213
Laboratory Animals (I978) 12,213-218
The incidence, pathogenesis and treatment of helminth infections in rhesus monkeys (Macaca mulatta) JENNY REMFR Y· University Laboratory of Physiology. Parks Road. Oxford. OX] 3PT
Summary In a survey of 259 newly-imported rhesus monkeys, 5 species of nematode parasites were commonly found. Of these 5 species, Oesophagostomum spp. and Strongyloides fulleborni were implicated in the deaths of 3 monkeys. 5 other nematode species, 2 cestode species and I trematode species were also found; their pathogenicity is discussed. The results of treatment of parasitic infections with 6 different drugs is reported. Thiabendazole at an initial dose of 100 or 133 mg/kg and repeated at 50 mg/kg was found to be the most effective treatment against Oesophagostomum spp. When rhesus monkeys are captured in the hills of Asia, they carry a variety of helminth parasites. So long as the monkeys are in otherwise normal health these parasites are not severely pathogenic, but transport and relocation in a new environment inevitably creates stresses which tend to lower the resistance of the animals, and the parasites can then cause disease and sometimes death. In this survey of all the monkeys coming to the Primate Quarantine Unit of Oxford University during an 18 month period, 5 species of intestinal nematodes were commonly found; less common were 5 more nematode, 2 cestode and an unidentified trematode species. The results were comparable with those of earlier surveys in the UK (Owen & Casillo, 1973) and the USA (Habermann & Williams, 1957; Reardon & Rininger, 1968). The pathogenicity of these parasites to rhesus monkeys has not been much studied, but the existing information has been reviewed by Ruch (1959) and Fiennes (1972). Several species are also parasitic to man, and the diseases caused are described in standard texts (e.g. Wilcocks & Manson-Bahr, 1972). 3 monkey deaths in the Oxford Unit were attributed to parasitism, and these cases are described. After a conditioning period of about 3 months, most of the monkeys were issued to University Departments for long-term experiments which involved handling. During conditioning, anthelmintic treatment was given • Present address: Universities Federation for Animal Welfare, 8 Hamilton Close, South Mimms, Potters Bar, Hertfordshire,EN6 3QD Received 2 November 1977. Accepted 24 April 1978.
with the aim of completely eradicating helminth parasites, both for animal health and to eliminate the risk of zoonotic infection. For treatment of helminthiasis, piperazine, phenothiazine and thiabendazole have been used extensively (see Flynn, 1973). However, there is now a range of newer drugs marketed for man or for domestic animals whose efficacy in non-human primates has been assumed but rarely tested. Results will be presented of the success obtained in treating newlyimported rhesus monkeys with some of those new anthelmintics. Materials and methods Animals Between October 1974 and July 1976, 259 rhesus monkeys (Macaca mulatta) were imported from northern India and received by Oxford University's Primate Quarantine Unit. Between capture and air shipment to London, the monkeys were held for about 4 weeks at the exporter's premises in Delhi in groups, pairs or singly. The majority of the monkeys were young males weighing 2·5-3·5 kg on arrival. Their ages, estimated by examination of the teeth and using the eruption times reported by Bowen & Koch (1970) for M. fascicularis (irus), were between 18 months and 3 years. A few females were imported: these were mostly mature (i.e. 5 years or over). All the monkeys were caged singly after arrival at Oxford.
Parasites Worms seen to be passed in the faeces were collected and identified. Faecal samples were collected for examination on the day after arrival, then at regular (usually weekly) intervals afterwards. Ova were concentrated by a variety of methods, but most frequently by the modified formol-ether concentration technique (Allen & Ridley, 1970) because protozoan cysts were being sought at the same time. Worms and ova were identified by size, shape, colour and special features, and referring to Flynn (1973), Georgi (1974) and Wilcocks & Manson-Bahr (1972). As a result of experience, Table 1 was compiled for the identification of nematode ova.
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Table I. Identification of nematode ova Species
Length (pm) range (n)
mean
Oesophagostomum spp. Strongyloides ful/ebomi Trichostrongylus spp. Streptopharagus pigmentatus Trichuris trichiura Ascaris lumbricoides Temidens deminutus Enterobius vermicularis observed spp. Physaloptera spp.
55-75 (32) 64·5 45-60 (35) 52· 7 80-100 (23) 87·8 37-42 (16) 38·8 55-60 (6) 57·2 40-50 (5) 47·4 Eggs indistinguishable from 50-60· 87 (1) 35 (1)
Anatrichosoma cutaneum
60-67·
sd
Width (pm) range (n)
4·9 32-52 (7) 4·1 32-35 (4) 5·5 45 (1) I·S 22-25 (3) 2·5 t 4·3 37 (I) Oesophagostomum 20-30·
t
Other features mean 42·1 33·0 23·7
20(1)
40-4S·
morulated in fresh faeces embryonated in fresh faeces morulated in fresh faeces embryonated and pigmented bipolar plugs; brown rough coat, bile-stained asymmetrical, embryonated asymmetrical, embryonated thick shell with double contour bipolar plugs
• Measurements quoted in Flynn (1973).
t Not measured.
