Pilobolus species and rapid translation of Dictyocaulus viviparus from cattle faeces

Research in Veterinary Science 1988,44, 178-/82

.Pilobolus species and rapid translation of Dictyocaulus viviparus from cattle faeces M. EYSKER, F. A. M. DE COO, Institute for Veterinary Parasitology and Parasitic Diseases, State University of Utrecht, Yalelaan 1, Utrecht, The Netherlands In a series of five laboratory experiments observations were made on the role of Pilobolus in the translation of infective lungworm larvae from faeces of cattle. The results indicated that a substantial proportion of the lungworm larvae present in the faeces may be translated from the faeces by this fungus within eight days at a temperature of 15°C. No clear relationship was observed between the numbers of Pi/obo/us and the translation of lungworm larvae. Further a longitudinal study on the occurrence of Pi/obo/us on faecal pats of grazing calves showed that between the beginning of July and the middle of September peak emergence of sporangia generally occurred within one week and most sporangia emerged within three weeks. From faecal pats which had been deposited at the end of September and the middle of October emergence of sporangia was lower and mainly occurred after two to four weeks.

of 7·5 em. The bottom and sides of the jars were covered with aluminium foil to prevent discharge of Pi/abo/us sporangia on to them and a Petri dish cover was placed on top. These cultures were put in an incubator at 15 ± 1°C and 12 hours illumination per day. After the growth of Pi/obolus sporangia began the Petri dish covers were replaced daily, the numbers of sporangia on these were counted and at least 10per cent of the sporangia were squashed to count the numbers of lungworm larvae on and inside the sporangia. Five of the cultures were also harvested daily from the seventh day, and all cultures from the ninth day onwards, by adding a layer of 1 em of water at approximately 20°C for one hour. This immersion was always done after the daily yield of Pi/abo/us sporangia had 'fired'.

TRANSLAnON of the larvae of the lungworm of cattle, Dictyocaulus viviparus, by the fungus Pilobolus was shown for the first time by Robinson (1962). More recently Jergensen et al (1982) and Somers et al (1985) observed that a much higher translation of lungworm larvae occurs when Pi/obo/us is present than when it is absent, thus the role of this fungus appears to be very important. The present study describes experiments to study the role of Pi/obolus in the translation.of D viviparus larvae and a longitudinal field study on the occurrence of Pi/obolus sporangia on faecal pats on pastures grazed by calves.

In this experiment five cultures of 30 g of faeces were made from calves 2 and 3, and five cultures of a mixture of 15 g of faeces from both calves. One day before collection of the faeces calf 3 had been fed experimentally an unknown number of Pi/obolus sporangia. Further procedures were as in experiment 1, except that harvesting of all cultures by immersion started on day 7.

Materials and methods Faeces of one naturally infected calf and four calves experimentally infected with D viviparus were used in a series of five experiments carried out between August and October 1985.

Experiment 1 From the faeces of the naturally infected calf (1) nine cultures of 30 g of faeces were made. Layers 1 cm thick were incubated in 200 ml glass jars with a height

Experiment 2

Experiments 3 to 5 These were replicates of experiment 2. In experiment 3 calves 2 and 3 were used again, and in experiments 4 and 5, calves 4 and 5. The longitudinal study of the occurrence of Pi/obolus sporangia on faecal pats of grazing calves was done as follows: on July 5 and 20, August 3, 17 and 31, September 14 and 28 and October 12, 1984, 15 freshly deposited faecal pats were marked. Twice a week between July 5 and October 20 and on October 27, November 2 and 16 the faecal pats were examined for the presence of Pi/obolus sporangia in crevices and on the surface. A score was given to each faecal pat by giving one point for crevices and, or, surface when low numbers of sporangia were present and two points when the crevices or surface were covered with 178

Role of PiloboIus in lungworm translation

179

TABLE 1: Mean numbers and standard deviations 1501 of Pi/obo/us sporangia IPI, mean numbers and so of Dictyocau/us species larvae present before culture (LPJOGI, translated by Pi/obo/us sporangia (LPI, harvested by immersion (L11 and recovered in both ways (LP+L1I, mean proportions of larvae translated by Pi/abo/us (LP/LPJOG%) and of larvae recovered in total (LP+LI/LPJOG%1 in experiments 1 to 5. In experiments 2 to 5 groups A, Band C represent the cultures of calves 2 or 4, which had not been inoculated with Pi/abo/us spores (AI, the mixed cultures (BI and the cultures of sporangia of calves 3 or 5 (CI

