中国科技论文在线 http://www.paper.edu.cn The Veterinary Journal 2003, 165, 73–77 doi:10.1016/S1090-0233(02)00124-7

Morphological Changes to Early Stage solium Cysticerci Following Oxfendazole Treatment

LIU YONG-JIE*, LI QING-ZHANG , HAO YAN-HONG *College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China; yCollege of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, People’s Republic of China

SUMMARY The progressive morphological changes to early stage cysticerci following the treatment of pigs with a single therapeutic dose of oxfendazole (30 mg/kg), are described. On Day 1 after treatment, no obvious changes occurred in the general appearance of the larvae but alternations were seen by electron microscope, with an apparent reduction in the number of microtriches, and a complete disappearance of the . Numerous granules were seen to have accumulated in the tegument cells. As treatment progressed, damage to the cysticerci was more serious and, by five days, all cysticerci were seen to be in an advanced stage of de- generation. By 45 days post-treatment, all cysts were calcified. These results suggest that oxfendazole is a highly effective drug against T. solium cysticerci in the early stages of development. Ó 2002 Elsevier Science Ltd. All rights reserved.

KEYWORDS: Oxfendazole; Taenia solium cysticerci; early stage; morphological changes.

INTRODUCTION treat mature T. solium cysticerci and good results have been achieved (Han, 1986). But clinical practice has In many countries, Taenia solium cysticerci are a indicated that the two drugs are less effective in public health hazard and an important cause of treating the larvae in the early stages and treatment economic loss, potentially eradicable. Mass treat- has to be repeatedly applied (Ma, 1997). ment of human populations with taeniacides has Oxfendazole (methyl 5[6]-phenylsulfinyl-2-benz- been performed with significant successes in reduc- imidazolecarbamate), a member of the benzimi- ing both the number of tapeworm carriers and new dazole group of , has broad-spectrum pig infections in the subsequent year (Cruz et al., activity against inhibited larval stages of gastroin- 1989; Diaz et al., 1991). However, parasite transmis- testinal roundworms, tapeworms, and lungworms in sion may continue due to incomplete chemotherapy many species (Marriner & Bogan, 1981). It coverage of human tapeworm carriers or because of has been shown to kill all viable T. solium cysticerci the immigration of tapeworm carriers into con- in infected pigs (Gonzalez & Falcon, 1997). trolled areas (Marshall, 1999). The objective of the present study was to see An extensive research program has been under- whether oxfendazole is effective against T. solium taken in many countries to develop an effective vac- cysticerci in the early stages and, so as to study the cine to prevent cysticercosis in pigs. But even with an mechanism of action of oxfendazole, to observe the effective vaccine, it is impossible to provide 100% morphological changes to the larvae after the in- protection. Once humans or pigs are infected with T. fected pigs have been treated with the drug. solium cysticercosis, drugs are indispensable. Both praziquantel and albendazole have been used to MATERIALS AND METHODS All animal protocols were approved by the Institu- Correspondence to: Y.-J. Liu, College of Veterinary Medicine, tional Animal Care and Use Committees of the Nanjing Agricultural University, Nanjing 210095, PeopleÕs Republic of China Tel.: 25-4442173. E-mail: liuyongjie72@ Northeast Agricultural University, Harbin. The yahoo.com.cn. method of euthanasia followed the recommenda-

