Veterinaty Research Communications, 13 (1989) 377-388 Copyright 6 Kluwer Academic Publishers bv - Printed in the Netherlands

THE EXTRAPARASITIC LIFE CYCLE OF TOXOCARA WW~ORUM IN THE VILLAGE ENVIRONMENT OF SRI LANKA

JA. ROBERTS Department of Parasitology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka

ABsrRAcr

Roberts, JA., 1989. The extraparasitic life cycle of T~xocaru vitulorum in the village environment of Sri Lanka. Veteritwy Research Communications, 13 (S), 377-388

The extraparasitic life cycle of Toxocura vitulorum of buffalo in the villages of Sri Lanka is related to observations on buffalo behaviour, experimental studies on the development and persistence of the eggs in soil and in wallows and the presence of eggs in village locations. Calf faeces on soil were rapidly incorporated by insect activity and the eggs developed only slightly slower than in the laboratory. Some infective eggs persisted 3-4 cm deep.for 17 months, finally dying during a prolonged hot, dry period. Eggs in a wallow developed intermittently over 16 months as it was flushed with rain water, and eventually died when the wallow dried out. When infected faeces were placed in water,

. decomposition caused some material to rise to the surface and eggs developed. In villages, eggs are ubiquitous where young calves are kept but survive best where there is moisture and shade around animal pens and wallows. Cows and calves acquire infection from infective ew in wallow water, soil and pasture, while calves may also be infected from contamination on the udder and teats of the cow. The larvae resulting from this infection do not mature until the infection is passed to the calf through the milk of the cow. At least 73% of village calves have patent infections and current treatment procedures do not reduce the prevalence.

The possibility that T. vitubrum is a cause of human visceral larva migrutzs is discussed.

Ktywor& bionomics, buffalo, epidemiology, To.wcara vitulorum, Sri Lanka

INTRODUCTION

Toxocara vitulomm is an ascarid parasite of the Asian buffalo (Bubalus bubalis) and cattle (Bos spp.). Mature parasites occur only in young calves which acquire the infection as third stage larvae, mainly through the milk of the dam (Warren, 1971) but possibly rarely in utero (Mozgovoi et al., 1973). The environment is contaminated by calves, usually when they are 3-8 weeks old, and infection is acquired from the environment by the dam. Second stage larvae develop in the eggs in lo-12 days at 29°C in moist conditions with adequate oxygen and the eggs are infective by 17 days (Enyenihi, 1%9a). The miniium temperature for full development is above 19°C (Enyenihi, 1972). The parasite is the major cause of buffalo calf mortality in the humid tropics (Dewan et al., 1979; Thienpont and De Keyser, 1981) but can cycle in temperate regions when intensive husbandry procedures provide the conditions for egg development and infection of cows (Thienpont and De Keyser, 1981).

The present study investigates the environmental conditions and husbandry procedures which maintain the infection in the buffalo population of the rural villages of Sri Lanka.

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MATERIALS AND METHODS

The experimental plots

The field experiments were conducted at the SAREC farm on the campus of the University of Peradeniya. The terrain is hilly with red-brown sandy latosolic soil. The experimental area was grassed and screened with dappled shade from tall shrubs for about half the day. The experimental area was protected from buffalo by a barbed wire fence.

The wallow was 1.3 m deep in the centre and 30 m2 in area. It was used at least once a day by about 20 buffalo. It was fed intermittently with water from a small stream. The night shed for the buffalo was 40 m away.

The experimental samples

Four aliquots of 100 g of calf faeces containing 185 000 eggs per gram were placed under wire mesh on pasture on which the grass had been clipped to a height of 3 cm.

10 g of faeces was placed in a tube of nylon mesh 25 cm long and 2.5 cm in diameter, with a pore diameter of 50 pm. The tube was sealed at the ends with artery forceps and placed in a 2 L plastic container, with 2 cm diameter holes on centres 5 cm apart punched through all six sides. The container was protected by tying it to a steel frame on a steep bank of the wallow, 40 cm below the surface. In the first few weeks samples were collected every day, and later they were collected monthly.