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sporangia. The 18 calves used for this experiment grazed the pastures from May to the middle of October. At the end of September grazing had become very scarce and hay was supplied ad libitum. Weekly, faecal cultures of all calves were made in Petri dishes and incubated in the same way as described earlier. The numbers of Pi/abo/us sporangia on these cultures were counted twice a week until no sporangia were seen. Meteorological data were obtained from the meteorological station at de Bilt, 3 km from the pastures, Results Table I shows that between, but also within, experiments a high variation occurred, particularly in the numbers of lungworm larvae harvested from Pi/abo/us sporangia. Translation by Pi/abo/us varied from very low in experiment 3 to 34 per cent of the total numbers of larvae which were present originally in group 4A in experiment 4, Table 1 does not show a clear influence of the numbers of Pi/obolus on translation, though the lowest translation was seen in the groups in experiment 3 and groups 2A and 2B which all showed a low Pi/abo/us growth. The

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147

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622 935 724 2061 1256

571 145 433 208 351 185

636 228 579 369 413 240

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numbers of Pilobolus in the groups in experiment 4 indicate that feeding with Pi/obolus sporangia did not result in a higher output of Pi/obolus in calf 5, In experiment 1 the immersion of the five cultures from day 7 onwards did not result in lower numbers of Pi/abo/us and lungworm larvae on days 8 and 9 in comparison to the four other cultures. The numbers of larvae which were harvested by immersion were higher than those translated by Pi/abo/us in all groups. However, in some cultures the numbers which were translated by the fungus were higher. Fig 1 shows that translation of lungworm larvae with Pilobolus sporangia occurred almost exclusively between four and eight days after the beginning of incubation. Further there is an indication that translation of large numbers of lungworm larvae slops earlier than the growth of Pi/abo/us sporangia. As larvae can be harvested in large numbers by immersion later on, the reason for this is not a depletion of lungworm larvae in the faecal cultures. The results of the longitudinal study on the occurrence of Pi/obolus sporangia in the field, the mean daily temperature and the rainfall are given in Fig 2. Until the middle of September peak emergence of sporangia was within one week in most cases and

M. Eysker, F. A. M. de Coo

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almost all sporangia will have emerged within three weeks. Only for the faecal pats of August 31 a more gradual emergence over a somewhat longer period was seen. The emergence of Pi/abo/us sporangia on faecal pats of September 28 and October 12 began later and was lower than earlier in the grazing season. Pi/abo/us was found more often only in the crevices on the faecal pats of July 20 and August I than in those from the other dates. The meteorological data show rainy periods between July 9 and 18, September 7 and October 12 and October 18 and 29. Some rain was also seen between July 26 and August 12, August 29 and September 5, October 12 and 18 and November 3 and 16. The mean daily temperature ranged between 13° and 24°C in July and August, between 8° and 20°C in September, between 6° and 14°C in October and between 3° and 12°C in November. The pattern of the growth of Pi/abo/us sporangia on the faecal cultures of the calves was uniform between the beginning of July and the end of September. After four days the cultures were densely covered with sporangia (over 5000 per culture), subsequently the numbers decreased rapidly and generally no sporangia were seen after two weeks. From the end of September onwards much lower numbers (generally less than 1000) were seen on the cultures and peak emergence was after seven days.

Discussion

The translation of Dictyocaulus viviparus larvae by Pi/abo/us in experiments 1 to 5 varied. One reason for this may be the very uneven distribution of larvae in the sporangia, which was also mentioned by Doncaster (1981) who found up to 50 larvae in a sporangium. When a relatively small proportion of the sporangia is searched for larvae, as in the present experiments, a substantial inaccuracy in the estimation of the numbers of larvae may occur. Nevertheless, the results strongly indicate that a substantial proportion of the lungworm larvae present in the faeces can be translated by Pi/abo/us. This supports the observations of Jergensen et al (1982) and Somers et al (1985), that a much higher translation of lungworm larvae occurs when Pi/abo/us is present on faecal pats than when it is absent. However, the very low translation in experiment 3 may imply that a high translation of lungworm larvae does not always occur. An important aspect of the present results is the observation that the lungworm larvae were translated by Pi/abo/us between four and eight days after the beginning of the experiment and predominantly when the first yields of Pi/abo/us emerged. This was surprising as the numbers of lungworm larvae which were still present in the faeces after seven or eight days were generally higher than the numbers translated by