1090-0233/02/$ - see front matter. Ó 2002 Elsevier Science Ltd. All rights reserved. 转载 中国科技论文在线 http://www.paper.edu.cn 74 THE VETERINARY JOURNAL, 165, 1 tions of the American Veterinary Medical Associa- motility in saline solution at 37 °C, more than 90% tion. Death was not an experimental end point. of the scolices were apparently alive. T. solium tapeworm proglottids were recovered In the treated group, at one day after treatment from the faeces of three patients. Species identifi- (31 days post-infection), no obvious changes had cation was made by counting the number of uterine occurred in the general appearance of the larvae, but branches and confirming the presence of hooklets. the host capsules had become thickened. At two days, The parasites were washed repeatedly with tap water a change in colour from white to yellowish-brown followed by distilled water to remove all visible fae- occurred in more than 95% of the cysts. The cyst cal material. fluid had gone and the oval cysticerci were elon- A total of 20, two-month-old Landrace pigs were gated. At five days, all of the cysts contained damaged selected for the study. They were considered to be cysticerci. The bladder walls were detached from the free from T. solium cysticerci infections since all were scolices and were seen to be in an advanced stage of very young and all had been purchased from two degeneration. At 10 days, similar but more marked animal farms with good sanitary conditions. Three injuries had occurred. Recognizable parts of larvae gravid proglottids, placed in gelatin capsules, were were not seen in any of the examined samples. Only a given orally to each animal in a safety pen. The little viscid fluid remained in the cysts. By 45 days were closely observed to be certain that all post-treatment (75 days post-infection), all muscle capsules were swallowed. All materials and waste appeared clear and only minuscule scars remained. were autoclaved after use. Earlier work had indicated that the age at which Ultrastructure T. solium cysticerci appeared was 19 days and the Untreated cysticerci. Bladder walls or scolices in de- mature age was 60 days (unpublished observations). veloping cysticerci were composed of tegument and As such, 30 days after infection was selected at the parenchyma. Microtrichia were distributed on the optimal time to treat animals. The first group of outer surface of the tegument (Fig. 1). The matrix animals (n ¼ 10) was treated orally with a single zone, full of vesicles, endoplasmic reticule and mi- dose of (30 mg/kg) oxfendazole (Gonzalez & Fal- tochondria, existed between microtrichia and basal con, 1997). The second group (n ¼ 10) served as membrane. Both microtrichia and matrix zone controls, and received no medication. comprised tegument, but the parenchyma was sep- After one, two, five, 10, and 45 days, two animals arated from the matrix zone by a basal membrane. in each group were killed. Three cysticerci from Muscles were dispersed in the parenchyma. The each animal were rapidly excised and rinsed with outer layer formed the circular muscle bundles and 0.1 M PBS (pH 7.2) three times. Small segments of the inner was composed of longitudinal muscle fi- the scolices and of bladder wall were sampled and bres. The cells in the parenchyma zone were highly then fixed in 2.5% glutaraldehycle solution. After heterogeneous, and consisted of parenchyma cells washing with the same PBS three times, the seg- (Fig. 2), tegument cells (Fig. 3), calcareous corpus- ments were fixed in 1% OsO4 for 1 h, dehydrated in cles forming cells and flame cells. Also a large a graded series of ethanol, and embedded in epon. number of glycogen granules and excretory ducts in Portions of the neighbouring muscle tissue of each various diameters were found in the parenchyma. animal were prepared in the same manner as the parasites. One-micrometre-thick sections were cut Treated cysticerci with oxfendazole and stained with uranium acetate and lead citrate On Day 1 after the treatment with oxfendazole, before examination under TEM-1200EX electron several prominent changes had occurred. A focal microscope. loss of microtriches was seen and the matrix zone had completely disappeared (Fig. 4). Degeneration of muscle cells was visible and the muscular bran- RESULTS ches were in an irregular order. Tegument cells had Macroscopic appearance become rounded with large areas of accumulated In the untreated animals, the 31-day old cysticerci granules, and the nuclei had been pushed towards were about 2.0–3.5 mm in diameter and had devel- the cell periphery. In some tegument cells, two oping scolices. With the duration of infection, the kinds of granules were found, which were respec- sizes of the milk-coloured cysticerci progressively tively electron-lucent and with an electron-dense increased. The surrounding host capsules were thin core (Fig. 5). In others, only electron-lucent and easy to remove. At 75 days, judging from their granules were observed and deterioration in form of 中国科技论文在线 http://www.paper.edu.cn

MORPHOLOGICAL CHANGES TO T. SOLIUM CYSTICERCI 75

Fig. 1. Tegument and parenchyma in control group, 10 k. MI: microtriches; MZ: matrix zone; MC: muscle. Fig. 2. Parenchyma cells in control group, 6000. Fig. 3. Tegument cells in control group, 10 k. Fig. 4. Day 1 following treatment with oxfendazole: microtriches were apparently decreased and matrix zone completely disappeared. Muscle bunches (MC) were in disorder, showing signs of degeneration, 4000. Fig. 5. Day 1 following treatment with oxfendazole: tegument cells were filled with numerous granules, electron-lucent, or with an electron-dense core, 5000. Fig. 6. Day 1 following treatment with oxfendazole: deterioration in form of bursts (arrow) were found at several spots in some tegument cells, 8000. Fig. 7. Day 1 following treatment with oxfendazole: lytic parenchyma cell, 12 k. Fig. 8. Day 2 following treatment with oxfendazole: only fibrous skeleton (F) and muscle cells (MC) were preserved, 5000. 中国科技论文在线 http://www.paper.edu.cn

