Mukaratirwa_173-180.indd INTRODUCTION Trichinellosis is a parasitic zoonosis manifesting in humans as a syndrome with specific clinical signs and symptoms of variable intensity (Kocieka 2000). The infection is contracted by eating raw or under- cooked infected meat resulting in various symptoms that range from mild subclinical to classic forms de- pending on the extent of invasion, the parasite load, the species of Trichinella involved and the immune response of the host (Clausen, Meyer, Krantz, Mo- ser, Gomme, Kayser, Albrectsen, Cui, Wang, Wu & Jin 1997; Taratuto & Venturiello 1997; Kocieka 2000). 173 Onderstepoort Journal of Veterinary Research, 75:173–180 (2008) Experimental infections of baboons (Papio spp.) and vervet monkeys (Cercopithecus aethiops) with Trichinella zimbabwensis and successful treatment with ivermectin S. MUKARATIRWA1*, B.M. DZOMA1, E. MATENGA1, S.D. RUZIWA1, L. SACCHI2 and E. POZIO3 ABSTRACT MUKARATIRWA, S., DZOMA, B.M., MATENGA, E., RUZIWA, S.D., SACCHI, L. & POZIO, E. 2008. Experimental infections of baboons (Papio spp.) and vervet monkeys Cercopithecus aethiops) with Trichinella zimbabwensis and successful treatment with ivermectin. Onderstepoort Journal of Veter- inary Research, 75:173–180 Experimental Trichinella zimbabwensis infections were established in three baboons (Papio sp.) and four vervet monkeys (Cercopithecus aethiops) and the clinical-pathological manifestations assessed. The infected animals showed clinical signs ranging from fever, diarrhoea, periorbital oedema and muscular pain in varying degrees. One baboon became blind due to the infection. Levels of creatinine phosphokinase and lactate dehydrogenase increased to reach a peak on Day 42 post-infection (pi) for both baboons and monkeys. Blood parameters such as packed cell volume, levels of red blood cells and white blood cells did not change significantly from the normal ranges except for the levels of eosinophils which peaked above the normal ranges at Day 28 and 56 pi in baboons and at Day 56 pi in monkeys. Two baboons and two monkeys died during the course of the experiment. They were emaciated and showed lesions such as ascites, hydropericardium, congested liver and enlarged gall bladder. Histopathological findings of various muscles included a basophilic transformation of muscle cells, the dis appearance of sarcomere myofibrils and basophilic sarcoplasm with the presence of Trichinella larvae in the sarcoplasm. These changes were mainly in the massetter and were of various intensities in the tail, gastrocnemius and biceps muscles. Five consecutive treatments with an oxfendazole-le- vamisole combination on surviving animals failed to clear the infection whereas ivermectin cleared the infection after one treatment in two monkeys and after two treatments in a baboon. Keywords: Baboons, Cercopithecus aethiops, ivermectin, levamisole, monkeys, oxfendazole, Papio spp., pathology, Trichinella zimbabwensis * Author to whom correspondence is to be directed. E-mail: mukaratirwa@ukzn.ac.za 1 Department of Paraclinical Veterinary Studies, Faculty of Vet- erinary Science, University of Zimbabwe, P.O. Box MP 167 Mount Pleasant Harare, Zimbabwe 2 Department of Animal Biology, University of Pavia, Piazza Botta 9, 27100 Pavia, Italy 3 Department of Infectious, Parasitic and Immunomediated Dis- eases, Istituto Superiore di Sanità, viale Regina Elena 299, 0061 Rome, Italy Accepted for publication 21 April 2008—Editor 174 Experimental infections of baboons and vervet monkeys with Trichinella zimbabwensis The description of a new non-encapsulated spe- cies, Trichinella zimbabwensis, by Mukaratirwa & Foggin (1999), Pozio Foggin, Marucci, La Rosa, Sacchi, Corona, Rossi & Mukaratirwa (2002), which affects both mammals and reptiles is a new addition to the already existing five encapsulated species in- fecting only mammals (Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella murrelli and Trichinella nelsoni) and the two non-encapsulated species Trichinella pseudospiralis occurring in both mammals and birds and Trichinella papuae occur- ring in both mammals and reptiles (Pozio, Owen, Marucci & La Rosa 2004a). The only report of a natural infection of T. zimbab- wensis is in farmed crocodiles (Crocodylus niloticus) in Zimbabwe (Foggin, Vassilev & Widdowson 1997), where animals are reared for their meat and