56

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1986) 80, 56-59

Giardia lamblia in children in day-care centres in southern Ontario, Canada, and susceptibility of animals to G. lamblia

PATRICK T. K. Woo AND W. BROCKLEY PATERSON Department of Zoology, College of Biological Science, University of Guelph, Guelph, Ontario, Canada NIG 2 WI

Abstract In 1982, Giardia lamblia cysts were found in the faeces of eight of 97 children (two to five years old)

in day-care centres in Guelph, Ontario, Canada. None of the infected children showed any of the classical signs of giardiasis. The survey was repeated in 1983 in the Kitchner-Waterloo area, Ontario. In this survey nine of 147 children were infected with G. lamblia. Experimentally established Giardiu-free colonies of mice, hamsters, rats, cats (adults and kittens) and dogs (adults and puppies) could not be infected with Giardia cysts from clinical and non-clinical patients. Also, kittens, hamsters, and mice could not be infected by trophozoites from an axenic culture. However, five-day-old suckling rats can be infected with G. Zamblia. Also, the Giardia-free rats and dogs are susceptible to their own Giardia (G. simoni and G. canis respectively). This proves that cats and dogs are not reservoirs of G. lamblia and consequently are not responsible for the spread of the infection in the human population.

Introduction Giardia lam&ha, an intestinal protozoan, has world-

wide distribution and its orevalence varies from 1% to 30% (PETERSEN, 1972). Iin the last 20 years there have been numerous reports which implicate Giurdia as a pathogen in man (see WOO, 1984). In the USA giardiasis is now considered to be the most common pathogenic intestinal parasite and the leading cause of diarrhoea due to protozoan infections in travellers (WOLF. 1975: 1978). It is also the most freauentlv reported intestinal parasite in Britain (KNIGHT & WRIGHT, 1978). In Canada, there were 4559 reported cases of giardiasis (ANON., 1984), and probably there are many more unreported cases. Outbreaks in communities have been traced to contaminated food (OSTERHOLM et al., 1981; BARNARD & JACKSON, 1984), water(SHAWetal., 1977; WRIGHTS?& 1977), person-to-person spread in day-care centres (BLACK et aE., 1977: KEYSTONE et al., 1978L institutions for the mentally retarded (YOELI et&, 1972; THACKER et al., 1979). homosexuals (MEYERS et al.. 1977). arnona campers (BARBOLJR etk., 1976) and s’kiers (MOORE e't al., 1969).

Although a great deal of experimental work has been done on transmission of Giardia between animal species, relatively little of that is conclusive. Although pets (e.g., cats and dogs) and wild animals (e.g., beavers) have been implicated as a source of cysts for human infections (PADCHENKO & STOLYARCHUK, 1969; DAVIES & HIBLER, 1979, HEWLETT et aE., 1982), it has not been demonstrated unequivocally that they are reservoirs of G. lamblia. Animals used in these studies were not from experimentally estab- lished Giardia-free colonies. and soecial mecautions were not taken in maintaihng these animals before and after they were inoculated with the human Giardia (see WOO, 1984).

The aims of the present study were to determine (i) the orevalence of non-clinical Giardia infections in children in day-care centres in two areas in southern Ontario, Canada and, (ii) the susceptibility of pet animals (experimentally established Giardia-free col-

onies) to human Giardia (cysts from clinical and non-clinical infections) by experimental infections.

Materials and Methods Survey of Giardia in day-care centres

Day-care centres in the Guelph and Kitchner-Waterloo areas (Ontario, Canada) were contacted in the spring of 1982 and 1983 respectively. Stool samples from children were individually placed in plastic containers with sodium acetate- formarm preservative. These were collected and brought back to the laboratory where they were examined using the zinc sulphate flotation technique. Parents of infected chil- dren were notified and were strongly advised to consult with their family physicians. Also, additional stool samples (fresh and with no preservative) were collected from the heavily infected children for experimental inoculation into animals after concentration.