The regular soil samples were 2 cm square and 5 cm deep. After two weeks and then every three months, separate samples 2 cm square and 1 cm deep were taken to a depth of 6 cm to determine the penetration and survival of eggs. The soil was replaced with uncontaminated soil.

Processing of samples

The soil was ground in water with 0.2% domestic detergent in a mortar and pestle. The fluid was decanted and centrifuged at 500 g. The sediment was thoroughly mixed with water saturated with NaCl and centrifuged at 1000 g. The surface layer was removed with a pipette, diluted with water and examined for eggs. Eggs were examined at a magnification of 125x and classified into developmental stages based on the illustrations of Chauhan and Pande (1981), namely:

1. One cell stage 2. 2-32 cell stage 3. Morula stage 4. First stage larva 5. Second stage larva

An additional category to suit this study was: 6. Dead embryonated eggs which could be distinguished by the presence of

vacuoles in the cytoplasm of the larva and distortion of the cuticle.

379

Eggs from the wallow site were removed and examined directly. At 3-monthly intervals the capacity for further development was assessed by washing the eggs by sedimentation and incubating them in 0.1 N H2S04 in petri dishes at room temperature of about 28°C.

The ability to hatch of eggs from soil and of incubated eggs from the wallow was tested at intervals. The eggs were washed by sedimentation and exposed to water saturated with Ca(OC1)2 until the shell was clear and flexible: this usually took about 40 min. A few larvae (~0.5%) had hatched by then. If the eggs had not yet reached this stage, fresh saturated Ca(OC1)2 was added. The decorticated eggs were then washed eight times by centrifugation at 50 g, four times in water and four times in phosphate buffered saline (PBS) at pH 7.2. The washed eggs in PBS were placed in an incubator at 385°C and CO2 was gently bubbled through for 30 min. The gassed eggs in PBS were left at 385°C 111 a 5% CO atmosphere. Most larvae hatched within two hours but they were left to hatch for 18 z ours.

Laboratory studies

10 g of calf faeces containing 37 000 eggs/g were placed in a 2 L container full of tap water on a bench in the laboratory. After 21 days the surface was skimmed and examined for eggs and eggs from the bottom of the container were also examined.

Meteorological data

Daily data from a site 1.5 km from the experimental plots provided the maximum temperature, soil temperatures under mown grass 5 cm deep at 08.30 h and 15.30 h, relative humidity at 15.30 h, evaporation and precipitation (Figure 1).

The village sites

Samples were collected from near village homes and buffalo holding areas and from village communal grazing and wallowing areas in the central western region of Sri Lanka. All samples were collected during the calving season. The climate is tropical with a rainfall of 1050 mm in two wet seasons. Samples were also collected from around homes and villages in a radius of 20 km from Peradeniya, where the climate is slightly cooler and the rainfall is 1300 mm in two wet seasons.

Soil samples were 10 cm square and 1 cm deep. Grass was collected from areas of 100 cm2 on grazing areas. It was clipped with large scissors as close to the ground as possible without disturbing the surface soil.

Wallow samples of 20 L were collected with 5 L buckets after the wallow had been agitated by buffalo or trampling. Samples were skimmed from the surface of large stagnant wallows with a sieve with a 75 micron mesh.

Cow and calf hair brushings were collected into a bowl with a stiff bristle brush, Udder washings were collected using a wash bottle containing water with 0.2%

detergent over a bowl, the skin being finally wiped finally with a thin polythene sheet.

380

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The village samples

The wallow samples were mixed with 0.2% domestic detergent and rapidly sedimented to remove dense materials. The dense material was washed and sedimented again. The pooled washings were filtered through a 50 pm pore sieve and washed on the sieve. The contents of the sieve were centrifuged in salt solution as described previously for soil samples. Surface samples were examined directly.

The soil samples were processed as described previously. Grass was placed in a 2 L container in water with 0.2% domestic detergent for

48 h and agitated occasionally. The fluid was then removed and the residue was washed with water. The combined fluids were sedimented under gravity and the sediment was examined for eggs.