skin. Experimental infections have shown that T. zimbab- wensis readily infects domestic pigs and laboratory mice and rats (Mukaratirwa & Foggin 1999; Mu ka- ratirwa, Nkulungo, Matenga & Bhebhe 2003) con- firming its chance to complete the entire life cycle irrespective of whether the host is homoiothermic or poikilothermic (Pozio, Marucci, Casulli, Sacchi, Mu- karatirwa, Foggin & La Rosa 2004b). However, the epidemiology and the public health significance of this parasite are still obscure. Non-human primates by virtue of their close relation- ship to humans are good models for the study of the clinical-pathological manifestations of trichinellosis and its treatment. The objective of this study was to evaluate the clinical-pathological manifestations of baboons (Papio spp.) and vervet monkeys (Cerco- pithecus aethiops) experimentally infected with T. zimbabwensis and their response to treatment. MATERIALS AND METHODS Experimental animals Two adult female (codes SFB and BFB) and one male (code SMB) baboons and two adult female (codes BFM and SFM) and two male (codes SMM and BMM) vervet monkeys were randomly selected for the study. These animals were born and bred at the animal house unit of the Faculty of Veterinary Science, University of Zimbabwe. They were reared on a commercial primate diet and water was avail- able ad libitum. A week before the infection, biop- sies were collected from the animals while they were under anaesthesia with a combination of xyla- zine (2 mg/kg) and ketamine (10 mg/kg) adminis- tered intramuscularly, from the massetter, biceps and gastrocnemius muscles to ensure that the ani- mals were free from Trichinella spp. infection. Restraint and experimental infection of animals The T. zimbabwensis used was an isolate derived from a naturally infected crocodile. It was maintained in the laboratory by serial passages in rats. First- stage larvae were collected from five skinned car- casses of infected rats following methods described by Pozio et al. (2002). Each experimental animal was anaesthetized with a combination of xylazine (2 mg/kg) and ketamine (10 mg/kg) intramuscularly. Two of the baboons were infected with doses ranging from 9 000–16 600 lar- vae per kg, whereas 17 600–25 000 larvae per kg were used to infect three of the monkeys (Table 1). One baboon and one monkey were kept as unin- fected controls. Sample collection and analysis Muscle biopsies and blood samples were collected while the animals were under anaesthesia at week- ly intervals after infection and at each time of collec- tion the rectal temperatures were recorded. The muscle biopsies were processed by the HCL-pepsin digestion method (Pozio et al. 2002) to detect the L-1 stage of T. zimbabwensis and the resulting lar- val counts were expressed as larvae per gram (lpg) of muscle. A portion of the muscle biopsy from each muscle was preserved in 3 % glutaraldehyde and processed for electron microscopy. The levels of creatinine kinase (CK) and lactate dehydrogenase (LDH) were determined in serum samples. The clinical signs were recorded daily until the end of the experiment. The animals were treated for 5 consecutive days with a combination of levamisole (40 mg/kg) and oxfendazole (50 mg/kg) and dexam- ethasone (5 mg/kg) on Days 35 and 51 post-infec- tion (pi) for the monkeys and baboons, respectively. An intramuscular injection of ivermectin (300 μg/kg) was administered after the levamisole-oxfendazole treatment. The efficacy of treatment was monitored through weekly collection of muscle biopsies from the masseter muscles to detect viable first stage lar- vae. Histological and ultrastructure studies of the nurse cell-parasite complex Muscle biopsies and muscles of animals which died during the study were cut into small sections and in 175 S. MUKARATIRWA et al. part fixed in 10 % buffered formalin and in part pre- served in glutaraldehyde for ultrastructural studies. For histology, fixed specimens were dehydrated us- ing different grades of alcohol, cleared in xylene em- bedded in paraffin wax at 58–60 °C and finally, 3 μm sections were stained by haematoxylin and eosin stain (Anderson & Gordon 1996). Sections were ex- amined by light microscopy using 5–40X magnifica- tion. Small pieces of the biopsy from the masseter mus- cle of a baboon, collected 51 days pi, were fixed for 4 h at 4 °C in 0.1 M