Establishment and maintenance of Giardia-free animals. Pregnant animals (cats and dogs) were housed in isolation

rooms at the Ontario Veterinary College, University of Guelph. The rooms were disinfected and steam-cleaned before use. They were designed and maintained for studying pathogenic organisms. Each room has a double door with a foot bath in between and only special clothing was worn in the building. As there were dogs and cats in other units in the buildine most of the Giardiu-free animals were looked after by a member of the research team. As a control, a treated bitch (see below) and her five puppies (no Giardia cysts were detected 21 days after parturition) were looked after by animal handlers who were also responsible for other dogs (used in other studies) in the building. No one was allowed into the Giardia-free rooms except two members of the research team. The inoculated animals were kept in separate rooms and Giardia-free colonies were fed and cleaned before the inoculated animals. The faeces of all animals were normally examined monthly for cysts.

Pregnant animals were given quinacrine hydrochloride (100 mg quinacrine base/dog; 50 mg quinacrine base/cat) every day for at least 10 days before parturition and every other day for 20 days after parturition. Before and during this treatment period, the faeces were examined for cysts by the zinc sulphate flotation technique (LENNETTE et. al., 1974) and wet-mount examination. The female was killed as soon as the young animals were weaned and the intestine examined for trophozoites (GRANT & Woo, 1978). Once

every two weeks, Giardia-free animals were examined for intestine when all the rats were killed 35 days after cysts during the first two months. inoculation.

Colonies of Giardia-free rats (Wistar), mice (Swiss- Webster) and hamsters were established and maintained as described earlier (GRANT & WOO, 1978).

Cyst preparation and inoculation into ammals The sucrose density gradient centrifugarion (ROBERTS-

THOMSON et al., 1976), was used to isolate cysts for experimental inoculation. This technique provides a high percentage of viable cysts as determined by excystation (BINGHAM et al., 1979). The number of cysts or trophozoites was enumerated in a haemocytometer (ARCHER, 1965) and diluted accordingly before being inoculated orally into experimental animals.

As positive controls, six rats were inoculated (two rats each with 100,000 cysts, two each with 10,000 cysts, and two each with 1,000 cysts) with G. simoni cysts from an infected rat. All the inoculated rats were passing cysts six days after inoculation; the number of cysts in the faeces of rats inoculated with 1,000 cysts was lower than those in rats inoculated with 100,000 cysts. No cysts were seen in the faeces of six uninoculated rats (negative controls).

Experiment 2: Three hamsters, two mice and four rats were each inoculated with at least one million G. lamblia cysts (Guelph 82-523). The faeces from each animal species were pooled before examinations at 12, 13, 20, 27, 32 and 41 days after inoculation. One cyst was seen in the faeces from the rats and mice at 12 days after inoculation; however, no cysts were seen in all susbequent exammations. Also, no trophozoites were seen in the small intestine when the rats and mice were killed 27 days after inoculation.

Examination of intestine for trophozoites Ar the end of each experiment, the small intestine of all

inoculated animals (with either cysts or cultures) and some uninoculated animals (controls) were examined for trophozo- ites. Rats, mice and hamsters were killed in chloroform, the small intestine removed, cut into 10 sections and suspended in mammalian saline. The contents and scrapings of 2 mm of each section were examined using an inverted microscope (eye-piece 10x ; objective 10x) for motile trophozoites.

Cats and dogs were killed with an overdose injection of sodium pentobarbital. The small intestine was removed, cut into about 30 sections and suspended in saline. The contents and scrapings of about 5 mm of each section were examined for trophozoites as described earlier.

Results A. Prevalence of Giardia in day-care centres in Guelph and Kitchner- Waterloo areas

In Tuly 1982, Giardia cysts were found in the faeces of eight bf 97 (8.3%) children from day-care centres in the Citv of Gueloh. The infected children (four males and fo& female;) were between two and f&e years old and were from four of the seven day-care centres. Three of the eight infected children were passing large numbers of cysts (easily detectable by wet-mount examination of faeces). According to the parents none of the infected children showed any of the classical clinical signs of the infection (e.g., loss of appetite, diarrhoea, stomach cramps). Freshly isolated cysts from two of these children were later inoculated into Giardia-free animals (BI, 1 and 2).

The survev was reseated in 1983 in the Kitchner- Waterloo aria. Giarka cysts were detected in the faeces of nine of 147 (6.1%) children examined. The infected children were between three and five years old and two of them were passing large numbers of cysts (detectable by wet-mount examination). Again, acccording to the parents the infected children did not seem to show any of the clinical signs of the infection.