Anthelmintic drugs Thiabendazole was used routinely for the initial treatment of nematode infections in all the early trials, then 5 other drugs were used for the eradication of specific parasites or for comparison with thiabendazole. Thiabendazole ('Thibenzole'; Merck, Sharp & Dohme Ltd, Hertford Road, Hoddesdon, Hertfordshire, EN 11 9BU) as a suspension for oral administration containing 13·3% w/v, i.e. approx. 133 mg/ml. Dosages recommended by the manufacturer vary between 44 mg/kg bodyweight for routine treatment of sheep, and a minimum of 110 mg/kg in severely infested cattle. Recommended dosage in man is 25 mg/kg twice daily for 2 days. We used doses of 50, 100 or 133 mg/kg, with a 2nd dose after 3 weeks. Administration was by stomach tube to monkeys under sedation induced with phencyclidine hydrochloride ('Sernylan'; Bioceutical Laboratories Inc., St. Joseph, Missouri 64502, USA). Thiabendazole is accepted by monkeys if mixed with the food, but this method was not fully tested. Mebendazole ('Telmin'; Janssen Pharmaceutica NV, Turnhoutsebaan 30, Beerse 2340, Belgium) supplied by special request as a 10% mixture in sucrose for oral administration. When required for use, the mixture was made up as a suspension in water to give a mebendazole concentration of 20 mg/ml. The dosage recommended by the suppliers (Crown Chemical Co. Ltd, Lamberhurst, Kent, TN3 8DJ) for horses is 510 mg/kg, but higher doses were recommended for monkeys. We used 20 mg/kg daily for 4 days, adding I ml of the suspension for each kg body weight to a water bottle containing about 150 ml water. The monkeys found the sweetness attractive and usually emptied the bottle within a few hours.
Levamisole ('Nemicide'; Imperial Chemical Industries Ltd, Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, SKIO 4TF) supplied as a 7·5% w/v solution for subcutaneous injection, i.e. approx. 75 mg/ml. Dosage recommended by the manufacturer for pigs and sheep is 7·5 mg/kg or, when given by mouth as 'Ketrax' for man, 2·5 mg/kg as a single dose. We used 7·5 mg/kg and 11·25 mg/kg. The monkeys were sedated, weighed, and the computed volume injected subcutaneously into the scruff of the neck. Thenium closylate and piperazine phosphate ('Ancaris'; Wellcome Foundation Ltd, 183 Euston Road, London, NW 1 2BP). Thenium is claimed to be effective against some hookworms (Ancylostoma and Uncinaria spp.), and piperazine is a time-honoured treatment for ascarids. The manufacturers recommend a dosage of I tablet containing 216·25 mg thenium and 260 mg piperazine per 4 kg for dogs, to be ·repeated 6 hr later. We used I tablet per monkey (weight 3·0-4·5 kg) as a 2nd-line treatment after thiabendazole, and did not repeat the dose. Administration was by mouth under sedation induced with phencyclidine, in which the swallowing reflex was retained. Dichlorvos ('Task'; Tasman Vaccine Laboratories (UK) Ltd, Eastern Way, Bury St Edmunds, Suffolk IP32 7AL) supplied as 10 mg tablets. Recommended dosage for dogs and cats is 10 mg/kg. The monkeys were dosed at this level or below, e.g. a monkey weighing 3·5 kg was given 3 tablets. Dichlorvos is a cholinesterase inhibitor and should not be used with other cholinesterase inhibitors such as phencyclidine. The tablets were therefore administered to the conscious animal at least 3 days after and 3 days before sedation, by crushing the tablets and concealing the powder in a banana.
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Helminths in rhesus monkeys
215
Niclosamide ('Yomesan'j Bayer (UK) Ltd, Eastern Way, Bury St Edmunds, Suffolk, IP327AH) as tablets for oral administration containing 0·5 g. Dosage recommended by the manufacturer is 1 tablet per 4 kg for dogs and cats, to be given on an empty stomach. We gave 1 tablet per monkey (weight 3·04·5 kg) to animals under sedation which did not inhibit the swallowing reflex.
Table
2. Incidence of helminth endoparasites in rhesus monkeys within 24 h after arrival in quarantine unit
No. animals passing worms or ova total Nematodes Oesophagostomum spp. Strongyloides Jul/eborni Trichuris trichiura Trichostrongylus spp. Streptopharagus pigmentatus Enterobius spp. Physaloptera tumeJaciens Ascaris lumbricoides Anatrichosoma cutaneum Unidentified Cestodes Bertiel/a studeri Hymenolepis nana Trematodes Unidentified Total monkeys screened
%
106 144 29 47 34 1 2 6 1 3
40·9 55·6 11·2 18·1 13·1 0·4
3 1
1·2 0·4
1 259
0·8
2·3 0·4 1·2
0·4 100
Strongyloides fulleborni, the commonest of the nematodes found in this survey, is also infective to man (Pampiglione & Ricciardi, 1971). The related S. stercora lis is an important parasite of man in the tropics. The species of Trichuris found in the rhesus monkey is thought to be the human whipworm, T. trichiura (Ruch, 1959). The Ascaris species found in the rhesus monkey is indistinguishable from the common large roundworm of man, A. lumbricoides. All the species mentioned above have direct life cycles, so infected animals and their excreta are a hazard to man. Under some circumstances, man may be a source of infection to monkeys (Orihel, 1970). Trichostrongylus spp. are common parasites of wild mammals and domestic ruminants. No positive identification was made in this survey. Streptopharagus pigmentatus was the largest of the common nematodes seen. Adults were vomited up by 2 monkeys and passed in the faeces by several. Females measured 58-60 mm and males 24-27 mm. The distinctive anatomy of the elongated buccal cavity, spirally twisted, is illustrated in Georgi (1974). Enterobius vermicularis, the common pinworm of man, has been reported from primates, but the ova seen in this survey were of another, unidentified species. The best way of demonstrating ova is said to be by applying sticky cellophane tape to the perianal region, then onto a microscope slide (Faust, Beaver & Jung, 1968): this was done for 66 monkeys but no eggs were found, and subsequently only the usual faecal concentration technique was used. Anatrichosoma cutaneum is parasitic in the nasal mucosa and, perhaps, the skin of monkeys. According
Results
to Long, Lichtenfals & Stooky (1976) it is fairly
Incidence The number of monkeys passing worms or ova of each species of parasite after arrival in the quarantine unit is shown in Table 2. The nomenclature of Oesophagostomum sp. is rather confused (Flynn, 1973). Moreover, the ova of Oesophagostomum spp. are similar to those of Ternidens deminutus, and since no adult Ternidens were ever found, all ova within the size range shown were recorded as Oesophagostomum. Both are infective to man (Wilcocks & Manson-Bahr, 1972). Adult Oesophagostomum were fairly commonly found in faecal samples. Hookworm eggs (Ancylostoma and Uncinaria spp.) are similar to Oesophagostomum eggs. No adult hookworms were found in the faeces or post mortem. Although Ancylostoma duodenale is a common parasite of man in India, it is thought to be rare in non-human primates (Nelson, 1965). Any ova present would have been recorded here as Oesophagostomum.