76 THE VETERINARY JOURNAL, 165, 1

bursts was found at some spots (Fig. 6). Parenchyma microvilli are reduced, the nutrient exchange be- cells were lytic (Fig. 7). No obvious alterations were tween larvae and surrounding environment would found in other cells. be hindered. In addition, the reduction of glycogen Excessive degenerative alterations were noted two granules indicated that necessary energy for para- days after treatment. Microtriches were completely sites’ survival could be severely restricted. absent from the absorptive border, leaving an ir- We have previously demonstrated the chemo- regularly lined surface (Fig. 8). Most characteristic therapeutic effectiveness of oxfendazole in the were the vacuoles present in the parenchyma. The treatment of mature cysticerci (unpublished data), remainder of the fibrous skeleton and muscle cells but the present findings indicate that the drug is could be recognized but tegument and cell types more effective against immature than mature cystic- were absent. In addition, glycogen granules were erci. The results of our observations showed that in reduced and the number of the microvilli in the immature cysticerci, both tegument and parenchyma excretory ducts had significantly decreased. were thin and connective tissue capsules did not form From five days after treatment onwards, ultra- around the larvae. Neither blood nor lymph circula- structural examinations of the cysticerci were not tion exists between the surrounding host capsules performed because no larvae were found in the and larvae, so the drug enters into the larvae only by cysts. At any time period during or after oxfendazole diffusion. Thus, we concluded that immature cystic- treatment no ultrastructural alterations were dem- erci are more vulnerable to oxfendazole, and it is onstrated in the surrounding non-infected muscle. possible that if the cysticerci are treated early with the drug, better results may be achieved. Early diagnosis of T. solium cysticercosis remains most important. DISCUSSION The fact that no ultrastructural alterations were The morphological observations described above noted in neighbouring non-infected muscle cells indicated the efficacy of oxfendazole in killing indicates that oxfendazole has a specific effect on the T. solium cysticerci in the early stages in a relatively infected area. These findings lead us to conclude short period of time. After treatment, the successive that oxfendazole creates non-viable conditions for morphological changes in immature cysticerci of the parasites by depriving them of their nutritional T. solium (30 days old) occurred. source and their protective surroundings. The most obvious ultrastructural changes were: (1) loss of the microtriches from the tegument ACKNOWLEDGEMENTS surface, (2) accumulation of numerous granules in tegument cells, (3) reduction of glycogen granules, This work was supported by the Nature Science Foun- (4) decrease in the microvilli in the excretory ducts. dation of Heilongjiang Province (No. C9906) and The ultrastructural changes revealed that a seri- Science and Technology Plan Program of Heilongji- ous block had occurred in the absorption, excre- ang (No. 98SG86SY10). We are grateful to Professor tion, and of the cysticerci. Like the Liu D.H. (Institute of cysticercosis, Jilin Agricultural microvilli in the intestine of mammals, microtriches University) for his skillful technical assistance. have the function of absorbing nutrients. The initial formation and the maintenance of microtriches REFERENCES occur with the cytoplsmic vesicles of the tegument CRUZ, M., DAVIS, A., DIXON,H.&PAWLOWSKI, Z. S. (1989). (Lumsden et al., 1974). The vesicles are produced in Operational studies on the control of Taenia solium the tegument cells and are then transported to the taeniasis/cysticercosis in Ecuador. Bulletin of World tegument by the cytoplasmic pathway. In this way a Health Organization 67, 563–6. continuous replenishment of the tegument surface DIAZ, C. S. P., CANDIL,R.A.&SUATE, P. V. (1991). coat takes place (Oaks & Lumsden, 1971). The loss Epidemiologic study and control of Taenia solium infections with praziquantel in a rural village of of microtrichia and the degeneration of tegument Mexico. American Journal of Tropical Medicine and caused by oxfendazole treatment may well be re- Hygiene 45, 522–31. lated to a block in the transportation of vesicles, GONZALEZ,A.E.&FALCON, N. (1997). Treatment of since numerous vesicles were found in the tegument porcine cysticercosis with oxfendazole: a dose-response cells. The microvilli in the excretory ducts do not trial. Veterinary Record 141, 420–2. HAN, Y. (1986). Comparion of albendazole and praziqu- only increase the surface area of absorption and antelum in treatment against taenia solium cysticercosis. promote the repeated uptake of fluid, but also Chinese Journal of Veterinary Science and Technology accelerate the excretion of waste material. Once the 16(Suppl),90–1. 中国科技论文在线 http://www.paper.edu.cn