The skin brushings and washings were left in water with 0.2% domestic detergent for 24 h with occasional agitation. They were then processed as for the grass samples.

Buffalo behaviour

Buffalo cows and their calves were observed in the neighbourhood of villages when the cows were grazing and the calves suckling or resting, and in villages when the cows were being milked. All observations were made from a distance, sometimes with field d asses.

RESULTS

Infected calf faeces on soil

The soil was moist when the faeces were applied. Within 24 h the faecal pats were broken up and mixed with the surface soil as a result of the activities of maggots and dung burying insects. All the dung had disappeared from the surface by 48 h and the soil was disturbed to a depth of 4 cm. Rain fell on 18 of the next 28 days, the total rainfall being 124 mm and the highest precipitation in one day being 22 mm. The eggs developed rapidly and relatively uniformly (Table I). The daily soil temperature 5 cm deep ranged from 23.4 to 29.6“C during that time.

Infective eggs were recovered from the site for 17 months although the proportion of degenerated eggs increased from six months onwards. The final complete loss of viability coincided with an extended dry period, which included the only period of prolonged and substantial excess of evaporation over rainfall during the study. The soil then dried out to a depth of 10 cm, while the temperature at 5 cm deep rose to over 40°C on one occasion and averaged 37.6”C at 15.30 h for one week.

Most viable eggs were 2-4 cm deep in the soil during the first nine months but a higher proportion of the survivors were deeper during the last six months. There were few viable eggs in the top 2 cm.

TABLE I Rate of development of Toxocara vitulorum eggs in calf faeces in soil in Sri Lanka in July 1985

Stage of development Time for 50% of eggs Proportion at each stage to attain stage (days) after 16 days (%)

1 cell cl 7 2 cell 2 0 4 cell 3 1 8 cell 5 0 16 cell 6 2 Morulla 6 1 Early embryo 7 0 First stage larva 9 2 Infective egg 14 87

Development of eggs in calffaeces in a wallow

The eggs in the wallow did not develop for four months and even then development was slow and intermittent, each spurt of development following a period of heavy rain which flushed out the wallow. By 10 months, 24% of eggs were undeveloped, 34% were at different stages of development, 28% were fully developed and the remainder were degenerated. At the end of the wet season after 12 months, only 5% of eggs were undeveloped, 44% were at different stages of development, 37% were fully developed and 4% were degenerated. After 16 months virtually all the eggs had developed, but one third of those which were identifiable had degenerated. By 18 months half had degenerated and then a long spell of dry weather dried out the wallow, killed the remaining viable eggs and terminated the experiment.

Changes in the proportions of eggs at different stages of development with time could be due to development or to deaths in other categories or to a combination of both. Eggs taken from the wallow at intervals of three months were therefore incubated in the laboratory to determine their potential for development. For at least 10 months all the eggs were capable of development but by 13 months 16% were incapable of full development.

Of the eggs classed as infective, 96% of those from soil and 94% from the wallow hatched in the laboratory.

Development of eggs placed under water in the laboratory

Eggs in faeces under water did not develop. However, a patchy surface layer formed and when it was skimmed off after 21 days it contained 73 fully developed eggs and

383

243 live developing eggs. Some eggs were on small rafts of organic material, others were single and attached to a small gas bubble, while some seemed to be supported only by the surface film.

Bufalo behaviour and husbandry

Swamp buffalo calves spend most of the day sleeping or near their mothers for their first three weeks of life, after which they also spend time with other calves in nurseries. They do not wallow but will go to the wallow area with their mothers. From 6-8 weeks of age they will stand in water up to about 25 cm deep but it is not usual for them to wallow until they are 5-6 months old. Adults prefer shallow mud wallows but calves wallow at a younger age if there is shallow open water available. Cows wallow for about two hours each day in the early or late afternoon.

Apart from suckling, physical interaction between cow and calf is slight. Systematic grooming by the cow of herself or of her calf was not observed, nor did calves groom unless they were irritated by skin lesions.