cacodylate buffer (pH 7.2) con- taining 2.5 % glutaraldehyde. The samples were then washed in the same buffer and post-fixed for 1.5 h at 4 °C with 1 % OsO4 in cacodylate buffer. All sam- ples were dehydrated in ethanol and embedded in Epon 812 for sectioning. For light microscopy, semi- thin sections (0.5 μm) were stained with 0.5 % tolui- dine blue. Thin sections (80 nm), stained with uranyl acetate and lead citrate, were examined under a Zeiss EM 900 transmission electron microscope. RESULTS Parasitological aspects The pre-infection muscle biopsies from all experi- mental animals were negative for Trichinella larvae. In the infected baboons that died, muscles with the highest lpg were the diaphragm, psoas, laryngeal, and tongue in this order (Table 1). In monkeys, the diaphragm, tongue and masseter had the highest lpg compared to the other muscles (Table 1). No relationship was observed between the infective dose, the severity of the disease and the lpg in both baboons and monkeys. Clinical manifestations The clinical signs observed are summarized in Table 2. The time between infection and onset of the first clinical signs ranged from 9 to 30 days pi. The rectal temperatures ranged between 38.4–40 °C in the ba- boons and 38.9–39.9 °C in the monkeys for a period of 8 weeks. TABLE 1 Number of larvae per gram (Lpg) of muscle in baboons and monkeys that died due to Trichinella zimbabwensis infection Larvae per gram of muscle Animal code Infective dose/kg Day of death pi Diaphragm Massetter Tongue Psoas Laryngeal muscle Digital flexus Hamstring muscle Eye SFB 16 600 50 621 260 820 965 545 552 910 37 SMB 16 400 49 9 987 465 888 1 819 1 600 287 479 144 BMM 17 600 35 255 ND 215 ND 135 75 115 ND BFM 25 000 30 100 168 165 ND ND 86 ND 1 ND = not determined pi = post infection TABLE 2 Clinical manifestations of baboons and monkeys infected with Trichinella zimbabwensis Animal code SFB SMB BFB BFM SFM SMM BMM Infective dose/kg 16 600 16 400 7 300 25 000 24 500 24 500 17 600 Clinical signs and symptoms Day of the first manifestation pi Fever Diarrhoea Depression Periorbital oedema Muscular pain Alopecia Blindness Death 9 16 12 30 30 26 38 49 dpi 11 16 12 30 30 26 no 50 dpi 16 16 12 30 30 26 no s 14 16 12 30 30 26 no 30 dpi 13 19 12 30 30 26 no s 18 21 12 30 30 26 no 36 dpi 10 21 12 30 30 26 no s pi = post infection, dpi = days post infection, no = not observed, s = survived 176 Experimental infections of baboons and vervet monkeys with Trichinella zimbabwensis Diarrhoea was observed in all infected animals from Day 16 pi (Table 2). Gradual depression and dete- rioration in body condition accompanied by alo- pecia, muscle pain on palpation and wasting were observed in all infected animals and these became marked from Day 26 pi. Blindness of both eyes was only observed in one baboon (SFB). Clinical biochemistry and blood parameters Levels of CK ranged from 197 to 3409 UI/ℓ in ba- boons and from 78 to 2679 UI/ℓ in the infected mon- keys. The levels of CPK in the infected baboons gradually increased from Day 0 to Day 28 pi and then increased tenfold to reach a peak on Day 42 pi. After the peak, there was a sharp drop to reach nor- mal levels from Day 56 pi (Fig. 1A and B). In infected monkeys, the CK levels slightly decreased from Day 0 pi to Day 28 pi before starting to increase with a peak on Day 56 pi. Levels of LDH ranged from 118 to 1587 UI/ℓ in ba- boons and from 203 to 1469 UI/ℓ in monkeys. In both monkeys and baboons, LDH reached a peak at day 42 pi. Packed cell volume (PCV) and white cell counts did not change significantly in the course of the infec- tion. However, the level of eosinophils peaked at Day 28 and 56 pi above the normal ranges in ba- boons and monkeys, respectively. Treatment The 5-day-treatment with oxfendazole-levamisole combination failed to kill Trichinella larvae; on the contrary, the single ivermectin treatment success- fully destroyed the larvae in all the surviving infected monkeys (BMM and SMM) but in the baboon (BFB) a low level of larvae was still detected in the biopsy (Table 3). In this animal, no living larvae were de- tected in another muscle biopsy collected after a second treatment with ivermectin. Histopathology and ultrastructure of muscle tissues All the animals which died due to the infection (SFB, SMB, BFM and FMM) were emaciated and showed ascites, hydropericardium, congested