the present study. whereas mebendazole, a closely related FBZ analog, is indicated One of the 7 infected baboons served as a positive control for Trichuris infection in humans.2,18 The purpose of the present and did not receive any anthelmintic medication; 3 infected study was to determine whether FBZ or MO was more effica- baboons received therapeutic doses of FBZ, while the remain- cious for treating baboons infected with T. trichiura. We found ing 3 received therapeutic doses of MO. Drug efficacy was FBZ eliminated T. trichiura eggs from the feces of baboons within determined by the reduction of T. trichiura eggs in host feces Received: 10 Jul 2006. Revision requested: 31 Jul 2006. Accepted: 8 Aug 2006. after treatment. 1Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma !NIMALS¬All animals were housed and cared for according State University, Stillwater, OK; 2Comparative Medicine, University of Oklahoma Health 3 to the standards detailed in the Guide for the Care and Use of Sciences Center, Oklahoma City, OK; Department of Biological Sciences, Tarleton State 15 University, Stephenville, TX. Laboratory Animals. Protocols for maintenance of the baboon *Corresponding author. Email: [email protected] colonies were approved by the University of Oklahoma Health

Anthelmintics for treating whipworms in baboons

Table 1. Baboon demographic data Time housed Animal Sex Age (y) Weight (kg) singly (mo) 13-369 Male 10.1 31.9 6 1201 Male 4.7 15.2 24 37-6 Female 11.0 18.8 6 2095 Male 10.1 33.5 4 885 Female 20.3 20.9 14 69-262 Male 7.7 23.9 12 B556 Male 8.6 30.1 72

Sciences Center Institutional Animal Care and Use Committee. Baboons used in the present study ranged from 4.7 to 20 y of age and were members of a breeding colony reared at the Okla- homa University Health Science Center. Baboons in the present Figure 2. The number of T. trichiura eggs shed from baboons before study had been individually housed for 4 to 72 mo (Table 1) but and after treatment with a 3-d course of FBZ. Treatment began on day prior to inhabiting single dwellings, the baboons were housed 0 (arrow). outdoors with other breeding-colony baboons. Baboons were fed a standard diet (Harlan Primate Diet 2055, Harlan Teklad, Madison, WI) supplemented 3 times weekly with fresh fruit offered to each baboon. Baboons in the MO treatment group and vegetables, as well as enrichment grains such as trail-mix received 1.0 mg/kg oral doses of milbemycin oxime (Interceptor and dry cereal. Potable water was available ad libitum. None tablets, Novartis, Greensboro, NC) at 30-d intervals for 3 mo. of the baboons used in the present study had ever been treated Each flavored Interceptor tablet was also ground into 25 ml of with an anthelmintic. applesauce, offered to each baboon, and immediately consumed. 3AMPLE¬COLLECTION¬AND¬CALCULATION¬OF¬THE¬NUMBER¬OF¬EGGS¬PER¬ Doses of FBZ and MO were based upon recommendations GRAM¬OF¬FECES¬%0' ¬Baboons infected with T. trichiura were by manufacturer as effective for treatment of domestic dogs identified by observation of eggs characteristic of whipworms infected with T. vulpis. One T. trichiura–infected baboon was during microscopic examination of feces (Figure 1). Quanti- randomly assigned as a vehicle control and received 25 ml of tative fecal examinations were accomplished using double commercially prepared applesauce. centrifugation with sugar flotation.21 Briefly, 5 g of fecal material $ETERMINATION¬OF¬ANTHELMINTIC¬EFlCACY¬As outlined by the was thoroughly mixed with 30 ml water, passed through a tea World Association for the Advancement of Veterinary Parasitol- ogy on the methods for the detection of anthelmintic resistance strainer, divided into two 15-ml conical tubes, and centrifuged 5 in a swinging bucket rotor at 176 t g for 10 min. Tubes were de- in nematodes of veterinary importance, descriptive statistics canted without disturbing the sediment and refilled with sugar as well as the percentage fecal egg count reduction (FECR) of solution (specific gravity, 1.27). To release the eggs from the plug T. trichiura eggs were reported. The percent FECR of T. trichiura was calculated as ([pretreatment EPG – post-treatment EPG/ of debris in the tube bottom, the sediment and sugar solution 5 were mixed thoroughly with an applicator stick. The tubes again pretreatment EPG] t 100). When a fecal egg count for an individual baboon increased after treatment, the percentage FECR were placed in a swinging bucket rotor and sugar solution was 10 added dropwise to each tube until a positive meniscus formed. was reported as 0. T. trichiura EPG for baboons that received A 22-mm square coverslip was placed over each tube, and the FBZ were compared using the Mann-Whitney rank sum test t whereas EPG for baboons that received MO were compared tubes were centrifuged at 176 g for 10 min. Coverslips were 19 removed and placed on a microscope slide; each 5-g sample with the Kruskal-Wallis one-way analysis of variance on ranks. yielded 2 coverslips. The number of Trichuris eggs shed per gram The Dunn method was used for multiple comparisons and Bon- ferroni adjustment for multiple comparisons was used as the of feces (EPG) was estimated by counting the whipworm eggs 19 visible under each coverslip at 100t magnification and dividing criterion for statistical significance. Analyses were performed that sum by the number of grams of feces used. with SigmaStat 3.1 statistical software package (Systat Software, We used the T. trichiura EPG value to approximate the abun- Point Richmond, CA). dance of gastrointestinal whipworms in each baboon. A baseline EPG was established for each baboon in the present study by 2ESULTS collecting and examining feces every other day for 20 d prior Pretreatment FEC of T. trichiura from infected baboons re- to treatment. Determination of EPG continued every other day vealed considerable variation in the number of eggs shed from for 30 d after treatment and then occurred weekly until 65 d individual baboons (Figures 2 and 3) as fecal egg counts ranged after treatment. from 0 to 418.4 EPG (Table 2). We performed 77 pretreatment !NTHELMINTIC¬TREATMENTS¬Baboons infected with T. trichiura fecal flotations to establish a baseline EPG and determine vari- were ranked qualitatively according to fecal egg count (FEC) as ance in the number of eggs shed per T. trichiura-infected baboon. low, medium, or high. One baboon from each FEC group was Only 1 of the 77 fecal samples examined prior to treatment was randomly allocated to 1 of the 2 treatment groups, comprised negative by flotation for T. trichiura eggs, corresponding to a of 3 baboons each. The 3 baboons in the FBZ treatment group false-negative rate of 1.3%. received 50.0 mg/kg doses of oral FBZ (Panacur, Intervet, Mills- Significant reductions in the number of T. trichiura eggs boro, DE) once daily for 3 consecutive days. We mixed each dose shed from baboons 13-369, 1201, and 37-6 (T 262.0, P 0.001; of Panacur, packaged as granulated FBZ, with approximately T 261.0, P 0.001; T 333.0, P 0.001, respectively) were 25 ml of applesauce and was immediately consumed when observed after 1 FBZ treatment course. The percent reduc-