Cows drank regularly from wallows but calves were not observed drinking water before they would stand in water at 6-8 weeks of age. Calves nuzzle soil and nibble grass within 2-3 days of birth and it is usual to find grit in the abomasum and small intestine in 2-day-old calves, which could have come from the teats of the cow. There is usually some vegetable material in the rumen from about 10 days old but systematic grazing and developed rumen function does not normally occur until the calf is 6-8 weeks old.

Most swamp buffalo are not milked but are used seasonally for paddy cultivation. Where there are grazing areas available, such as on coconut plantations, employees may be allowed to graze a limited number of buffalo, or a buffalo owner may come to some arrangement with the plantation owner or manager. Depending on the circumstances, the buffalo may be free, supervised or tethered to palms and moved during the day. Young calves may be tethered at the worker’s house or be with the cows. Cows from outside the estate usually return to the owner’s home for the night and the calves would not accompany them to the estate until they were 4-6 weeks old. Herds of lo-30 animals are not uncommon under these husbandry conditions. Where grazing is not available, a farmer may keep one or two buffalo in a small open-sided thatched cover near his house. They are fed by some combination of tethered grazing and cut and carry of grasses and tree leaves. The calf usually remains tethered in the shed until it is lo-14 weeks old, although sometimes the calf is with the mother from about 10 days of age.

Excretion of T. vitulomm eggs by calves in villages

Faecal samples were rollected from calves believed to be in the 3-8-week-old age group and, where possible, information on treatment history was obtained. Of the 131 calves 72% were infected with a mean egg count of 35 400 and a maximum of 240 000, and 65% of the calves had been treated: 57% had received traditional medicine and 43% had received a western anthehnintic. Of the treated calves, 76% had patent infections, while 70% of untreated calves had patent infections.

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Husbandry conditions predisposing to mortality associated with T. vitulorum infections

On four occasions small groups of calves were examined where some had died and others were sick. In all cases faecal egg counts were ~30 000 eggs/g but in addition the cows were being milked and the calves did not have water or supplementary feed. No deaths occurred after the calves were treated for toxocarosis and husbandry was improved.

T. vitulomm in the environment of smali dairy hem3

A high level of contamination with eggs built up in the milking area and calf pens. The mortality of eggs was also high. There was a large build-up of eggs in soil around the wallows and the survival rate here was higher. Where there was free water in the wallows, there was a substantial build-up of infective eggs but this did not occur in small mud wallows. Infective eggs were recovered from the surface of stagnant wallows but most had degenerated. Some infective eggs were recovered from grass samples from village grazing areas but most of these were degenerated (Table II).

Cows and calves kept by the village houses

The build-up of dung and moisture in this situation was obvious, so effort was concentrated on the build-up of eggs on the bodies of the cows and calves. The hair and skin retained many infective eggs in spite of a high proportion of degenerated eggs.

DISCUSSION

Penetration of eggs from faeces into the soil, mainly through insect activity, rapidly places them in a favourable environment and thp, rate of development to infectivity was only four days longer than for eggs in the laboratory (Chauhan and Pande, 1981). All the soil to 5 cm deep was processed together but the relatively even rate of development indicates that sufficient oxygen was available at all levels. By three months, 85% of the infective eggs were more than 2 cm deep and, by 15 months, 94% were more than 3 cm deep. This may have been partly due to eggs being transported by water and animal activity and partly to differential killing by higher temperatures and lower humidities in the upper layers (Storey and Phillips, 1985). The availability to buffalo of eggs 2-3 cm deep would be limited but some would be returned to the surface by animals such as ants and earthworms. Around the wallows, calf pens and milking areas, where soil was compacted by the activities of adult buffalo and moisture was available, infective eggs were present in the top ‘1 cm of soil and were transferred to the skin and hair coat of cows and calves. Calves must have consumed many infective eggs in these situations but whether such larvae can survive in females until they have their first calf at about four years of age is not known. The longest documented persistence of larvae in the tissues of a cow is five months (Warren, 1971). Cows in these situations will also acquire larvae from feed contaminated with