common, though only 1 ovum was found in this survey. Eggs are transmitted in nasal mucus; some are swallowed and may be found in the faeces as in this case. Segments of the tapeworm Bertiella studeri, about 1 em wide, were commonly seen in the faeces of infected animals. The proglottid of Hymenolepis nana is small and easily missed in the dropping tray, but the embryonated egg is distinguished under the microscope by the presence of 3 pairs of small hooks. Pathogenicity Parasitism undoubtedly contributed to the morbidity of the newly-imported monkeys; coughing and diarrhoea were common during the 1st 3 weeks of quarantine, but the cOIftribution of parasitism was difficult to quantify. No specific clinical signs were found to correlate with specific parasitisms. Unthriftiness was often associated with parasitic burdens, but it
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216
was difficult to establish was effect.
which was cause and which
Oesophagostomiasis. Nodules contaInmg live Oesophagostomum larvae were frequently found in the wall of the caecum and colon of monkeys which died They were found also on the serosal (outer) surface of the large intestine, and in the omentum and mesenteries as a result of aberrant migration of the larvae. This was sometimes associated with local infection, abscess formation and adhesions, most commonly in the region of the ileo-caecal junction. In this survey, 2 deaths were associated with oesophagostomiasis. Monkey A. Young male. No anthelmintic treatment had been given. Post-mortem findings were Oesophagostomum worms in the lumen and nodules in the walls of the large intestine; an abscess within the omentum in the region of the ileo-caecal junction with adhesions between caecum and jejunum; congestion of liver, kidney and spleen; peritonitis; pneumonia with pleuritic adhesions in both lungs; pericarditis. Diplococcus (Streptococcus) pneumoniae was recovered from heart blood. It is thought (Fiennes, 1972) that the lesions caused by migrating larvae may become infected by colonic bacteria, leading to ulceration, perforation, peritonitis and septicaemia. The present case appeared to illustrate this. Monkey B. Young male. 50 mg/kg thiabendazole had been given 16 days before death. Post-mortem findings were similar to monkey A, with the addition of 2 intussusceptions, and the presence of hundreds of Oesophagostomum spp. and a few Trichuris trichiura worms in the lumen of the large intestine.
Strongyloidiasis. The pathological effects of Strongyloidesfulleborni (Flynn, 1973) are divided into 3 phases: the invasive phase when infective larvae penetrate the skin or buccal mucosa (this does not appear to cause irritation in monkeys); the migratory phase when infective larvae are carried in the blood to the heart and lungs, break into the alveoli, enter the bronchi, and are coughed up and swallowed, causing symptoms varying from a cough to bronchopneumonia; the intestinal phase when the swallowed larvae penetrate the glandular epithelium of the small intestine, causing symptoms varying from diarrhoea to peritonitis. 55% of the newly-imported monkeys were passing ova of Strongyloides, and a high percentage of these must have been undergoing the migratory and intestinal phases of the infection, thus contributing to the incidence of respiratory and intestinal disease. To see the larvae, it is necessary to examine histological sections oflung and small intestine. This was not done. However, ulceration of the intestine was sometimes found post mortem, as in the following case. Monkey C. Young male. Recently treated for pneumonia, and anthelmintic treatment (50 mg/kg
thiabendazole) was given 18 days before death. Postmortem findings were a small abscess on the caecum; blood-staining of omentum and peritoneal fluid; ulceration of the mucosal surface of the small and large intestines. No nodules or worms were seen, but the monkey had been passing Strongyloides ova. The pneumonia was partly resolved. It was thought that the peritonitis was due to the perforation of a Strongyloides-induced ulcer.
Trichuriasis in man causes great irritation and is thought to be a cause of rectal prolapse (Wilcocks & Manson-Bahr, 1972). In the rhesus monkey, rectal prolapse and intussusception were found from time to time; worms such as Trichuris may have been a cause (e.g. in monkey B) but often worms were absent. Streptopharagus pigmentatus lives in the stomach; its pathogenicity is unknown. 2 cases of vomiting were associated with it, but no other clinical signs.
No clinical signs were attributed to the other
parasites listed.