livers and en- larged gall bladders. Histopathological findings of various muscles included a basophilic transforma- tion of muscle cells, the disappearance of sarcom- ere myofibrils and a sarcoplasm that was basophilic. The changes also included pale, swollen muscle fi- bres with the presence of Trichinella larvae in the sarcoplasm, and the loss of myofibrils in affected muscles. These changes were mainly in the mas- seter, and were of various intensities in the tail, gas- trocnemius and bicep muscles. Biliary fibrosis, eosi- nophilic myositis, lymphocytic enteritis and dilatation of lymphatic vessels of the large and small intes- tines were also observed in SFB. In muscles infected with T. zimbabwensis, clear mod ifications of the muscle fibre architecture were observed. The myofilaments appeared modified in a nurse-cell-like structure (Fig. 2A). The thick colla- gen capsule was absent, and no inflammatory reac- tion was observed around the nurse cell-larva com- plex. Transmission electron micrographs revealed the ultrastructural changes in the muscle fibres. In the nurse-cell-like structure the contractile elements 3 500 3 000 2 500 2 000 1 500 1 000 500 0 IU /ℓ 0 14 28 42 56 60 Days post-infection Baboon (n = 2) A LDHCK 3 500 3 000 2 500 2 000 1 500 1 000 500 0 IU /ℓ 0 14 28 42 56 60 Days post-infection Monkey (n = 2) B FIG. 1 Kinetics of creatinine phosphokinase (CK) and lactate dehydrogenase (LDH) in a baboons BFB and SMB (A) and monkeys SFM and BMM (B) infected with Trichinella zimbabwensis 177 S. MUKARATIRWA et al. FIG. 2 Larvae of Trichinella zimbabwensis in muscles of a baboon 51 days post infection A Longitudinal section of an intracellular larva (l), surrounded by a nurse-cell-like structure (nc). Note the absence of the typical collagen capsule. No inflammatory cells were ob- served around the infected muscle fibres. mc = normal muscle cell. Bar = 200 μm B TEM micrograph showing a Trichinella larva (l) surrounded by a nurse-cell-like structure (nc). Note the loss of contractile elements. Bar = 5 μm C Details of the nurse-cell-like structure (nc) showing a hypertrophic nucleus with the nu- cleolus (nu) in the transcriptional phase. Bar = 5 μm D Details of the outer zone of the nurse-cell-like structure (nc) showing bands of collagen fibres (arrows). Mc = normal muscle cell, n = fibroblast nucleus, nc = nurse-cell-like struc- ture. Bar = 5 μm A B C D 178 Experimental infections of baboons and vervet monkeys with Trichinella zimbabwensis were replaced by a sarcoplasmic reticulum (Fig. 2B). At higher magnification, in the cytoplasm of this structure, characterized by the presence of a smooth and rough endoplasmic reticulum, it was possible to observe hypertrophic nuclei each of which contained a prominent nucleolus (Fig. 2C). This detail was in- dicative of the presence of an intense transcriptional activity. At the peripheral zone of the nurse-cell lar- va complex, bundles formed by irregularly arranged collagen fibres were observed (Fig. 2D). DISCUSSION In this study, T. zimbabwensis was able to establish in both baboons and monkeys and the clinical signs observed did not differ from those already reported in humans infected with the non-encapsulated spe- cies T. pseudospiralis (Ranque, Faugére, Pozio, La Rosa, Tamburrin, Pellissier & Brouqui 2000). The normal rectal temperatures of baboons and monkeys are 36–39 °C and 36–40 °C, respectively (Poole 1987). In the course of trichinellosis in hu- mans, fever is one of the most common sign ex- pected (Clausen et al. 1997) and in this study it was recorded in both infected baboons and monkeys. The observation of diarrhoea in infected animals in this study around Day 16 pi could be related to the early stages of intestinal invasion which manifests clinically as diarrhoea of several days’ duration and abdominal pains (Kociecka 2000). The pathophysi- ology of trichinellosis in the small intestines includes restricted absorption, disturbed motility of the intes- tines, diarrhoea or constipation and the presence of cellular infiltrates in the lamina propia (Ruitenberg, Elgersma, Kruizing & Leenstra 1977; Castro & Bul- lick 1983; Gustowska, Ruitenberg, Elgersma & Ko- ciecka 1983; Ruitenberg & Buys 1986). In humans infected with T. spiralis, pathomorphology of the in- testinal mucosa includes lesions to the epithelium involving