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Spread of infective Dictyocaulus viviparus larvae in pasture and to grazing cattle: Experimental evidence of the role of Pilobolus fungi
Veterinary Parasitology, 10 (1982) 331--339 Elsevier Scientific Publishing Company, Amsterdam - - P r i n t e d in The Netherlands
331
S P R E A D O F I N F E C T I V E D I C T Y O C A U L U S V I V I P A R US L A R V A E I N PASTURE AND TO GRAZING CATTLE: EXPERIMENTAL EVIDENCE OF THE ROLE OF PILOBOLUS FUNGI
R.J. J O R G E N S E N *
Institute o f Veterinary Microbiology and Hygiene, Royal Veterinary and Agricultural University, Bu'lowsvej 13, DK 1870 Copenhagen V (Denmark) H. RO NNE and C. HELSTED
Zoological Laboratory, University of Copenhagen, Universitetsparken 15, DK-2100, Copenhagen 0 (Denmark) A.R. I S K A N D E R
Animal Health Research Institute, Dokki, Cairo (Egypt) (Accepted for publication 17 February 1982)
ABSTRACT J~brgensen, R.J., R~bnne, H., Helsted, C. and Iskander, A.R., 1982. Spread of infective Dictyocaulus viviparus larvae in pasture and to grazing cattle: experimental evidence of the role of Pilobolus fungi. Vet. Parasitol., 10: 331--339. Four calves experimentally infected with Dictyocaulus viviparus were made Pilobolusfree by hygienic measures and by feeding them irradiation sterilized feed. Two of the calves were orally administered laboratory cultured Pilobolus sporangia daily. As a result, the faeces from one pair contained D. viviparus larvae and Pilobolus spores, and the faeces from the other pair contained D. viviparus larvae, but no Pilobolus spores. T w o identical plots were used for deposition of the two kinds of faeces, and one of them remained free o f Pilo bolus fructification. Herbage sampling and the use of tracer calves revealed that on this plot the larval contamination and the infectivity of the pasture were greatly reduced. A mean larval count of 1321 near the faecal pats (0--5 cm) in the plot where Pilobolus was observed was reduced to 69 per kg of herbage on the Pilobolus-free plot. At a distance o f 100 cm from the pats, a reduction from 99 to 3 larvae per kg herbage was found. Each plot was grazed by four parasite-free tracer calves for 3 days. During the subsequent stabling period o f these calves, the lungworm larval excretion of those from the Pilobolusfree plot was reduced by 90% and the clinical symptoms were milder than those which grazed the plot which contained the fungus. The mean post m o r t e m worm counts after 4 weeks o f stabling showed a reduction from 167 to 25 worms. A more marked effect of Pilobolus fungi on the transmission of D. viviparus infection is to be expected under field conditions where calves are grazing more selectively than in the present study.
*Present address: Institute of Internal Medicine, Royal Veterinary and Agricultural University, Biilowsvej 13, DK-1870 Copenhagen V, Denmark.
0304-4017/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing Company
332 INTRODUCTION The free-living stages of the cattle lungworm Dictyocaulus viviparus are known to possess a relatively poor migratory ability, and their motility is correspondingly low (Taylor, 1951; Soliman, 1952; Michel and Rose, 1954; Roses 1956) and of a relatively short duration in the absence of host stimuli (J¢rgensen, 1980a). However, outbreaks of verminous bronchitis among grazing cattle m a y result from the sudden availability of large numbers of infective stages on the herbage (Michel and Ollerenshaw, 1963; Duncan et al., 1979; J~rgensen, 1980b, c, 1981). A possible explanation of this apparent discrepancy may be sought in the report of Robinson (1962a) who showed that D. viviparus larvae may be transmitted b y the violent discharge of the sporangia of the coprophilous Pilobolus spp. fungi, the so-called Pilobolus gun. Robinson believed that this interesting p h e n o m e n o n represents a significant factor in the epidemiology of parasitic bronchitis, and he reported on the presence of Pilobolus spores in 95% of bovine faeces f r o m British cattle (Robinson, 1962b). Since then no original observations have appeared on this relationship, except the recent laboratory studies b y Doncaster (1981). Danish field studies concerned with the epidemiology of D. viviparus have shown that during the grazing season the translocation of larvae takes place in approximately one week (JCrgensen, 1980b, c, 1981). In one of these studies an increase in the pasture larval contamination was observed around noon (J~brgensen, 1980c), concomitant with the circadian periodicity of Pilobolus sporanglal discharge (Buller, 1934; Ingold, 1971, 1978). As a result of these observations it was decided to determine the role of Pilobolus spp. in the epidemiology of D. viviparus under field conditions. MATERIALS AND METHODS
Calves Four Jersey calves weighing 150--170 kg each were artificially infected orally with 1000 D. viviparus 3rd stage larvae each. The faeces produced b y these donor calves were monitored and collected for field use. Eight one-year-old black pied Danish Friesian bull calves with a mean b o d y weight o f 323 kg (range 262--421 kg) were used as tracers. They were bought from one farm where t h e y had been intensively fed indoors since birth. The calves were equally divided according to b o d y weight into t w o groups of 4 calves each (Groups 1 and 2).