Treatment The effect of treating the naturally-infected rhesus monkeys with various drugs is shown in Table 3. The numbers of worms originally present in the animals were not shown, nor could they be deduced from the egg counts. The numbers of worms left after treatment were not known either, since few animals died. Therefore the results are expressed simply in terms of the percentage of infected animals which stopped passing ova after treatment, which continued clear at each sampling over several weeks, and which were still clear at the end of the conditioning period (3 months). From these results it appears that thiabendazole remains the most effective drug against Oesophagostomum, particularly at the higher dosage. The repeat dose is necessary to kill recently-developed adults, because larvae are resistant to treatment. Levamisole is the most effective drug against Strongyloides. However, the dose levels were high, and in man would be expected to cause side-effects such as nausea, headache and abdominal pain (Pene & Delmont, 1973). Mebendazole was active against Trichuris as well as the other common nematodes. However, a repeat treatment would be necessary for eradication of Strongyloides. This drug is considerably more expensive than the others. Dichlorvos was a useful specific drug against
Trichuris. Thenium was not significantly better than thiabendazole for the eradication of Streptopharagus. Anyway, Streptopharagus is self-limiting, because the life cycle requires an arthropod intermediate which seems to be absent in the UK.
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Helminths in rhesus monkeys
217
Table 3. Activity of ant helm in tics on the common
parasites
animals treated
% clear
animals treated
% clear
animals treated
24 45
92 96
38 56
87 96
8 12
25 33
a
a
100
90
I 12
10
1 10
75
70 60 75
12 9 9
83 100 100
10 5 4
For treatment against Trichostrongylus, thiabendazole, dichlorvos, mebendazole and levamisole were each 100% effective at the dosages described. Levamisole was 100% effective against Enterobius and mebendazole against Ascaris in the few cases treated. For treatment of cestodes, niclosamide was 100% effective in the few cases treated. No adverse side-effects were observed with any of the drugs given.
Discussion The results show that available anthelmintics are effective against the nematodes commonly parasitizing rhesus monkeys. In the 3 deaths attributed to parasitism in this survey, 1 (monkey A) had not been treated and the other 2 had been inadequately treated. This compares with 9 deaths due to enteritis in the same series, 13 to respiratory disease, and 3 cases of tuberculosis which were killed. At the end of the conditioning period (about 3 months), the monkeys were passed to users free from all helminth parasites except the occasional Trichuris or Strongyloides, though in order to achieve this repeated treatment was often necessary. The best method of administering anthelmintics is a matter of opinion. A sedated animal can be given an accurately measured dose by stomach tube, though occasionally vomiting occurs and treatment has to be repeated. Similarly, an accurately calculated dose can be injected. However, if animals are not sedated for examination and treatment until they have been in quarantine for several days, the parasitic damage may already have been done. Where a delay is likely it may be preferable to give the anthelmintic in the food and risk that the full dose will not be taken, or to inject into the conscious animal a dose calculated from the estimated bodyweight. The anthelmintic chosen will depend partly on the route of administration to be used, but since Oeso-
ofrhesus
Trichuris
Oesophagostomum Strongyloides
Thiabendazole 2 x 50 mg/kg Ix 100+lx500r I x 133 + I x 50 mg/kg Dichlorvos Thenium-piperazine Mebendazole Levamisole I x 7·5 mg/kg 2 x 7·5 mg/kg 2 x 11·25 mg/kg
nematode
4
% clear
100
a
monkeys
Streptopharagus
Trichostrongylus
animals treated
animals % treated clear
% clear
9 10
56 80
6 3
83 100
13 16
100 100
1 1
100
II
100
5 6 3
100 100 100
a
phagostomum was the most pathogenic of the nematodes encountered, thiabendazole in high dosage, or mebendazole in extended dosage, is to be preferred for the initial treatment of newly-imported animals. Subsequent treatment of these animals, or of animals already conditioned when brought in, will depend on the parasites found. If Trichuris or Streptopharagus are present in large numbers, treatment with dichlorvos or mebendazole is desirable, whereas for Strongyloides levamisole is the most effective. Acknowledgements I wish to thank Mr Ramsay Hovell for making this survey possible, Mr G. Richardson and Mr R. Weston for their help, and Mr T. Richards for his care for the monkeys. I thank the Wellcome Trust for a Veterin-
ary Research Fellowship. Drugs were kindly donated
by Crown Chemical Co. Ltd and Tasman Laboratories (UK) Ltd.
Vaccine
References
Allen, A. V. H. & Ridley, D. S. (1970). Further observations on the formol-ether concentration technique for faecal parasites. Journal of Clinical Pathology 23, 345-346. Bowen, W. H. & Koch, G. (1970). Determination of age in monkeys (Macaca irus) on the basis of dental development. Laboratory A nimals 4,113-123. Faust, E. C., Beaver, P. C. & Jung, R. C. (1968). Animal agents and vectors of human disease, 3rd ed. Philadelphia: Lea & Febiger. Fiennes, R. N. T. W. (ed.) (1972). Pathology of simian primates. Basel: Karger. Flynn, R. J. (1973). Parasites of laboratory animals. Ames: Iowa State University Press. Georgi, J. R. (1974). Parasitology for veterinarians, 2nd ed. Philadelphia: Saunders. Habermann, R. T. & Williams, F. P. (1957). Diseases seen at necropsy of 708 Macaca mulatta (rhesus monkey) and Macaca philippinensis (cynomolgus monkey). American Journal of Veterinary Research 18,419-426.