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infected soil. If there is acquired resistance to infection by eggs, it is likely to be modulated by pregnancy and lactation in the same way as other helminth infestations (Lloyd, 1983), so that resistance would be reduced, larval migration would recommence and infection would be transmitted to the calf through the mammary gland. Seventy two percent of the calves were excreting eggs and this is probably an underestimate, as some would not have been in the 3-8 weeks age group in which most patent infections occur. Although 65% had been treated, neither traditional nor western anthelmintics had reduced the proportion of patent infections. Inactive reagents and treatment at the wrong time may explain this failure. Where deaths were observed during the study they confirmed the interaction between T. vitulorum infection and poor nutrition (Lee, 1955; Enyenihi, 1%9b).

The persistence of a pool of undeveloped eggs in wallow water may be important for the survival of the parasites under otherwise unfavourable circumstances. However, eggs would normally be agitated in the wallow by the activities of the buffalo and many would rise to the surface as observed in the laboratory experiment, presumably as a result of anaerobic microbial activity. The present study was undertaken during the calving season. The mean gestation period of the swamp buffalo is 316 days (Perera and de Silva, 1985) and most conceptions occur 2-5 months after the peak of rainfall (Lundstrom et al., 1982), so most calves are dropped at, or shortly after, the peak of rainfall in the wet season. These conditions are favourable for the calf but they are also favourable for survival, development and transfer of T. vitulorum eggs. Furthermore, they ensure that there will be calves contaminating the environment while there are cows in late pregnancy. Adult buffalo drink at least 12 L per day and eat approximately 8 kg of dry matter per day, so the intake of infective eggs would be more than enough for transmission to the calf. However, the proportion which establish and the duration of their persistence in the maternal host is not known.

Infective eggs of T. vihdorum appear to have a capacity for survival comparable to that of other ascarids. However, the temperatures at which infective eggs of this species survived for long periods were higher than in studies with other ascarids (O’Donnell et al., 1984), which may be part of the adaptation to a tropical environment. Survival of T. vitulorum in the field for more than two years has been reported (Thienpont and De Keyser, 1981).

There was a high mortality of eggs exposed to direct sunlight on grass or on the surface of wallow water. Clearly there is ample opportunity for development and transmission of T. vitulorunt in the village buffalo environments studied. During unfavourable periods the parasite can persist deep in the soil and deep in wallows as undeveloped or developed eggs or, for some presently unknown period, in the tissues of the maternal host. There is no practical way of preventing contamination of the village environment by calves with patent infections, nor is there a practical way of decontaminating the environment. However, if treatment can be scheduled to prevent infections becoming patent in calves, then the calves will be protected against the pathogenic effect of the mature parasites and the level of contamination of the environment will decrease.

T. canis and T. cati cause visceral larva migrans in humans (Beaver, 1956; Soulsby, 1983). Infective eggs are ingested and the aberrant visceral migration of the second stage larvae in an unusual host can have serious consequences (Zinkham, 1978). Vkcerd larva migrans due to Toxocara spp. occurs in Sri Lanka (Fernando et al.,

387

1970) but the role, if any, of T. vifzalomm is not known. Clearly there is abundant opportunity for transmission to humans. The possibility of visceral larva migrans being caused by consumption of third stage larvae from buffalo milk has been suggested (Gautam et al, 1976) but this is unlikely as that stage stays in the lumen of the intestine of its normal host, whereas the second stage larva from the infective egg migrates through the intestine of the normal host.

ACKNOWLEDGEMENTS

The cooperation of the village people in the study was rewarding. The Field Assistants of the milk cooperatives interpreted, guided and assisted.

The assistance of Dr W.A.I. Subasingha in the laboratory is appreciated. The hospitality and support of Professor S.T. Fernando, Dean of the Faculty of

Veterinary Medicine and Animal Science, University of Peradeniya is gratefully acknowledged.

The project is funded by the Australian Centre for International Agricultural Research with contributions from FAO/IAEA and SAREC.