B. Susceptibility of Giardia-free animals to G. lamblia, G. simoni and G. canis

I. Inoculation of human Giardia cvsts from non- clinical cases:

_ ,

Experiment 1: Eight adult rats were inoculated with varying numbers (two rats each with 862,500 cysts; two each with 86,250 cysts; two each with 8625 cysts; and two each with 862 cvsts’, of G. lamblia cvsts (Guelph 82-507). Seven days ifter inoculation,-& cyst was seen in one of the two rats inoculated with 86,250 cysts while none were seen in the other inoculated rats nor in the control rats. No cysts were seen in any of the rats at 14, 21 and 35 days after inoculation. No trophozoites were seen in the small

Although no cysts were seen in the hamsters 12 days after inoculation, one cyst (pooled faeces) was seen on day 13. No cysts were seen in subsequent examinations and no trophozoites were seen when the hamsters were killed on day 41.

II. Inoculation of human Giardia cysts from clinical cases into animals

Exberiment I: Three adult hamsters (each given 100,doO cysts), four young adult rats (each iiven 100,000 cysts), five adult mice (each given 50,000 cysts), and two cats (each given 200,000 cysts) were inoculated with G. lamblia (designated No. 10M) isolated from a hospital patient in Montreal, Quebec. According to Dr. G. Faubert (Institute of Parasitol- ogy, M&ill University, Quebec) the cysts were infective to the Mongolian gerbil.

No cysts were seen in the faeces of the inoculated animals at 6, 13,20,35, and 43 days after inoculation. Also, no trophozoites were seen in the small intestine when the hamsters, rats and mice were killed at 43 days. The cats were examined at 35, 43, 63 days and at irregular intervals for a further six months. No cysts were detected and no trophozoites were seen when they were killed.

Experiment 2: Fresh cysts from another patient (Montreal hospital; supplied by Dr. G. Faubert) were inoculated into Giardia-free animals. About 300.000 cysts were inoculated into each of four five-week:old puppies, three lCmonth-old dogs, three four-week- old kittens, and two 13-month-old cats. Also, each of ten five-day-old suckling rats was inoculated with about 5,000 cysts. The controls (Cl controls; looked after by members of the research team) consisted of two five-week-old puppies (same litter), one three- week-old kitten (same litter) and five suckling rats (same colony). Another group of five puppies (C2 controls; looked after by the animal handlers), housed in a separate room, was used to determine the possibility of accidental contamination of Giardia-free animals by animal handlers during routine mainte- nance of the animals.

No cysts were detected in the faeces of cats and

58 G. lamblia IN CHILDREN AND SUSCEPTIBILITY OF ANIMALS

dogs at 7, 14,21 and 28 days after inoculation of cysts; also no trophozoites were seen in the small intestine at 28 days when the cats were killed. Similarly, no trophozoites were detected when the dogs were killed at 30 days after inoculation.

Cysts were detected in the faeces of suckling rats at 12. 14 and 16 davs after inoculation: also lame numbers of trophdzoites were seen in the smill intestine when they were killed on the 16th day. However, no cysts were detected in the faeces of the lactatine female (mother of inoculated sucklina rats) and fivecontrol suckling rats (kept separately) during the same period and no trophozoites were seen when they were killed.

No cysts were detected in the faeces of the Cl controls and no trophozoites were seen when they were killed. However, Giardia (presumably G. canis) cysts were detected m the faeces of one of the C2 control puppies at 2 1 days (after they were maintained by the animal handlers) and all were passing cysts at 28 days. Cyst excretion continued for over 200 days with irregular fluctations. One of the dogs was still passing cysts 360 days later and trophozoites were seen when it was killed.

Experiment 3: The susceptibility of suckling rats to G. lamblia was repeated with another eight five-day old suckling rats. Each rat was inoculated with approximately 3,500 cysts isolated from a clinical infection (supplied by Dr. J. Yang, Tropical Disease Unit, Toronto General Hospital, Ontario). A few trophozoites were seen in the small intestine of the two suckling rats that were killed three days after inoculation. Increasing numbers were seen in rats killed 6, 9 and 12 days after inoculation. No trophozoites were seen in the four control suckling rats from the same colony kept in a separate cage.