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Long, G. G., Lichtenfals, J. R. & Stooky, J. L. (1976). Anatrichosoma cynomolgi (Nematoda: Trichinellidae) in rhesus monkeys, Macaca mulatla. Journal oJ Parasitology 62, 111-115. Nelson, G. S. (1965). The parasitic helminths of baboons with particular reference to species transmissible to man. In The baboon in medical research (ed. H. Vagtborg), pp. 441-470. Austin: University of Texas Press. Orihel, T. C. (1970). The helminth parasites of non-human primates and man. Laboratory Animal Care 20,395-401. Owen, D. & Casillo, S. (1973). A preliminary survey of the nematode parasites of some imported Old-World monkeys. Laboratory Animals 7,265-269.
Pampiglione, S. & Ricciardi, M. L. (1971). The presence of Strongyloides Julleborni von Listow, 1905, in man in Central and East Africa. Parassitologia 13,257-269. Pene, P. & Delmont, J. (1973). Action du L-tetramisole dans Ie traitement de !'ancylostomiase. Medecine d'AJrique noire 20,41-43. Reardon, L. V. & Rininger, B. F. (1968). A survey of parasites in laboratory primates. Laboratory Animal Care 18,577-580. Ruch, T. C. (1959). Diseases oJ laboratory primates. Philadelphia: Saunders. Wilcocks, C. & Manson-Bahr, P. E. C. (1972). Manson's tropical diseases, 17th ed. London: Bailliere Tindall.
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Laboratory Animals (I978) 12,213-218
The incidence, pathogenesis and treatment of helminth infections in rhesus monkeys (Macaca mulatta) JENNY REMFR Y· University Laboratory of Physiology. Parks Road. Oxford. OX] 3PT
Summary In a survey of 259 newly-imported rhesus monkeys, 5 species of nematode parasites were commonly found. Of these 5 species, Oesophagostomum spp. and Strongyloides fulleborni were implicated in the deaths of 3 monkeys. 5 other nematode species, 2 cestode species and I trematode species were also found; their pathogenicity is discussed. The results of treatment of parasitic infections with 6 different drugs is reported. Thiabendazole at an initial dose of 100 or 133 mg/kg and repeated at 50 mg/kg was found to be the most effective treatment against Oesophagostomum spp. When rhesus monkeys are captured in the hills of Asia, they carry a variety of helminth parasites. So long as the monkeys are in otherwise normal health these parasites are not severely pathogenic, but transport and relocation in a new environment inevitably creates stresses which tend to lower the resistance of the animals, and the parasites can then cause disease and sometimes death. In this survey of all the monkeys coming to the Primate Quarantine Unit of Oxford University during an 18 month period, 5 species of intestinal nematodes were commonly found; less common were 5 more nematode, 2 cestode and an unidentified trematode species. The results were comparable with those of earlier surveys in the UK (Owen & Casillo, 1973) and the USA (Habermann & Williams, 1957; Reardon & Rininger, 1968). The pathogenicity of these parasites to rhesus monkeys has not been much studied, but the existing information has been reviewed by Ruch (1959) and Fiennes (1972). Several species are also parasitic to man, and the diseases caused are described in standard texts (e.g. Wilcocks & Manson-Bahr, 1972). 3 monkey deaths in the Oxford Unit were attributed to parasitism, and these cases are described. After a conditioning period of about 3 months, most of the monkeys were issued to University Departments for long-term experiments which involved handling. During conditioning, anthelmintic treatment was given • Present address: Universities Federation for Animal Welfare, 8 Hamilton Close, South Mimms, Potters Bar, Hertfordshire,EN6 3QD Received 2 November 1977. Accepted 24 April 1978.
with the aim of completely eradicating helminth parasites, both for animal health and to eliminate the risk of zoonotic infection. For treatment of helminthiasis, piperazine, phenothiazine and thiabendazole have been used extensively (see Flynn, 1973). However, there is now a range of newer drugs marketed for man or for domestic animals whose efficacy in non-human primates has been assumed but rarely tested. Results will be presented of the success obtained in treating newlyimported rhesus monkeys with some of those new anthelmintics. Materials and methods Animals Between October 1974 and July 1976, 259 rhesus monkeys (Macaca mulatta) were imported from northern India and received by Oxford University's Primate Quarantine Unit. Between capture and air shipment to London, the monkeys were held for about 4 weeks at the exporter's premises in Delhi in groups, pairs or singly. The majority of the monkeys were young males weighing 2·5-3·5 kg on arrival. Their ages, estimated by examination of the teeth and using the eruption times reported by Bowen & Koch (1970) for M. fascicularis (irus), were between 18 months and 3 years. A few females were imported: these were mostly mature (i.e. 5 years or over). All the monkeys were caged singly after arrival at Oxford.
Parasites Worms seen to be passed in the faeces were collected and identified. Faecal samples were collected for examination on the day after arrival, then at regular (usually weekly) intervals afterwards. Ova were concentrated by a variety of methods, but most frequently by the modified formol-ether concentration technique (Allen & Ridley, 1970) because protozoan cysts were being sought at the same time. Worms and ova were identified by size, shape, colour and special features, and referring to Flynn (1973), Georgi (1974) and Wilcocks & Manson-Bahr (1972). As a result of experience, Table 1 was compiled for the identification of nematode ova.
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Table I. Identification of nematode ova Species
Length (pm) range (n)
mean
Oesophagostomum spp. Strongyloides ful/ebomi Trichostrongylus spp. Streptopharagus pigmentatus Trichuris trichiura Ascaris lumbricoides Temidens deminutus Enterobius vermicularis observed spp. Physaloptera spp.