REFERENCES

Beaver, P.C., 1956. Larva migrans. Experimental Parasitology, $587-621 Chauhan, P.P.S. and Pande, B.P., 1981. Observations on the embryonic development of bubaline and

bovine strains of Neoascaris vitulorum (Goeze, 1782) Travassos, 1927 eggs. Indian fournal of Animal Science, 51,439-445

Dewan, ML., Hossain, MI. and Baki, MA., 1979. Pathological investigations on the mortality of buffalo calves of Bangladesh. Batglad~h Veterinary Journal, 13, l-7

Enyenihi, UK, 1%9a. Studies on the epizootiology of Neoascaris vitulorum in Nigeria. Journal of Helminthology, 43,3-10

Enyenihi, UK, 1969b. Pathogenicity of Neoascarir vitulomm infections in calves. BuZletin of Epizootic Diseases ofAfrica, 17,171-K%

Enyenihi, UK, 1972. Studies on the bionomics and epizootiology of Neoascaris vitulorum in Nigeria: the effect of temperature on development, longevity and infectivity of Neoascti vitulorum eggs. Journal of the West Ajiican Science Association, 17,25-33

Fernando, S.T., Vasudevan, B., Hamtya, M.H.M., Panditha-Gunawardene, I.K.T. and Samarsinghe, H.T., 1970. Precipitin reactions in monkeys (Macaca sinica) experimentally infected with Toxocara canis and in children with visceral larva migrans syndrome. Journal of Comparative Pathology, 80,407-415

Gautam, O.P., Malik, P.D. and Singh, D.K, 1976. Neoascaris vitulotum larvae in the colostrum milk of buffaloes. Indian Journal of Public Health, 20, X33-184

Lee, RP., 1955. The anthelmintic efficiency of piperazine adipate against Neoascatis vitubmm (Goeze, 1782). Veterinary Record, 67,146-149

Lloyd, S., 1983. Effect of pregnancy and lactation upon infection. Veterinary Immunology and Immunopathology, 4,153-176

Lundstrom, K, Abeygunawardena, H., de Silva, L.NA. and Perera, B.MA.O., 1982. Environmental influence on calving interval and estimates of its repeatability in the Murrah buffalo in Sri Lanka. Animal Reproduction Science, 5, 99-109

Mozgovoi, AA., Shakhmatova, V.I. and Shikhov, RM., 1973. Experimental study of the life cycle of Neourcti vitulorum, a pathogenic nematode of ruminants. In: V.G. Gagarin (ed), Roblemy obshchei iprikkdnoi gel’mintologii. Izdatel’stvo ‘Nauka’, Moscow, pp. 105-112

O’Donnell, C.J., Meyer, KB., Jones, J.V., Benton, T., Kaneshiro, E.S., Nichols, J.S. and Shaefer, F.W., 1984. Survival of parasite eggs upon storage in sludge. Applied and Environmental Microbiology, 48, 618-625

Perera, B.MA.0. and de Siha, L.NA., 198.5. Gestation length in indigenous (Lanka) and exotic (Murrah) buffaloes in Sri Lanka. Buflalo Jouhal, 1,83-87 - - . ’

Soulsbv, E.J.L.. 1983. Toxocariasis. British Veterinarv Journal. l39.471-475 Stoieyi. G.W. and Phillips, RA., 1985. The s&iv-al of parasite eggs throughout the soil profile.

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Thienpont, D. and De Keyser, H., 1981. Toxocariasis of cattle in Belgium. In: P. Nansen, RJ. Jorgensen and EJ.L. Soulsby (eds), Epidemiology and Control of Nematodiasis in Catde, (Martinus Nijhoff, The Hague) pp. 569-582

Warren, E.G., 1971. Observations on the migration and development of Toxocara vitulorum in natural and experimental hosts. International Journal for Parasitology, 1,85-99

Zinkham, W.H., 1978. Vhxrral hrva migrans. American Journal of Diseases of Childhood, 132,627~633

(Accepted: 22 May 1989)