III. Inoculation of human Giardia trophozoites from culture into animals

An axenic culture of human Giardia (supplied by Dr. G. Faubert; designated WB) was inoculated into Giardia-free animals. Two eight-week-old kittens and two adult hamsters were each given 100,000 motile trophozoites and five adult mice were each given 80,000 trophozoites. The faeces of the animals were examined 6, 13,20,27, and 28 days after inoculation. No cysts were seen at any of the examinations. Also, no trophozoites were seen in the small intestine when the hamsters and mice were killed at 28 days after inoculation. No cysts were seen in the faeces of the two kittens at 40.57.67 davs and at irreaular intervals throughout the year: Also;no trophozo:tes were seen when the cats were killed eight months after inocula- tion.

Discussion In our survey, 6 to 8% of children in day-care

centres were passing Giardia cysts. This falls within the range of 4 to 20% found in young schoolchildren in the USA. In a recent study (HEALY, 1978) the faeces of 78 children were examined bimonthly using wet-mount and formalin-ether concentration techni- ques. The percentage of infected children varied between 5 and 16% durine a 12-month ueriod. DESOWITZ & WIEBENGA (1975) using wet:mount examination found 4 to 5% of children in a Hawaiian school infected with Giardia. However, WEINER et al. (1959) found the prevalence to be 8 to 20% in

Philadelphia. They used polyvinyl-alcohol fixative, formalin-ether concentration technique and stained slides in their survey.

In addition to Giardia we also found children infected with other intestinal parasites as did HEALY (1978) in his survey in the USA.

In an earlier review (WOO, 1984) it was pointed out that although quite a few studies had been conducted on the susceptibility of animals to G. lamblia most are contradictory or inconclusive because animals were not from exnerimentallv established Giardia-free col- onies and ‘special precautions were not taken in maintaining these animals before and after inoculation with cysts. In the present study all these precautions were taken and it clearly shows that hamsters, adult rats, cats (kittens and adults) and dogs (puppies and adults) from Giardia-free colonies cannot be infected with G. lamblia (cysts from clinical and non-clinical cases). WOO (1984)in his review concluded that there is some evidence in the literature “.. .to suggest that dogs may be important reservoirs...“; however, he suggested that the work be repeated with animals from experimentally established Giardia-free colonies and proper precautions taken to prevent contamina- tion before and after inoculation of cysts. The present study demonstrates quite conclusively that cats and dogs are not reservoirs of G. lamblia and consequently are not responsible for spreading the disease. The positive controls (BI,l and BII,Z) showed that the animals were susceptible to their own Giardia spp. (rats to G. simoni; dogs to G. canis), this indicating that the nrocedure for establishing Giardia-free col- onies did not affect their suscep&bility. Cysts from symptomatic and asymptomatic individuals were used in the present study-because it is not known if suscentibilitv of animals is affected bv the source of cysts.* Cysts-from non-clinical cases (BI,l and BI,2) were isolated and inoculated into experimental anim- als within three hours after collection.

Although all precautions to prevent contamination were carefully explained to the animal handlers, all five Giardia-free puppies (C2 controls in BII,Z) were passing cysts, presumably G. canis, within 28 days after they were handed over for routine maintenance. These dogs were passing cysts, with irregular flucta- tions for over 200 days while those maintained by members of the research team were not infected. This shows that accidental contamination can occur when Giardia-free animals are maintained routinely by animal handlers and helps to explain some of the inconclusive results reported in the literature. KESSEL (1929) found eight of 15 kittens and one of three dogs were passing cysts after they were given G. Zamblia cysts. There was no data on the source or dose of the cyst and neither were experimental controls men- tioned in the study. More recently, PADCHENKO & STOLYARCHUK (1969) reported an outbreak of G. canis in a colony of dogs. Those that were apparently free of the infection were inoculated with cysts from children. The inoculated dogs were passing cysts between five and 29 days. DAVIES & HIBLER (1979) inoculated two groups of two dogs each with G. lamblia cysts. In one group both dogs were passing cysts six days after inoculation and “...the in-group control was positive seven days later.” Similarly, the second group of inoculated dogs were passing cysts at eight days after inoculation and the in-group control was