55-75 (32) 64·5 45-60 (35) 52· 7 80-100 (23) 87·8 37-42 (16) 38·8 55-60 (6) 57·2 40-50 (5) 47·4 Eggs indistinguishable from 50-60· 87 (1) 35 (1)
Anatrichosoma cutaneum
60-67·
sd
Width (pm) range (n)
4·9 32-52 (7) 4·1 32-35 (4) 5·5 45 (1) I·S 22-25 (3) 2·5 t 4·3 37 (I) Oesophagostomum 20-30·
t
Other features mean 42·1 33·0 23·7
20(1)
40-4S·
morulated in fresh faeces embryonated in fresh faeces morulated in fresh faeces embryonated and pigmented bipolar plugs; brown rough coat, bile-stained asymmetrical, embryonated asymmetrical, embryonated thick shell with double contour bipolar plugs
• Measurements quoted in Flynn (1973).
t Not measured.
Anthelmintic drugs Thiabendazole was used routinely for the initial treatment of nematode infections in all the early trials, then 5 other drugs were used for the eradication of specific parasites or for comparison with thiabendazole. Thiabendazole ('Thibenzole'; Merck, Sharp & Dohme Ltd, Hertford Road, Hoddesdon, Hertfordshire, EN 11 9BU) as a suspension for oral administration containing 13·3% w/v, i.e. approx. 133 mg/ml. Dosages recommended by the manufacturer vary between 44 mg/kg bodyweight for routine treatment of sheep, and a minimum of 110 mg/kg in severely infested cattle. Recommended dosage in man is 25 mg/kg twice daily for 2 days. We used doses of 50, 100 or 133 mg/kg, with a 2nd dose after 3 weeks. Administration was by stomach tube to monkeys under sedation induced with phencyclidine hydrochloride ('Sernylan'; Bioceutical Laboratories Inc., St. Joseph, Missouri 64502, USA). Thiabendazole is accepted by monkeys if mixed with the food, but this method was not fully tested. Mebendazole ('Telmin'; Janssen Pharmaceutica NV, Turnhoutsebaan 30, Beerse 2340, Belgium) supplied by special request as a 10% mixture in sucrose for oral administration. When required for use, the mixture was made up as a suspension in water to give a mebendazole concentration of 20 mg/ml. The dosage recommended by the suppliers (Crown Chemical Co. Ltd, Lamberhurst, Kent, TN3 8DJ) for horses is 510 mg/kg, but higher doses were recommended for monkeys. We used 20 mg/kg daily for 4 days, adding I ml of the suspension for each kg body weight to a water bottle containing about 150 ml water. The monkeys found the sweetness attractive and usually emptied the bottle within a few hours.
Levamisole ('Nemicide'; Imperial Chemical Industries Ltd, Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, SKIO 4TF) supplied as a 7·5% w/v solution for subcutaneous injection, i.e. approx. 75 mg/ml. Dosage recommended by the manufacturer for pigs and sheep is 7·5 mg/kg or, when given by mouth as 'Ketrax' for man, 2·5 mg/kg as a single dose. We used 7·5 mg/kg and 11·25 mg/kg. The monkeys were sedated, weighed, and the computed volume injected subcutaneously into the scruff of the neck. Thenium closylate and piperazine phosphate ('Ancaris'; Wellcome Foundation Ltd, 183 Euston Road, London, NW 1 2BP). Thenium is claimed to be effective against some hookworms (Ancylostoma and Uncinaria spp.), and piperazine is a time-honoured treatment for ascarids. The manufacturers recommend a dosage of I tablet containing 216·25 mg thenium and 260 mg piperazine per 4 kg for dogs, to be ·repeated 6 hr later. We used I tablet per monkey (weight 3·0-4·5 kg) as a 2nd-line treatment after thiabendazole, and did not repeat the dose. Administration was by mouth under sedation induced with phencyclidine, in which the swallowing reflex was retained. Dichlorvos ('Task'; Tasman Vaccine Laboratories (UK) Ltd, Eastern Way, Bury St Edmunds, Suffolk IP32 7AL) supplied as 10 mg tablets. Recommended dosage for dogs and cats is 10 mg/kg. The monkeys were dosed at this level or below, e.g. a monkey weighing 3·5 kg was given 3 tablets. Dichlorvos is a cholinesterase inhibitor and should not be used with other cholinesterase inhibitors such as phencyclidine. The tablets were therefore administered to the conscious animal at least 3 days after and 3 days before sedation, by crushing the tablets and concealing the powder in a banana.
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215
Niclosamide ('Yomesan'j Bayer (UK) Ltd, Eastern Way, Bury St Edmunds, Suffolk, IP327AH) as tablets for oral administration containing 0·5 g. Dosage recommended by the manufacturer is 1 tablet per 4 kg for dogs and cats, to be given on an empty stomach. We gave 1 tablet per monkey (weight 3·04·5 kg) to animals under sedation which did not inhibit the swallowing reflex.