the brush border, lamina propia and smooth muscles of the jejunum, deformation of villi, stimu- lated enterocyte proliferation at villi margins, hyper- plasia in the crypts of Lieberkuhn and the presence of massive cellular infiltrates in the mucosal sublayer (Castro & Bullick 1983). Lesions may persist until Day 65 pi (Kociecka 1981a; Gustowska et al. 1983). In this study, similar intestinal lesions were noted from a baboon that died on Day 49 pi. Periorbital and facial oedema is a common clinical sign of T. spiralis and T. pseudospiralis trichinellosis (Kociecka 1981a) and this agrees with the findings from this study where all affected animals manifested perior- bital oedema. The observation that the intensity of clinical disease and the muscle larval yields were not proportional to the initial infective doses given to the animals might suggest host factors that determine disease resist- ance since the parasite and environment factors were similar for all the animals. The experimental infection of Swiss CD1 mice re- veals the existence of structural changes of the in- fected muscle cells 4 months pi. These structures were very similar to those observed in crocodiles 18 months pi (Pozio et al. 2002). In the baboon infected with the same species of Trichinella, the myofila- ments of the infected muscle cell had lost their in- tegrity and appear transformed in a nurse-cell-like structure surrounded by a bundle of collagen fibres irregularly arranged (a true collagen capsule being absent) 51 days pi. These results suggest that T. zimbabwensis is able to infect different mammalian species in which it reproduces the same morpho- logical changes as have been described in reptiles (Pozio et al. 2002, 2004b). The choice of treatment for trichinellosis in humans varies according to the clinical severity of the infec- tion and the strain or species of Trichinella involved (Andrews, Ainsworth & Abernethy 1994). Drugs ad- ministered to infected humans include anthelmintics, glucocorticosteroids, immuno-modulating drugs and preparations which compensate protein and elec- trolyte deficits (Kociecka 2000). In this study, since TABLE 3 Larvae per gram (lpg) of masseter muscle following treatment with oxfendazole-levamisole combination and subsequent use of ivermectin in three monkeys and one baboon infected with Trichinella zimbabwensis Animal code lpg after oxfendazole- levamisole treatment lpg after one ivermectin treatment lpg after second ivermectin treatment BMM SMM *SFM BFB 11.1 9.5 6.4 44.1 0 0 – 7.5 – – – 0 * Died 36 days post infection 179 S. MUKARATIRWA et al. adult worms had long been expelled the treatment was targeted at the larvae in muscles. Injectable ivermectin was able to clear the infection after 7 days following a single dose unlike the oxfendazole- levamisole combination which failed to clear the in- fection in both the monkeys and baboons when ad- ministered for 5 consecutive days. However, it was not determined whether the effects of ivermectin were potentiated by the earlier use of the combina- tion of oxfendazole-levamisole or were due solely to the anthelmintic effects of the drug. Disturbances in blood muscle enzyme activities are related primarily to activities of those enzymes that are muscle bound, and these include CK, LDH and occasionally aspartate aminotransferase (AST). This follows the damage of the muscle cell by the L-1 resulting in an increase in permeability of the muscle cell membranes (Boczon, Winiecka, Kociecka, Hadas & Andrezejewska 1981). In 75–90 % of human cases of trichinellosis, an increase in the activity of CK was noted between Weeks 2 and 5 pi and involved an increase of several fold and LDH levels fluctuated from Week 1 to Week 6 pi. In this study, LDH and CK peaked around Day 42 pi in both baboons and monkeys although there were phases where the levels of both enzymes dropped. This is unsurprising since the increase in CK and LDH activity in blood is not correlated to the clinical severity of trichinellosis (Boczon et al. 1981) and there could also be some host differences in react- ing to different Trichinella spp. The peak around Day 42 pi of both enzymes could be related to the fact that almost all of the parasites are expected to be in the muscles by that period resulting in marked muscle membrane damage (Boczon et al. 1981). The settling of