inlected seven days later. HEWLETT et al. (1982) infected three of eight dogs with cysts and two of three dogs with trophozoites from axenic culture. According to them no cysts were found in the faeces of the dogs for at least three weeks before inoculation of uarasites: however. 68% of the 37 dogs thev examined had natural Giardia infections. W> agree with BEMRICK (1984) who seriously questioned the validity of the results of previous studies on the susceptibility of dogs to human Giardia. In the present study, we were unable to infect two eight- week-old kittens, two adult hamsters and five adult mice with an axenic culture (BIII).

Similarly, studies on the susdeptihility of aduit rats to G. lamblia are contradictory (see WOO, 1984) because animals used were not from experimentally established G&d&free colonies. In the present study, Giardia-free adult rats and hamsters could not be infected. This confirmed an earlier study (GRANT & WOO, 1978) where Giardia-free rats and mice were used. Although adult rats could not be infected, suckling rats were susceptible consequently they were used to determine the viability of cysts used for inoculation into some of the cats and dogs (BII, 2).

Acknowledgements This study was supported by the Ontario Ministry of

Health and the Natural Science and Engineering Research Council of Canada. We are grateful to Dr. G. Faubert, Institute of Parasitology, McGill University, Quebec, and Dr. J. Yang, Tropical Disease Unit, Toronto General Hospital, Ontario, for providing Giardia cysts and trophozo- ites from clinical cases. Mrs. K. Davidson-Taylor helped us maintain the Giardia-free animals.

References Anon. (1984). Notiable diseases summary. Canada Dtseoses

Weekly Report, 10, 18-19. Archer, R. K. (1965). Haematological Techniques for Use on

Animals. Oxford: Blackwell Scientific Publications, 135

BarpbEur, A. G., Nichols, C. R. & Fukushima, T. (1976). An outbreak of giardiasis in a group of campers. Amencan Toumal of Tronical Medicine and Hveiene. 25. 384-389.

Barnard, R. J. & Jackson, G. J. (1984). ?%ard& la&blia: The transfer of human infections by foods. In: Giardia and Giardiasis. Erlandsen, S. L. & Meyer, E. A. (Editors). Plenum Press, pp. 365-378.

Bemrick, W. J. (1984). Some perspectives on the transmis- sion of giardiasis. In: Giardia and Giardiasis. Erlandsen, S. L. & Meyer, E. A. (Editors). Plenum Press, pp. 379-400.

Bingham, A. K., Jarroll, E. L., Meyer, E. A. & Radulescu, S. (1979). Induction of excystation and comparison of dye-exclusion versus excystation. In: Waterborne Trans- mission of Giardiasis. Jakubowski, W. & Hoff, J. C. (Editors). Cincinnati: U. S. Environmental Protection Agency, Environmental Research Centre, pp. 217-229.

Black, R. E., Dykes, A. C., Sinclair, S. I’. & Wells, J. G. (1977). Giardiasis in day-care centers: evidence of person-person transmission. Pediatrics, 60, 486-491.

Davies, R. B. & Hibler, C. I’. (1979). Animal reservoirs and ‘_ ‘.

cross-species transnussion of Grardia. In: Warerbome Transmission of Giardiasis. Jakubowski, W. & Hoff, J C. (Editors). Cincinnati; U.S. Environmental Protection Agency, Environmental Research Centre, pp. 104-126.

Desowitz? R. S. & Wiebenga, N. H. (1975). A survey of intestmal parasites in Oahu school children. Hawatt Medical J&nal, 34, 21-23.

Grant, D. R. & Woo, I’. T. K. (1978). Comparative studies of Giardia in small mammals in southern Ontario. II. Host specificity and infectivity of stored cysts. Canadian 3oumal of Zoology, 56, 1360-1366.

Healy, G. R. (1978). The presence and absence of Giardia lamblia in studies on parasite prevalence in the U.S.A.

In: Waterborne Transmission of Giardiasis. Jakubowski, W. & Hoff. I. C. (Editors). Cincinatti; U.S. Environ- mental Pro&on ’ Agency, Environmental Research Centre, pp. 92-103.