Table
2. Incidence of helminth endoparasites in rhesus monkeys within 24 h after arrival in quarantine unit
No. animals passing worms or ova total Nematodes Oesophagostomum spp. Strongyloides Jul/eborni Trichuris trichiura Trichostrongylus spp. Streptopharagus pigmentatus Enterobius spp. Physaloptera tumeJaciens Ascaris lumbricoides Anatrichosoma cutaneum Unidentified Cestodes Bertiel/a studeri Hymenolepis nana Trematodes Unidentified Total monkeys screened
%
106 144 29 47 34 1 2 6 1 3
40·9 55·6 11·2 18·1 13·1 0·4
3 1
1·2 0·4
1 259
0·8
2·3 0·4 1·2
0·4 100
Strongyloides fulleborni, the commonest of the nematodes found in this survey, is also infective to man (Pampiglione & Ricciardi, 1971). The related S. stercora lis is an important parasite of man in the tropics. The species of Trichuris found in the rhesus monkey is thought to be the human whipworm, T. trichiura (Ruch, 1959). The Ascaris species found in the rhesus monkey is indistinguishable from the common large roundworm of man, A. lumbricoides. All the species mentioned above have direct life cycles, so infected animals and their excreta are a hazard to man. Under some circumstances, man may be a source of infection to monkeys (Orihel, 1970). Trichostrongylus spp. are common parasites of wild mammals and domestic ruminants. No positive identification was made in this survey. Streptopharagus pigmentatus was the largest of the common nematodes seen. Adults were vomited up by 2 monkeys and passed in the faeces by several. Females measured 58-60 mm and males 24-27 mm. The distinctive anatomy of the elongated buccal cavity, spirally twisted, is illustrated in Georgi (1974). Enterobius vermicularis, the common pinworm of man, has been reported from primates, but the ova seen in this survey were of another, unidentified species. The best way of demonstrating ova is said to be by applying sticky cellophane tape to the perianal region, then onto a microscope slide (Faust, Beaver & Jung, 1968): this was done for 66 monkeys but no eggs were found, and subsequently only the usual faecal concentration technique was used. Anatrichosoma cutaneum is parasitic in the nasal mucosa and, perhaps, the skin of monkeys. According
Results
to Long, Lichtenfals & Stooky (1976) it is fairly
Incidence The number of monkeys passing worms or ova of each species of parasite after arrival in the quarantine unit is shown in Table 2. The nomenclature of Oesophagostomum sp. is rather confused (Flynn, 1973). Moreover, the ova of Oesophagostomum spp. are similar to those of Ternidens deminutus, and since no adult Ternidens were ever found, all ova within the size range shown were recorded as Oesophagostomum. Both are infective to man (Wilcocks & Manson-Bahr, 1972). Adult Oesophagostomum were fairly commonly found in faecal samples. Hookworm eggs (Ancylostoma and Uncinaria spp.) are similar to Oesophagostomum eggs. No adult hookworms were found in the faeces or post mortem. Although Ancylostoma duodenale is a common parasite of man in India, it is thought to be rare in non-human primates (Nelson, 1965). Any ova present would have been recorded here as Oesophagostomum.
common, though only 1 ovum was found in this survey. Eggs are transmitted in nasal mucus; some are swallowed and may be found in the faeces as in this case. Segments of the tapeworm Bertiella studeri, about 1 em wide, were commonly seen in the faeces of infected animals. The proglottid of Hymenolepis nana is small and easily missed in the dropping tray, but the embryonated egg is distinguished under the microscope by the presence of 3 pairs of small hooks. Pathogenicity Parasitism undoubtedly contributed to the morbidity of the newly-imported monkeys; coughing and diarrhoea were common during the 1st 3 weeks of quarantine, but the cOIftribution of parasitism was difficult to quantify. No specific clinical signs were found to correlate with specific parasitisms. Unthriftiness was often associated with parasitic burdens, but it
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was difficult to establish was effect.
which was cause and which
Oesophagostomiasis. Nodules contaInmg live Oesophagostomum larvae were frequently found in the wall of the caecum and colon of monkeys which died They were found also on the serosal (outer) surface of the large intestine, and in the omentum and mesenteries as a result of aberrant migration of the larvae. This was sometimes associated with local infection, abscess formation and adhesions, most commonly in the region of the ileo-caecal junction. In this survey, 2 deaths were associated with oesophagostomiasis. Monkey A. Young male. No anthelmintic treatment had been given. Post-mortem findings were Oesophagostomum worms in the lumen and nodules in the walls of the large intestine; an abscess within the omentum in the region of the ileo-caecal junction with adhesions between caecum and jejunum; congestion of liver, kidney and spleen; peritonitis; pneumonia with pleuritic adhesions in both lungs; pericarditis. Diplococcus (Streptococcus) pneumoniae was recovered from heart blood. It is thought (Fiennes, 1972) that the lesions caused by migrating larvae may become infected by colonic bacteria, leading to ulceration, perforation, peritonitis and septicaemia. The present case appeared to illustrate this. Monkey B. Young male. 50 mg/kg thiabendazole had been given 16 days before death. Post-mortem findings were similar to monkey A, with the addition of 2 intussusceptions, and the presence of hundreds of Oesophagostomum spp. and a few Trichuris trichiura worms in the lumen of the large intestine.
Strongyloidiasis. The pathological effects of Strongyloidesfulleborni (Flynn, 1973) are divided into 3 phases: the invasive phase when infective larvae penetrate the skin or buccal mucosa (this does not appear to cause irritation in monkeys); the migratory phase when infective larvae are carried in the blood to the heart and lungs, break into the alveoli, enter the bronchi, and are coughed up and swallowed, causing symptoms varying from a cough to bronchopneumonia; the intestinal phase when the swallowed larvae penetrate the glandular epithelium of the small intestine, causing symptoms varying from diarrhoea to peritonitis. 55% of the newly-imported monkeys were passing ova of Strongyloides, and a high percentage of these must have been undergoing the migratory and intestinal phases of the infection, thus contributing to the incidence of respiratory and intestinal disease. To see the larvae, it is necessary to examine histological sections oflung and small intestine. This was not done. However, ulceration of the intestine was sometimes found post mortem, as in the following case. Monkey C. Young male. Recently treated for pneumonia, and anthelmintic treatment (50 mg/kg
thiabendazole) was given 18 days before death. Postmortem findings were a small abscess on the caecum; blood-staining of omentum and peritoneal fluid; ulceration of the mucosal surface of the small and large intestines. No nodules or worms were seen, but the monkey had been passing Strongyloides ova. The pneumonia was partly resolved. It was thought that the peritonitis was due to the perforation of a Strongyloides-induced ulcer.
