Effect of temperature and relative humidity on survival of eggs and infective larvae of Ostertagia circumcincta

Veterinary Parasitology, 49 ( 1 9 9 3 ) 2 1 9 - 2 2 7 0 3 0 4 - 4 0 1 7 / 9 3 / $ 0 6 . 0 0 © 1993 - Elsevier Science Publishers B.V. All fights reserved

219

Effect of temperature and relative humidity on survival of eggs and infective larvae of Ostertagia

circumcincta V.S. Pandey *,a, A. Chaerb, A.

Dakkak b

alnstitute of Tropical Medicine, Nationalestraat 155, B-2OOOAntwerp, Belgium blnstitut Agronomique et Vdtdrinaire Hassan II, Department of Parasitology, BP6202, Rabat-Instituts, Morocco (Accepted 1 December 1992)

Abstract Survival of Ostertagia circumcincta eggs in faeces kept at 4 ° C and of third stage larvae (L 3 ) kept in distilled water at - 5 0 , - 2 5 , - 10, 4, 16, 25 and 35 °C was studied. The effect of relative humidity ( R H ) of 95, 75, 50 and 30% at 16, 25 and 35°C on the survival of L3 was also studied. The survival of eggs at 4°C was high during the first week, followed by a gradual decline to 10% by Day 22 of storage. The L3 survived for 7 weeks at - 50°C, 9 weeks at - 2 5 ° C , 13 weeks at - 10°C, and 7 weeks at 35°C. By 16 weeks, at the termination of the experiments, 74%, 76% and 23% of L3 were still alive at 4°C, 16°C and 25°C, respectively. At all temperatures tested, the L 3 survived longer at RH of 30 and 50% than at 75 and 95%. At 35 °C, L3 survived up to 6 weeks at 30%, 5 weeks at 50% and 1 week at 75 and 95% RH. At 16 and 25°C and RH of 30 and 50%, over 70% of L3 were still alive at the end of experiments at the seventh week. At 25 ° C, 40% of L3 survived for up to 5 weeks at 75% RH and 5% survived for 6 weeks at 95% RH. At 16°C at the end of experiment at the seventh week, 25% of L3 were still alive at 75% RH and 56% were still alive at 95% RH. It is concluded that the L 3 of O. circumcincta are very resistant to temperature and RH.

Introduction Trichostrongylid nematodes are subjected to various environmental factors during the free living period of their life. Temperature and humidity are the two main factors which influence their survival in the external environment. In a study of the effect of temperatures between 5 and 30°C on the survival of third stage infective larvae (L3) of several genera of nematodes, Boag and Thomas (1985 ) found L3 of Ostertagia spp. to be the most resistant. Similarly, Ostertagia spp. appear to be more cold tolerant than many other trichostrongylids (Furman, 1944; Pandey, 1972; Jasmer et al., 1987). *Corresponding author.

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Although the results obtained under controlled conditions in the laboratory cannot be applied as such to explain ecology of the free living stages in the field (Smith et al., 1986 ), they are useful in understanding the ecological patterns and may explain differences in the epidemiology of these trichostrongylid parasites. In spite of the fact that Ostertagia circumcincta, an important parasite of sheep, has a wide distribution mainly in temperate and mediterranean type climates, very little work has been done on the effect of temperature and humidity on the free living stages of this parasite. Recently, Jasmer et al. ( 1987 ) studied survival of the L 3 of the Washington isolate of O. circumcincta exposed to low temperatures for very short periods. The objective of the present work was to study the effect of temperature and relative humidity (RH) on the survival of eggs and infective larvae of the Weybridge isolate of O. circumcincta. The effects of temperature on development of free living stages of this isolate were reported previously (Pandey et al., 1989). Materials and methods

The isolate of O. circumcincta used in the present work was obtained from Weybridge, UK, and was originally isolated in 1965. The parasite was maintained in worm-free, 5 m o n t h old Timhadit lambs as described previously by Pandey et al. (1989).

Eggs The faeces from lambs carrying a monospecific infection of O. circumcincta (300-500 eggs g-1 faeces) were collected in faecal bags attached to the animal for 6 h. The faeces were broken and homogenised so that the distribution of eggs in the sample would be as even as possible. Ten-gram samples were weighed and their consistency adjusted by adding charcoal or water as needed so that the whole mass was moist and crumbly. They were then placed in Petri dishes 9.5 cm in diameter. Several replicates were prepared from the same bulk of faeces. Three of these Petri dishes, which served as controls, were cultured at 25 °C for 8 days to determine the number of infective larvae that could be recovered under o p t i m u m conditions. The other Petri dishes were transferred to 4 ° C. For all these cultures, filter papers were placed inside the Petri dish lids and were moistened regularly to keep the humidity at 100%. If necessary, tap water was sprinkled over the faecal cultures to maintain the moist and crumbly consistency. Daily triplicate samples were removed, cultured and incubated under the same o p t i m u m conditions as control cultures, at 25°C for 8 days. They were then Baermannised overnight and infective larvae counted. The numbers thus obtained were compared with those from the control samples and percentage survival calculated.