Stabling and feeding o f donor calves After the first week following artificial infection with D. viviparus, the donor calves were thoroughly washed and transferred to individual, steam
333 sterilized stable compartments. They were fed sterilized lucerne pellets and sterilized calf concentrate in order to clean t h e m f r o m Pilobolus spores.
Sterilization o f feed and faeces Feed for the four d o n o r calves was placed in polyvinylchloride (PVC) bags in portions o f 2--4 kg. The bags were flattened to a m a x i m u m height of 4 cm and exposed to ionizing irradiation on b o t h sides at a dose of 4 Mrad, which is k n o w n to be lethal to microorganisms (K. Sehested, 1979, personal communication). A total of 600 kg of feed was sterilized. Fifty kg of fresh bovine faeces were similarly sterilized.
Cu Ituring o f Pilo bolus sporangia Fifty grams of faeces f r o m parasite-free cattle were kept in non-transparent commercial plastic flower pots on wet mineral wool. Each pot was provided with a transparent plastic lid, kept at r o o m temperature n o t exeeding 25°C, and exposed to artificial illumination during day-time. Sporangia were harvested f r o m the lids and mixed with 0.5% liquid agar at 40°C. The mixtures were allowed to set to a semi-liquid consistency in syringes. The syringes were stored at 4°C until use. Two of the d o n o r calves were later orally administered these sporangia in agar in order to contaminate their faeces with Pilobolus spores.
Hots A two-year<~ld ungrazed rye grass pasture was cut one week before the c o m m e n c e m e n t o f the field experiment, leaving a rather coarse and open 15cm high pasture. Two square plots (Plots 1 and 2) of 750 m 2 were each fenced off, leaving a distance of 12 m between them.
Pilobolus contamination o f the faeces The faeces produced by the four donor calves were collected twice daily after the first week of infection, and the weight recorded. Each batch of faeces was checked for the presence of Pilobolus as described above, and for the presence, and concentration of D. viviparus larvae by the m e t h o d of Nevenic et al. (1962). After one week of feeding sterilized feed, the faeces of the four calves were free of Pilobolus spores. Three weeks after artificial infection, two of the donors (Pair 1) received 1000 Pilobolus sporangia per os daily while the remaining two calves (Pair 2) were not given the fungi. During this period, the daily faecal production f r o m each pair was stored separately overnight in closed PVC bags. During the following day, the a m o u n t of faeces and the total number o f D. viviparus larvae in the two portions were standardized by (1) transferring Pilobolus-free faeces with a relatively high concentration of larvae to the
334 other portions, (2) adding sterilized faeces and/or (3) disposal of surplus faeces. Thus the two portions of faeces were similar, except for the presence (Batch 1) or absence (Batch 2) of Pilobolus spores.
Deposition o f faeces on plots Placement on the plots of the faeces standardized as above t o o k place from 8--24 June, 1979. It was carried out manually by dropping portions of approximately 500 g f r o m a height of l m . On the 24th of June, a total of 104 dung pats or 72 kg o f faeces containing 2.2 million D. viviparus larvae had been deposited on Plot 1. Faeces of equal a m o u n t s from Pair 2 (Batch 2) were similarly deposited on Plot 2.
Herbage examination for infective D. viviparus larvae Herbage samples were collected daily from areas near the faecal pats (0--5 cm) as well as at a distance of 100 cm, and at a distance of more than 100 cm on both plots. The samples were processed separately and examined for D. viviparus larvae by t h e bile--agar technique described by J¢rgensen (1975). Infective Dictyocaulus larvae were identified using Iskander and J~brgensen (1980) criteria, and t h e n u m b e r of larvae were expressed per kg of fresh herbage.
Grazing by tracer calves Based on the larval counts obtained from Plot 1, it was decided to graze the two groups o f tracer calves on their respective plots for 3 days only. The calves were t u r n e d o u t on the 25th of June, 1979, and during the following 3 days, the calves were tethered individually. They were moved once or twice daily, and their behaviour was observed.