Trichuriasis in man causes great irritation and is thought to be a cause of rectal prolapse (Wilcocks & Manson-Bahr, 1972). In the rhesus monkey, rectal prolapse and intussusception were found from time to time; worms such as Trichuris may have been a cause (e.g. in monkey B) but often worms were absent. Streptopharagus pigmentatus lives in the stomach; its pathogenicity is unknown. 2 cases of vomiting were associated with it, but no other clinical signs.
No clinical signs were attributed to the other
parasites listed.
Treatment The effect of treating the naturally-infected rhesus monkeys with various drugs is shown in Table 3. The numbers of worms originally present in the animals were not shown, nor could they be deduced from the egg counts. The numbers of worms left after treatment were not known either, since few animals died. Therefore the results are expressed simply in terms of the percentage of infected animals which stopped passing ova after treatment, which continued clear at each sampling over several weeks, and which were still clear at the end of the conditioning period (3 months). From these results it appears that thiabendazole remains the most effective drug against Oesophagostomum, particularly at the higher dosage. The repeat dose is necessary to kill recently-developed adults, because larvae are resistant to treatment. Levamisole is the most effective drug against Strongyloides. However, the dose levels were high, and in man would be expected to cause side-effects such as nausea, headache and abdominal pain (Pene & Delmont, 1973). Mebendazole was active against Trichuris as well as the other common nematodes. However, a repeat treatment would be necessary for eradication of Strongyloides. This drug is considerably more expensive than the others. Dichlorvos was a useful specific drug against
Trichuris. Thenium was not significantly better than thiabendazole for the eradication of Streptopharagus. Anyway, Streptopharagus is self-limiting, because the life cycle requires an arthropod intermediate which seems to be absent in the UK.
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Table 3. Activity of ant helm in tics on the common
parasites
animals treated
% clear
animals treated
% clear
animals treated
24 45
92 96
38 56
87 96
8 12
25 33
a
a
100
90
I 12
10
1 10
75
70 60 75
12 9 9
83 100 100
10 5 4
For treatment against Trichostrongylus, thiabendazole, dichlorvos, mebendazole and levamisole were each 100% effective at the dosages described. Levamisole was 100% effective against Enterobius and mebendazole against Ascaris in the few cases treated. For treatment of cestodes, niclosamide was 100% effective in the few cases treated. No adverse side-effects were observed with any of the drugs given.
Discussion The results show that available anthelmintics are effective against the nematodes commonly parasitizing rhesus monkeys. In the 3 deaths attributed to parasitism in this survey, 1 (monkey A) had not been treated and the other 2 had been inadequately treated. This compares with 9 deaths due to enteritis in the same series, 13 to respiratory disease, and 3 cases of tuberculosis which were killed. At the end of the conditioning period (about 3 months), the monkeys were passed to users free from all helminth parasites except the occasional Trichuris or Strongyloides, though in order to achieve this repeated treatment was often necessary. The best method of administering anthelmintics is a matter of opinion. A sedated animal can be given an accurately measured dose by stomach tube, though occasionally vomiting occurs and treatment has to be repeated. Similarly, an accurately calculated dose can be injected. However, if animals are not sedated for examination and treatment until they have been in quarantine for several days, the parasitic damage may already have been done. Where a delay is likely it may be preferable to give the anthelmintic in the food and risk that the full dose will not be taken, or to inject into the conscious animal a dose calculated from the estimated bodyweight. The anthelmintic chosen will depend partly on the route of administration to be used, but since Oeso-
ofrhesus
Trichuris
Oesophagostomum Strongyloides
Thiabendazole 2 x 50 mg/kg Ix 100+lx500r I x 133 + I x 50 mg/kg Dichlorvos Thenium-piperazine Mebendazole Levamisole I x 7·5 mg/kg 2 x 7·5 mg/kg 2 x 11·25 mg/kg
nematode
4
% clear
100
a
monkeys
Streptopharagus
Trichostrongylus
animals treated
animals % treated clear
% clear
9 10
56 80
6 3
83 100
13 16
100 100
1 1
100
II
100
5 6 3
100 100 100
a
phagostomum was the most pathogenic of the nematodes encountered, thiabendazole in high dosage, or mebendazole in extended dosage, is to be preferred for the initial treatment of newly-imported animals. Subsequent treatment of these animals, or of animals already conditioned when brought in, will depend on the parasites found. If Trichuris or Streptopharagus are present in large numbers, treatment with dichlorvos or mebendazole is desirable, whereas for Strongyloides levamisole is the most effective. Acknowledgements I wish to thank Mr Ramsay Hovell for making this survey possible, Mr G. Richardson and Mr R. Weston for their help, and Mr T. Richards for his care for the monkeys. I thank the Wellcome Trust for a Veterin-
ary Research Fellowship. Drugs were kindly donated
by Crown Chemical Co. Ltd and Tasman Laboratories (UK) Ltd.
Vaccine
References
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