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LUMSDEN, R. D., OAKA,J.A.&MUELLER, J. F. (1974). Brush MARSHALL, W. L. (1999). Eradication of Taenia solium border development in the tegument of the tapeworm, cysticercosis: a role for vaccination of pigs. International spirometra mansonoides. Journal of Parasitology 60, 209–26. Journal for Parasitology 29, 811–7. MA, Y. X. (1997). My opinions on prevention and cure of OAKS,J.A.&LUMSDEN, R. D. (1971). Cytological studies Taenia solium cysticerci in China. Henan Journal of on the absorptive surfaces of cestodes. V. Incororation Preventive Medicine 8, 314–5. of carbohydrate-containing macromolecules into tegu- MARRINER,S.&BOGAN, J. A. (1981). Pharmacokinetics of ment membrane. Journal of Parasitology 57, 1256–68. oxfendazole in sheep. American Journal of Veterinary Research 42, 1143–5. (Accepted for publication 10 April 2002)

doi:10.1016/S1090-0233(02)00182-X

Book Review There is some repetition within the text with, for example, two cases involving nasal polyps in the cat. The illustrations are generally of a very high quality Clinical Atlas of Ear, Nose, and Throat Diseases in including clinical lesions, line diagrams, and views Small Animals: the Case-Based Approach of various endoscopic examinations. Some of the Merchant, S., Mortellaro, C., and White, R.A.S., Hannover, photomicrographs of the histopathology and cytol- SchluuterscheGmbH€ & Co., 2002, 208 pp, £64 (hard) ISBN ogy specimens are limited in quality and provide 3-87706-621-6 limited information especially about the cell types involved. Also in one illustration you almost have to This well-produced atlas is a truly international guess where the dog is located in an oxygen cage/ effort to make ENT disease in small animals accessi- chamber! The references at the end of each chapter ble to the reader. The intended audience includes vary widely in number. Some references are some- the so-called ‘‘progressive’’ veterinarian and student. what dated including the fifth edition of Muller and The book is liberally illustrated by the German pub- Kirk’s Small Animal Dermatology. This has been up- lisher and draws upon the experience of three sur- dated with a sixth edition in 2001 and Greene’s In- geons: Dr. Cheryl S. Hedlund, Professor Carlo M. fectious Disease text has also been updated. A large Mortellaro, and Dr. R.A.S. White from the USA, Italy, chapter is devoted to ear conditions but the various and the UK, respectively, a dermatologist (Professor conditions listed and discussed at the end of the S. Merchant) and an internist (Dr. J. Taboada) both chapter are somewhat uncommon to rare and not from the USA. The book is composed of six chapters likely to be seen by many clinicians. In the chapter that cover the ear, nasal, and paranasal cavities and on oral disease the intraoral eosinophilic granuloma sinuses, the larynx, the trachea, the oral cavity, and seems to be regarded as an entity of unknown aeti- the pharynx. It is clearly stated that the book is not ology although there is a reference in one table to intended to be a comprehensive treatise on the areas looking for evidence of ectoparasite infestation. given. The chapter contents include an introduction, Many dermatologists would consider this condition a series of cases and a discussion that covers other in part to be attributable to allergic skin conditions. conditions that may affect the area discussed, and The cost seems quite high until one considers finally a short list of references. that it is a well-bound hardback book with a lot of The most significant feature of this atlas is that colour illustrations. Although the intention is not to each chapter covers from five to 16 individual cases in be comprehensive the authors have covered a lot of which the reader is invited to use each case as a conditions and demonstrate how one might ap- learning exercise by self-assessment as they read proach a variety of clinical conditions involving this through each case. So the reader may compare their part of the body. Beyond dentistry there are few own approach to the diagnosis and treatment of each other texts that cover this area of the body and so case with that made by authors. Alternatively, the this book should prove useful to the student and reader may simply refer to the table of contents where veterinarians with an interest in small animal med- the presenting problem for each case is matched with icine and particularly ENT surgery. a specific diagnosis. If the reader turns to a particular disease they wish to study then the case presentation is still problem-orientated so that each case is somewhat comparable with that seen in general practice. AIDEN FOSTER