Vol 46, No 2 Journal of the American Association for Laboratory Animal Science March 2007

stopped shedding T. trichiura eggs within 6 d after treatment, and whipworm eggs were not detected again for the duration of the study (Figure 2). Reductions in T. trichiura FEC were observed in baboons 885 and 69-262 (H 9.450, P 0.024; H 14.368, P 0.002, respectively) after treatment with MO. The Dunn multiple comparison procedure revealed significant decreases (73.0% and 79.4%, respectively) in the number of T. trichiura eggs shed from baboons 2095 and 69-262 (Q 2.727, P 0.05; Q 3.266, P 0.05, respectively) after the second treatment of MO but at no time in the present study were T. trichiura eggs completely eliminated from the feces (Figure 3). Differences in the number of T. trichiura FEC shed from MO-treated baboon 2095 and the untreated control (B556) were not greater than would be expected from random sampling variability.

$ISCUSSION Figure 3. The number of T. trichiura eggs shed from baboons before and after treatment with MO. Baboons were treated with MO on days FECR tests provide an estimation of anthelmintic efficacy by 0, 30, and 60 (arrows). enabling comparison of worm egg counts from animals before and after treatment. Although the FECR test was originally tion in T. trichiura eggs shed by each of the 3 baboons treated developed for detecting anthelmintic resistance in domestic with FBZ was, respectively, 97.2%, 96.3%, and 99.1% (Table 2). animals,5,10 we have used this assay to determine the efficacy of All baboons that received a single therapeutic course of FBZ FBZ and MO in the treatment of baboons infected with T. trichi-