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Third stage infective larvae (L3) The infective larvae were obtained by culturing the faeces at 25°C for 8 days. After Baermannisation, the larvae were washed three times in distilled water. Only freshly harvested larvae were used in all experiments.

Effect of temperature on L3 Two hundred L 3 in 5 ml of distilled water were placed in Petri dishes and kept at - 50, - 25, - 10, 4, 16, 25 or 35 oC. At weekly intervals, three samples were removed from the temperature cabinets and allowed to equilibrate for 12 h if they had been stored at temperatures below 0 °C or for 6 h if they had been stored at temperatures of 4 °C or above. The criteria of viability was the motility of the larvae as reported earlier (Todd et al., 1970; Pandey, 1972; Jasmer et al., 1987 ). Infective larvae demonstrating spontaneous movement, while viewed with a dissecting microscope, were counted as alive. The larvae that did not move during the observation were counted as non-viable and considered dead.

Effect of relative humidity on L3 Different relative humidity ( R H ) gradients between 30 and 95% were obtained by the use of potassium hydroxide (Table 1 ) according to Solomon (1951 ). Glass desiccators containing 200 g of KOH solution served as humidity chambers. A known n u m b e r of L3 (200___ 12) in 0.5 ml of distilled water was placed on plastic dishes and allowed to dry at room temperature (approximately 22 °C) overnight. If necessary, a fan was used to accelerate drying. The following day the dishes were transferred to different RH chambers, which were placed in 16, 25 and 35 °C incubators. At weekly intervals, three test samples TABLE 1 Amount of KOH required to obtain different relative humidities at 16, 25 and 35 °C Amount of KOH required to prepare 100 g of solution in water (g)

Relative humidity (%)

Temperature ( ° C)

7 22.25 33.7 34.7 42.3 42.8

95 75 50 50 30 30

16,25,35 16,25,35 16,25 35 16,25 35

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were removed from RH chambers; a few drops of distilled water added to cover the dry area where L 3 w e r e placed and left at room temperature (approximately 22 ° C) for 10-12 h. The L 3 showing spontaneous movement when viewed by dissecting microscope were counted as alive. The differences in the survival of L 3 at different temperatures and relative humidities were analysed by t-test using the PC software package Statgraphics (STSC, USA). Results

Survival of eggs Eighty percent of the eggs kept at 4°C survived for the first 4 days, followed by a rapid decline in survival until Day 8 (22% survival) and a gradual slow decline thereafter (Fig. 1 ). Only 11% of eggs kept at 4°C for 22 days produced L 3.

Survival of Ls at different temperatures At 4, 16, 25 and 35°C (Fig. 2), the survival of L3 was high (80-99%) until the fifth week of storage with no significant difference between the temperatures. Thereafter, the survival curve at 4 and 16 °C followed a similar trend with slow mortality, whereas at 25 and 35 °C, the mortality was much higher (P<0.001). All the L3 w e r e dead by the eighth week at 35°C. By the end of the experiment at Week 16, the mortality of L3 at 25°C, 16°C and 4°C was 77%, 24% and 26%, respectively (Fig. 2 ). At lower temperatures the survival % survival of eggs 100

80

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Fig. 1. Percentage survival of eggs ofO. circumcincta in faeces kept at a constant temperature of 4°C.

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KS. Pandey et al./Veterinary Parasitology 49 (1993.) 219-227 % of living L3 100

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7 8 9 10 Time (weeks)

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Fig. 2. Percentage survival of infective larvae (L3 ) of O. circumcincta in water at constant temperatures of 4, 16, 25 and 35°C. % of living L3 100 - A - -60 C, -'A'- -2s O ~ - :iO C

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Fig. 3. Percentage survival of infective larvae (L3) of O. circumcincta at constant temperatures o f - 10, - 2 5 and -50°C. o f L3 was longest at - 10°C and shortest at - 5 0 ° C (Fig. 3). The m a x i m u m survival was 13 weeks at - 1 0 ° C (6%), 9 weeks at - 2 5 ° C (0.8%) and 7 weeks at - 5 0 ° C (0.7%). The difference in rates o f survival between - I 0 , - 5 0 and - 2 5 °C was highly significant ( P < 0.001 ).

Survival of L3 at different relative humidities The rates o f survival of L 3 at different R H at 16°C, 2 5 ° C and 35°C are presented in Figs. 4, 5 and 6, respectively. At all temperatures, in general, the

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v.s. Pandey et al./Veterinary Parasitology 49 (1993) 219-227 Survival(%)

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Fig. 4. Percentage survival of infective larvae (L 3) of O. circumcincta at different relative humidities and a constant temperature of 16 ° C.