Trichinella larvae in the muscles re- sults in the basophilic transformation of the muscle cells, encapsulation of the larvae and the develop- ment of a capillary network surrounding the affected cell (Gabryel & Blotna 1969; Gabryel, Gustowska & Blotna-Filipiak 1995). These changes appear early and persist for as long as the encapsulated larva remains viable. In this study the basophilic changes were observed together with an eosinophilic infiltra- tion around the affected cell. The muscles that were preferred most by the parasite were the diaphragm, psoas, laryngeal, tongue and masseter muscles in that order. The masseter muscle, however, had the greatest histopathological changes attributable to the parasite. Trichinella pseudospiralis and T. spira- lis larvae have been shown to have the massetter muscle as their main predilection sites in monkeys (Kociecka 1981b), and this probably explains the high histopathological changes in the massetter muscles from our animals. Leukocytosis appears early and rapidly increases between Weeks 2 and 5 of the disease and sub- sides in parallel to clinical signs while eosinophilia persists (Kociecka 2000). However, an extremely severe course of trichinellosis may be accompanied by eosinopenia and/or lymphopenia which is a man- ifestation of immunosuppression (Dupoy-Camet, Paugam, Picard & Ancelle 1994). In our study, eosi- nophilia was noted, albeit between Weeks 4 and 8 pi. However, eosinophilia has been reported to re- gress slowly and may persist from several weeks to 3 months pi, and no relationship has been noted between the clinical course of disease and eosi- nophilia (Dupoy-Camet et al. 1994). The clinical, parasitological, pathological, biochemi- cal and haematological picture of T. zimbabwensis infection in the baboons and monkeys in this study closely simulates those reported for T. pseudospira- lis and T. spiralis infection in humans and other pri- mates (Clausen et al. 1997; Taratuto & Venturiello 1997; Ranque et al. 2000). The fact that T. zimbab- wensis could successfully infect non-human pri- mates to give clinical features characteristic of other Trichinella species in humans could have relevant implications in human infection as chances of hu- mans getting infected with T. zimbabwensis are pres ent in Zimbabwe where crocodile meat is con- sumed. ACKNOWLEDGEMENTS We thank the University of Zimbabwe for financial support and staff of the University Animal House and Parasitology Section, Department of Paraclinical Veterinary Studies for their assistance in handling the animals and processing of the samples. REFERENCES ANDERSON, G. & GORDON, C.K. 1996. Tissue processing, in Theory and practice of histological techniques, 4th ed., edited by J. Bancroft & A. Stevens. Churchill Livingstone. ANDREWS, J.R., AINSWORTH, R. & ABERNETHY, D. 1994. Trichinella pseudospiralis in humans: description of a case and its treatment. Transactions of the Royal Society of Trop- ical Medicine and Hygiene, 88:200–203. BOCZON, K., WINIECKA, J., KOCIECKA, W., HADAS, E. & ANDREZEJEWSKA, I. 1981. The diagnostic value of enzy- matic and immunological tests in human trichinosis. Tropen- med Parasitology, 32:109–114. CASTRO, G.A. & BULLICK, G.R.1983. Pathophysiology of gas- trointestinal phase, in Trichinella and trichinosis, edited by W.C. Cambell. Vol. 22. New York: Plenum Press. 180 Experimental infections of baboons and vervet monkeys with Trichinella zimbabwensis CLAUSEN, M.R., MEYER, C.N., KRANTZ, T., MOSER, C., GOMME, G., KAYSER, L., ALBRECTSEN, J., CUI, J., WANG, Z.Q., WU, F. & JIN, X.X. 1997. Epidemiological and clinical studies in an outbreak of trichinosis in central China. Annals of Tropical Medicine and Parasitology, 91:481–488. DUPOY-CAMET, J., PAUGAM, A., PICARD, F. & ANCELLE, T. 1994. Lymphopenie au cours de la trichinose. Presse Medi- cale, 23:95. FOGGIN, C.M., VASSILEV, G.D. & WIDDOWSON, M.A. 1997. Infection with Trichinella in farmed crocodiles (Crocodylus niloticus) in Zimbabwe. Abstract book on the 16th International Conference of the World Association for the Advancement of Veterinary Parasitology, 10–15 August 1997, Sun City, South Africa. Abstract No. 110. GABRYEL, P. & BLOTNA, M. 1969. Ultrastructural significances of the altered metabolism of muscle fibres infected by Trichinella spiralis. Wiadomosci Parazytologizne, 15:673– 