Table 2. Descriptive statistics and percentage reduction of Trichuris trichiura egg counts from baboons Fenbendazole Milbemycin oxime Control Baboon 13-369 1201 37-6 2095 885 69-262 B556 Pretreatment Sample size 11 11 11 11 11 11 11 Mean q standard error 10.9 q 2.0 35.4 q 5.7 255.8 q 35.6 3.6 q 0.8 36.6 q 5.1 141.6 q 20.7 22.5 q 4.3 95% Confidence interval 6.4 – 15.4 22.7 – 48.1 176.6 – 335.0 1.9 – 5.3 25.2 – 48.0 95.3 – 187.8 13.0 –32.0 Median 10.0 32.8 282.4 3.4 36.2 138.6 20.0 Range 1.2 – 19.8 11.4 – 78.6 89.4 – 418.4 0.0 – 9.2 12.6 – 69.4 61.6 – 279.0 7.6 – 46.4 After 1st treatment Sample size 18 18 18 12 12 12 12 Mean q Standard Error 0.3 q 0.2 1.3 q 1.3 5.3 q 5.3 7.7 q 1.3 41.6 q 7.2 118.4 q 14.6 16.4 q 3.1 95% Confidence interval –0.2 – 0.8 –2.3 – 6.3 –5.8 – 16.4 4.8 – 10.6 25.7 – 57.5 86.2 – 150.4 9.5 –23.3 Median 0.0 0.0 0.0 7.2 33.5 120.4 13.3 Range 0.0 – 4.2 0.0 – 23.4 0.0 – 94.6 0.6 – 16.2 16.2 – 102.4 57.2 – 244.8 1.4 – 35.6 % Reduction 97.2 96.3 99.1 0.0 0.0 16.4 27.1 After 2nd treatment Sample size – – – 4 4 4 4 Mean q standard error ––– 11.6 q 3.3 9.9 q 2.9 29.2 q 9.7 17.6 q 5.4 95% Confidence interval – – – 1.0 – 21.6 0.7 – 19.1 –1.5 – 59.9 0.3 – 34.9 Median – – – 10.8 11.5 23.7 14.4 Range – – – 4.4 – 20.4 2.2 – 14.4 13.0 – 56.4 8.6 – 32.8 % Reduction – – – 0.0 73.0 79.4 21.8 After 3rd treatment Sample size – – – 2 2 2 2 Mean q standard error ––– 5.6 1.2 7.3 3.1 41.7 12.7 8.8 5.2 95% Confidence interval – – – –9.6 - 20.8 –32.1 – 46.7 –119.7 – 203.1 –58.1 – 74.9 Median – – – 5.6 7.3 41.7 8.8 Range – – – 4.4 – 6.8 4.2 – 10.4 29.0 – 54.4 3.6 – 14.0 % Reduction – – – 0.0 80.0 70.6 60.8