Role o/Pilobolus in lungworm translation

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Pi/obolus. Perhaps some differences in activity of the larvae determine if they can climb the sporangiophores of Pi/obolus and subsequently be ejected or remain in the faeces. Even though the methods of examination were not very accurate the results of the field study show that peak emergence of Pilobolus sporangia in summer also occurs after approximately one week and that most sporangia develop within three weeks. This was also seen on faecal pats of July 20 and August 17 when no rain fell during the first six and 12 days respectively. However, the finding of Pi/obolus sporangia only in the crevices in many faecal pats from these dates clearly indicates the influence of dry weather on the growth of Pi/obolus sporangia. There is no clear explanation for the low growth during the first week of Pilobolus sporangia on the faecal pats of August 31, but the longer emergence from these faecal pats may be explained by the extremely wet weather from the second week of September. The low yield of Pi/obolus on the faecal pats of September 28 and October 12 in part was due to the very wet weather conditions leading to flooding of the pasture and many of the faecal pats. Another explanation is the low numbers of Pilobolus spores in the faeces of

181

the calves in that period. The reason for this probably was that the calves ingested less Pi/abo Ius spores as they were mainly fed on hay. The results of the faecal cultures in that period also indicated a somewhat slower growth of sporangia. This and the lower temperatures may explain why the emergence of sporangia was delayed. Comparison of the Pi/obolus growth on pasture and on faecal cultures of the same calves shows that on the cultures the growth generally was more abundant and, as the emergence of Pilobolus stopped earlier, more rapid. The main reason for this probably was that in the field a crust, preventing an abundant Pi/obolus growth, often developed within a couple of days on the surface of the faecal pat, particularly under dry conditions. This possibly implies that translation of Dictyocaulus larvae by Pi/obolus sporangia is less efficient under field conditions than was measured in experiments 1 to 5. Nevertheless, the results also strongly suggest that in the field in summer a rapid translation of lungworm larvae will result from Pi/obolus. In the autumn and perhaps also in the spring translation by Pi/obolus may be less rapid. Jergensen (1981) mentioned that a rapid translation is a crucial factor in the epidemiology of lungworm infections as it implies that subsequent generations of lungworms will follow each other with intervals of approximately four to five weeks, thus possibly leading to a rapid build up of pathogenic infections in susceptible animals. Some field studies in 1985 and 1986 also clearly demonstrated translation of lungworm larvae within a week after patency (Eysker and van Miltenburg, in press). Jacobs et al (1985) also mentioned this very rapid translation. Surprisingly Jorgensen et al (1982) did not link this rapid translation to Pilobolus, though they demonstrated the quantitative role of this fungus. It is unlikely that a massive translation of the sluggish lungworm larvae from faeces can occur within a week in any other way than by Pi/obolus sporangia, except perhaps occasionally by immersion of faecal pats as a result of heavy rainfall. Thus, probably one of the most important aspects of the role of Pilobolus in the epidemiology of lungworm infections is that it is the main cause of this rapid translation. Experiments I to 5 also showed that large numbers of lungworm larvae could be harvested by immersion of the cultures, when translation of larvae by Pilobolus had become very low. Under field conditions this may mean that a reservoir of viable larvae remains in the faecal pat after translation by Pi/obolus has stopped. These larvae may be translated more gradually in other ways than by Pi/obolus sporangia. Further studies on the translation of lungworm larvae, and particularly the significance of Pi/obolus, are clearly necessary.

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M. Eysker, F. A. M. de Coo

References DONCASTER, c. c. (1981) Parasitology 82, 421-428 EYSKER, M. & VAN MILTENBURG, L. (In press) Veterinary Parasitology JACOBS, D. E., FOX, M. T. & KOYO, F. A. (1985) Veterinary Record 116, 492-496 J0RGENSEN, R. J. (1981) Acta Veterinaria Scandinavica Supplementum 76, 1-77

Jli'!RGENSEN, R. J., R0NNE, H., HELSTED, C. & ISKANDER, A. R. (1982) Veterinary Parasitology 10, 331-339 ROBINSON, J. (1962) Nature 193, 353-354 SOMERS, C. J., DOWNEY, N. E. & GRAINGER, 1. N. R. (1985) Veterinary Record 116, 657-660

Received February 27, 1987 Accepted April 9, 1987