Hewlett, E. L., Andrews, J. S. Jr., Ruffier, J. & F. W. Schaefer. F. W. III. (1982). Exoerimental infection of mongrel-dogs with G&d& lam&a cysts and cultured trophozoites. Journal of Infectious Disease, 145, 89-93.

Kessel, J. F. (1929). Experimental giardiasis in kittens and puppies. Journal of Parasitology, 16, (Suppl.), 99-100.

Keystone, J. S., Krajden, S. & Warren, M. R. (1978). Person to person transmission of Gtardia lamblia in day-care nurseries. Canadian Medical Association Jour- nal, 119, 241-248.

Knight, R. & Wright, S. G. (1978). Intestinal protozoa. Progress report. Gut, 19, 241-248.

Lennette, E. H., Spaulding, E. H. & Truant? J. P. (1974). Manual of Clinical Microbiology, (2nd edit.). Washing- ton, D.C., USA: American Society of Microbiologists.

Lopez-Brea, M. (1982). Giardia lamblia: incidence in man and dogs. Transactions of the Royal Socieg of Tropical Medicine and Hygiene, 76, 565.

Meyers, J. D., Kuharic, H. A. & Holmes, K. K. (1977). Giardia lamblia infection in homosexual men. British Journal of Veneral Diseases, 53, 54-55.

Moore, G. T., Cross, W. M., McGuire, D., Mollohan, C. S., Gleason, N. N., Healy, G. R. & Newton, L. H. (1969). Epidemic giardiasis at a ski resort. New England 3oumal of Medicine, 281, 402-407.

Osterholm, M. T., Forfang, J. C., Ristinen, T. L., Dean? A. G., Washburn, J. W., Godes, J. R., Rude, R. A. & McCullough, J. G. (1981). An outbreak of foodborne giardiasis. New EnglandJoumal of Medicine, 304, 24-28.

Padchenko, I. K. & Stolyarchuk, N. G. (1969). On the possible circulation of Iambha in nature. Progress in i’rotozoology, 3, 311-312.

Petersen. H. (1972). Giardiasis (Lambliasis). Scandinavian Joumd of ‘Gastroenterology, 7-(Suppl. (4), pp. 1-44.

Roberts-Thomson, I. C., Stevens, D. P., Mahmoud, A. A. F., & Warren, K. S. (1976). Acquired resistance to infection in an animal model of giardiasis. Gastroencer- 010&71, 57.

Shaw,P.K.,Brodsky,R.E.,Lyman,D.O.,Wood,B.T., Hibler, C. P., Healy, G. R., Macleod, K. I. E., Stahl, W., & Schultz, M. G. A. (1977). A communitywide outbreak of giardiasis with evidence of transmission by a municipal water supply. Annals of Internal Medicine, 87, 426-432

Thacker, S. B., Simpson, S., Gordon, T. J., Wolfe, M., & Kimball, A. M. (1979). Parasitic disease control in a residential facility for the mentally retarded. American Journal of Public Health, 69, 1279-1281.

Weiner, D., Brooke, M. M., & Witkow, A. (1959). Investigation of parasitic infections in the central area of Philadelphia. An&can Journal of Tropical Medicine and Hygiene, 8, 625-629.

Wright, R. A., Spencer, C. H., Brodsky, R. E. & Vernon, T. M. (1977). Giardiasis in Colorado: an epidemiologic study. American Journal of Epidemiology, 105, 330-336.

Wolfe, M. S. (1975). Giardiasis. Journal of the American Medical Associarion, 233, 1362.

Wolfe, M. S. (1978). Giardiasis. Ne-r.u England Journal of Medicine, 298, 3 19-32 1.

Woo, P. T. K. (1984). Evidence for animal reservoirs and transmission of Giardia infection between animal species. In: Giardia and Giardiasis. Erlandsen, S. L. & Meyer, E. A. (Editors). Plenum Press, pp. 341-361.

Yoeli, M., Most, H., Hammond, J. & Scheinesson, G. P. (1972). Parasitic infections in a closed community: Results of a IO-year survey in Willowbrook State School. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 764-776.

Accepted for publication 13th December. 1984