Anthelmintics for treating whipworms in baboons

ura. As demonstrated by the significant reduction and eventual 2EFERENCES absence of T. trichiura eggs from infected captive baboons, FBZ 1. Blagburn BL, Hendrix CM, Lindsay DS, Vaughn JL, Helper DI, was more effective than MO in treating baboons naturally in- Wright JC. 1992. Efficacy of milbemycin oxime against naturally fected with T. trichiura. acquired or experimentally induced Ancylostoma spp. and Trichuris FBZ is a benzimidazole agent that exerts nematocidal activity vulpis infections in dogs. Am J Vet Res 53:513–516. by selectively binding to nematode tubulin, disrupting mitosis 2. Bowman DD, Carl RC, Eberhard ML. 2003. Georgis’ parasitology and mitochondrial function.2 Mebendazole, in the same drug for veterinarians, 8th ed. St Louis (MO): Elsevier Science. family as FBZ, is indicated for treatment of T. trichiura in humans. 3. Bowman DD, Legg W, Stansfield DG.2002. Efficacy of moxidec- FBZ, effective in eradicating T. trichiura eggs from the feces of tin 6-month injectable and milbemycin oxime/lufenuron tablets against naturally acquired Trichuris vulpis infections in dogs. Vet baboons in the present study, is also effective in whipworm Ther 3:286–289. treatment of other animals, as well as nematode, trematode, and 4. Bundy DAP, Cooper ES. 1989. Trichuris and trichuriasis in humans. 2,17 cestode parasites of domestic and wild animals. Adv Parasitol 28:107–173. MO is a macrocyclic lactone similar to ivermectin in structure 5. Coles, GC, Bauer C, Borgsteede FHM, Geerts S, Klei TR, Taylor and activity, binding with high affinity to glutamate-gated chlo- MA, Waller PJ. 1992. World association for the advancement of ride channels, causing paralysis and death of the nematode.2 In veterinary parasitology (W.A.A.V.P.) methods for the detection of comparison with FBZ, MO was not as effective in reducing or anthelmintic resistance in nematodes of veterinary importance. Vet eliminating the number of T. trichiura eggs shed from infected Parasit 44:35–44. 6. Flynn RJ. 1973. Parasites of laboratory animals. Ames (IA): Iowa baboons. MO has been reported to have 95% efficacy in treating State University Press. 1,3,9 dogs infected with T. vulpis but whether the lack of efficacy 7. Hahn NE, Proulx D, Muruthi PM, Alberts S, Altmann J. 2003. in present study baboons is due to differences in species of host Gastrointestinal parasites in free-ranging Kenyan baboons (Papio or whipworm remains to be determined. cynocephalus and P. anubis). Int J Primatol 24:271–279. Generally speaking, intestinal nematode infection is treated 8. Hennessy A, Phippard AF, Harewood WJ, Horam CJ Horvath with at least 2 anthelmintic doses, the first given to eradicate JS. 1993. Helmintic infestation complicated by intussusception in adult worms and the second to eliminate immature forms that baboons (Papio hamadryas). Lab Anim 28:270–273. survived the primary dose. Timing of the second dose should 9. Horii Y, Otsuka Y, Tateishi M, Makimura S, Kusano K. 1998. Anthelmintic efficacy of milbemycin oxime against Trichuris vulpis coincide with the interval between infection and egg production, in dogs. J Vet Med Sci 60:271–272. typically 14 to 21 d. However, adjustments must be made in 10. Kaplan RM, Klei TR, Lyons ET, Lester G, Courtney CH, French scheduling the second dose when treating whipworms because DD, Tolliver SC, Vidyashankar AN, Zhao Y. 2004. Prevalence of Trichuris has a longer prepatent period than most intestinal anthelmintic-resistant cyathostomes on horse farms. J Am Vet Med nematodes.2 The prepatent period of T. trichiura in baboons is Assoc 225:903–910. currently unknown but is estimated to be approximately 60 d in 11. Kuntz RE, Meyers BJ. 1967. Parasites of the Kenya baboon: ar- humans.4 Regardless of prepatent period, however, the efficacy thropods, blood protozoa, and helminths (Kenya, 1966). Primates of any anthelmintic program is dependent upon preventing 8:75–82. 12. Munene E, Otsyula M, Mbaabu DAN, Mutahi WT, Muriuki reinfection. SMK, Muchemi GM. 1998. Helminth and protozoan gastrointesti- Baboons in the present study were singly housed in sanitized nal tract parasites in captive and wild-trapped African non-human cages, but baboons housed in unsanitized colonies would likely primates. Vet Parasitol 78:195–201. require more than a single 3-d course of FBZ to eradicate whip- 13. Murray S, Stem C, Boudreau B, Goodall J. 2000. Intestinal worm infection. T. trichiura eggs require 28 d at 25pC to reach parasites of baboons (Papio cynocephalus anubis) and chimpan- infectivity and cages used to house baboons in the present study zees (Pan troglodytes) in Gombe National Park. J Zoo Wildl Med were disinfected twice a week with 50pC water, temperatures 31:176–178. lethal to developing T. trichiura eggs and larvae. Baboons 1201 14. Myers BJ, Kuntz, RE. 1965. A checklist of parasites reported from the baboon. Primates 6:137–194. and B556 had been (and continue to be) housed singly for 24 and 15. National Research Council. 1996. Guide for the care and use of 72 mo (respectively) prior to treatment. The shortest period of laboratory animals. Washington (DC): National Academy Press. single-housing for a baboon (2095) of the present study prior to 16. Ooi HK, Tenora F, Itoh K, Kamiya M. 1993. Comparative study treatment was 4 mo (Table 1). Therefore, results from the present of Trichuris trichiura from non-human primates and from man and study seem to indicate that T. trichiura infection in baboons is their difference with T. suis. J Vet Med Sci 55:363–366. fairly long-lived. The life-span of adult T. trichiura in humans 17. Plumb DC. 2005. Plumb’s veterinary drug handbook, 7th ed. Ames is currently unknown but has been estimated at 3 y.4 (IA): Blackwell Publishing. Because Papio spp resemble humans physiologically, bio- 18. Roberts LS, Janovy J Jr. 2005. Gerald D Schmidt and Larry S Roberts’ Foundations of parasitology, 7th ed. New York: McGraw chemically, and anatomically, it should come to no surprise that Hill. baboons are being used for biomedical purposes. The impor- 19. Sokal RR, Rohlf FJ. 1997. Biometry, 3rd ed. San Francisco (CA): tance of efficacious anthelmintics for treating and controlling WH Freeman and Company. T. trichiura is critical to maintaining healthy baboon colonies, 20. Soulsby EJL. 1982. Helminths, arthropods, and protozoa of do- providing quality baboons for research, and reducing the threat mesticated animals, 7th ed. Philadelphia (PA): Lea and Febiger. of zoonotic disease to human handlers. 21. Zajac AM, Conboy GA. 2006. Veterinary clinical parasitology, 7th ed. Ames (IA): Blackwell Publishing. !CKNOWLEDGMENTS The authors wish to thank Gail Goodson and Tammy McKnight, who care for these baboons on a daily basis. This project was funded through National Institutes of Health grant P40 RR12317 (to Gary L White).