675. GABRYEL, P., GUSTOWSKA, L. & BLOTNA-FILIPIAK, M. 1995. The unique and specific transformation of muscle cells in- fected with Trichinella spiralis. Basic Applied Myology, 5: 231–238. GUSTOWSKA, L., RUITENBERG, E.J., ELGERSMA, A. & KO- CIECKA, W. 1983. Increase of mucosal mast cells in the je- junum of patients infected with Trichinella spiralis. Inter- national Archives of Allergy and Applied Immunology, 71: 304–308. KOCIECKA, W. 1981a. Relationship between the clinical picture of trichinosis, the species or strain of Trichinella and the in- tensity of invasion. I. Clinical studies. Wiadomosci Para- zytolo gizne, 27:399–442. KOCIECKA, W. 1981b. Relationship between the clinical picture of trichinosis, the species or strain of Trichinella and intensity of invasion. II. Experimental studies. Wadomosci Para zytolo- gizne, 27: 443–482. KOCIECKA, W. 2000. Trichinosis: human disease, diagnosis and treatment. Veterinary Parasitology, 93:365–383. MUKARATIRWA, S. & FOGGIN, C.M. 1999. Infectivity of Tri- chinella sp. isolated from Crocodylus niloticus to the indige- nous Zimbabwean pig (Mukota). International Journal for Parasitology, 29:1129–1131. MUKARATIRWA, S., NKULUNGO, E., MATENGA, E. & BHE- BHE, E. 2003. Effect of host age in the distribution of adult Trichinella zimbabwensis in the small intestines of golden hamsters (Mesocricetus auratus) and Balb C mice. Onder- stepoort Journal of Veterinary Research, 70:169–173. POOLE, T. (Ed.). 1987. The care and management of laboratory animals. Harlow, Essex: Longman Scientific and Technical. POZIO, E., FOGGIN, C.M., MARUCCI, G., LA ROSA, G., SACCHI, L., CORONA, S., ROSSI, P. & MUKARATIRWA, S. 2002. Trichinella zimbabwensis n. sp. (Nematoda), a new non-encapsulated species from crocodiles (Crocodylus nilo- ticus) in Zimbabwe also infecting mammals. International Journal for Parasitology, 19:1787–1799. POZIO, E., OWEN, I.L., MARUCCI, G. & LA ROSA, G. 2004a. Trichinella papuae in saltwater crocodiles (Crocodylus poro- sus) of Papua New Guinea: A potential source of human in- fection. Emerging Infectious Diseases, 10:107–1509. POZIO, E., MARUCCI, G., CASULLI, A., SACCHI, L., MU KA RA- TIRWA, S., FOGGIN, C.M. & LA ROSA, G. 2004b. Trichinella papuae and Trichinella zimbabwensis induce infection in ex- perimentally infected varans, caimans, pythons and turtles. Parasitology, 128:333–342. RANQUE, S., FAUGÉRE, B., POZIO, E., LA ROSA, G., TAM- BURRIN, A., PELLISSIER, J.F. & BROUQUI, F. 2000. Trichi- nella pseudospiralis outbreak in France. Emerging Infectious Diseases, 6:43–547. RUITENBERG, E.J., ELGERSMA, A., KRUIZING, N. & LEEN- STRA, F.1977. Trichinella spiralis infection in congenitally athymic (nude) mice: parasitological, serological and haema- tological studies with observations on intestinal pathology. Immunology, 33:581–587. RUITENBERG, E.J. & BUYS, J. 1986. Eosinophils and mononuclear cells as effector cells in a Trichinella spiralis infection; cell biological and biochemical aspects and the use of biological response modifiers. Wiadomosci. Parazytolo- gizne, 32:219–231. TARATUTO, A.L. & VENTURIELLO, S.M. 1997. Trichinosis. Brain Pathology, 7:663–672. << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (Europe ISO Coated FOGRA27) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts false /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 600 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.00000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 600 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.00000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 600 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.00000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /CHS /CHT /DAN /DEU /ESP /FRA /ITA /JPN /KOR /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.) /NOR /PTB /SUO /SVE /ENS () /ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames true /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides false /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks false /AddPageInfo false /AddRegMarks false /ConvertColors /ConvertToCMYK /DestinationProfileName () /DestinationProfileSelector /DocumentCMYK /Downsample16BitImages true /FlattenerPreset << /PresetSelector /MediumResolution >> /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ] >> setdistillerparams << /HWResolution [600 600] /PageSize [595.276 841.890] >> setpagedevice