Survival

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Fig, 5. Percentage survival of infective larvae (L3) of O. circumcincta at different relative humidities and a constant temperature of 25 ° C.

L 3 survived better at 50 and 30% R H t h a n at 75 and 95% R H ( P < 0 . 0 5 0.01 ). At all R H the death rate was fastest at 35 °C with n o L3 surviving more than 1 week at the R H o f 95 a n d 75% and o n l y very small n u m b e r s surviving until Week 6 at lower R H (Fig. 6 ) . At 2 5 ° C (Fig. 5) a n d 1 6 ° C (Fig. 4 ) over 70% o f L 3 survived until Week 7 at R H o f 30 a n d 50%. The difference in the survival t i m e o f L 3 at 75 a n d 95% R H was n o t significant at any o f the three temperatures.

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V.S. Pandey et al./Veterinary Parasitology 49 (1993) 219-227 Survival (%) 100 -~-

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Fig. 6. Percentagesurvival of infectivelarvae (L3 ) of O. circumcincta at differentrelativehumidities and a constanttemperatureof 35°C.

Discussion As at 4°C 95% of eggs of O. circumcincta hatch to first stage larvae within 3 to 5 days (Pandey et al., 1989) and the mortality of first stage larvae, as shown for Ostertagia ostertagi (Pandey, 1972 ) is very high between 5 and 20 days, the low recovery of L3 from the eggs of O. circumcincta at 4 ° C could be due to heavy mortality of newly hatched larvae. However, at 4 °C the O. ciro cumcincta eggs appear to be more resistant than those ofHaemonchus contortus which survive for only 4 days (Todd et al., 1976). However, O. circumcincta eggs are less resistant than O. ostertagi eggs, which showed 100% viability until Week 6, with a few eggs able to survive for up to 50 weeks (Pandey, 1972). Survival of L3 of O. circumcincta at lower temperatures ( - 10, - 2 5 and - 5 0 ° C ) (Fig. 3) was much less than at the higher temperatures (Fig. 2). The low survival at - 2 5 and - 5 0 ° C is of less practical significance because such low temperatures for prolonged periods at ground level, where L3 are normally found during the winter, is rare in most areas where O. circumcincta is common. However, their survival for up to 3 months at - 10 °C is of practical importance in temperate cold climates. Jasmer et al. (1987) examined L3 of the Washington isolate of O. circumcincta at - 10, - 15 and - 18 °C for up to 15 h and found 79-88% survival, which shows that the Weybridge isolate is more resistant to low temperatures than the Washington isolate. The Weybridge isolate was also more resistant than a Californian isolate of O. circumcincta, as Furman (1944) reported maximum survival of L3 for up to 2 weeks at - 6 ° C and for 3 weeks at 37°C. The L3 of O. circumcincta also survived longer than L3 of O. ostertagi (Pandey, 1972 ).

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The fact that at 4 and 16°C, two-thirds and at 25°C, one-fifth, of the L 3 survived for up to 16 weeks shows that, like O. ostertagi (Pandey, 1972), O. circumcincta can survive for longer periods. These findings also confirm the results of Boag and Thomas (1985) who, after studying L3 of 12 nematode species at 5-30°C concluded that O. circumcincta came second only to O. ostertagi for survival at the temperatures tested. The L3 of O. circumcincta were found to be more resistant to desiccation than those of O. ostertagi (Pandey, 1976 ). Similar conclusions were drawn in field experiments where both O. ostertagi and O. circumcincta were exposed to identical field conditions in Australia (Young, 1983 ). In general, at lower RH of 30 and 50%, the survival of L3 was better at all temperatures. In other trichostrongylids such as Trichostrongylus colubriformis (Andersen and Levine, 1968) and O. ostertagi (Pandey, 1976), it has been shown that desiccated L3 survive better than non-desiccated L3. Smith et al. (1986), in their model on O. ostertagi using field and laboratory data, showed that the survival of pre-infective stages in the field are about twice as long as in the laboratory. The isolate of O. circumcincta used in the present study has been passaged in lambs for several generations and therefore it may be possible that its behaviour would not be exactly the same as the field strains of O. circumcincta. However, our results corroborate field work in Morocco, where studies using tracer lambs demonstrated that the infective larvae of O. circumcincta do not survive on pastures during the hot dry months of July and August (Ouhelli et al., 1981; Pandey et al., 1990). Furthermore, as the ideal temperature for development of eggs to infective larvae is 16 °C (Pandey et al., 1989) and a high percentage of L 3 survive for over 4 months at 4 and 16 ° C, it would appear that O. circumcincta is a parasite better adapted to cooler climates (Pandey et al., 1980). The survival of L 3 for 3 months at - 10 °C and for a much longer period at 4 ° C, indicates that in the cold temperate regions some of these larvae would be able to overwinter on pasture (Gibson and Everett, 1972 ) and initiate new infection in spring when animals are turned out onto pasture (Waller and Thomas, 1978 ).

References

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