Stabling and post mortem worm counts of tracer calves The tracer calves were moved indoors on 29th June, 1979, and stabled conventionally for four weeks before slaughter. The faecal larval excretion was determined f r o m 19th July to 26th July. At post mortem, the lungs were perfused with water according to the m e t h o d of Inderbitzin (1976), and the bronchial tree was opened. Worms present in the washings and worms observed in t h e bronchial tree were collected and counted. RESULTS
Observations on Pilobolus fructification Observations carried out daily on the experimental plots during the afternoons revealed no Pilobolus fruitbodies on faeces deposited on Plot 2. On
335
Plot 1, fruitbodies were observed for the first time on the 20th of June, 1979, on faeces deposited 8, 10, and 12 days previously. During the following week, growth and fructification was observed to occur daily on Plot 1. Pasture larval counts The results o f the examination o f herbage samples for presence of infective D. viviparus larvae are shown in Table I. It appears that the larval concentration close to faecal pats was greatly reduced on Plot 2 (average 94.8%). At a distance o f 100 cm or more, the reduction reached 97% and 91.5%, respectively. TABLE I
Dictyocaulus viviparus larvae o n h e r b a g e f r o m p l o t s i n f e c t e d with, and free f r o m , Pilobolus spp. f u n g i
Distance P l o t
N u m b e r of l a r v a e / k g of fresh h e r b a g e
from faecal pats
June
(cm)
24
Total July
25
26
27
28
29
30
1
2
3 4
<5
1 2
423 9
489 28
217 20
192 12
1321 69
100
1 2
53 0
25 0
8 3
13 0
99 3
>100
1 2
2 0
0 0
8 0
29 0
9 0
6 0
5 5
0 0
0 0
0 0 0 0
59 5
Behaviour o f tracer calves During t h e first day on pasture, the calves were excited. The faeces previously deposited b y hand was trampled and therefore partly spread into the surrounding pasture. During the next t w o days, the calves started to graze, apparently at random. The a m o u n t o f herbage ingested appeared to be limited. Clinical and post mortem findings Coughing was n o t e d among 3 of the calves of Group 1 during the 3rd and 4th week o f stabling, whereas only one calf which had grazed the Pilobolusfree field coughed during the same period. The mean respiratory rates are shown in Fig. 1, from which a slight b u t consistent difference in respiratory rates appears. The difference was most pronounced during relatively warm weather conditions during the middle of July.
336
RESP./MIN.
/\
40
•
•
\
/
/
rl
0
'
• 4
~T
,
[] 6
,
i 8
.
, I0
o
. 12
.
\
G~lq
, 14
,
, 16
.
.
. 18
.
. 20
.
.
.
.
.
22
24
26
JULY
Fig. 1. Mean respiratory rates (resp./min) of the two groups of tracers and difference (Group 1-2) in mean respiratory rates between 2; = - - c , G r o u p 1 - - 2 .
t h e g r o u p s . • - - =, G r o u p 1; • - - - • , G r o u p
LARVAEIO / g 400
300
200
1
I00
0
18
20
22
24
26 JULY
Fig. 2. M e a n l a r v a l c o u n t s p e r 1 0 g o f f a e c e s ( l a r v a e / 1 0 g) o f t h e t w o t r a c e r g r o u p s . V e r t i c a l l i n e s i n d i c a t e r a n g e i n c o u n t s o f i n d i v i d u a l c a l v e s . • - - • , G r o u p 1; • - - - • , G r o u p 2.
Results o f faecal analysis for presence of D. viviparus larvae are shown in Fig. 2. It appears that both groups had patent infections, but that the number of larvae excreted by Group 2 was only approximately 10% of the larval excretion of Group 1.
337 During t h e period f r o m turning out until slaughter, Group 2 gained an average o f 26 kg compared with an average loss of 0.25 kg among the calves of Group 1. Post m o r t e m determinations of the number of worms in the tracer calves revealed seven times higher worm counts in Group I compared with Group 2. Individual worm counts are presented in Table II. TABLE II Numbers of worms recovered at necropsy from the tracer calves Calf no.
Group 1 1
Worm
counts
2
Group 2 3
4
146 77 102 343
Mean
167
5
6
7
26 11 4
8 58
25
DISCUSSION This experiment shows t h a t the pasture contamination with infective D. vivi. parus larvae as well as the infectivity of the pasture was greatly reduced in the absence o f Pilobolus fungi. It thus appears t h a t in the presence of Pilobolus fungi, the bulk o f infective larvae which were dispersed f r o m the faecal pats and which became available to grazing calves were Pilobolus translocated. Trichostrongyle species were n o t included in the present investigation. Whether Pilobolus spp. play a role in the translocation of such larvae under grazing conditions is u n k n o w n , although Bizzell and Ciordia (1965) observed Pilobolus translocation of Cooperia punctata and Trichostrongylus colubriformis larvae f r o m faecal cultures. In t h e present experiment, the lungworm larvae f o u n d in some pasture samples picked on t h e Pilobolus-free field as well as the infections acquired on that field could n o t have been dispersed by Pilobolus fungi since no fruit bodies were observed on the field despite careful daily observations of all deposited faecal pats. It is most likely t h a t the larval translocation on Plot 2 resulted f r o m mechanical spread of larval contaminated faecal material to the surrounding herbage by the bodies and hooves of the tethered and excited tracer calves. It is also likely t h a t the apparently non-selective grazing resulted in a relatively high intake of larvae spread in this manner. Therefore, the results o f the present experiment do not indicate that the spread of bovine dictyocaulosis is totally dependent on the presence and dispersal of Pilobolus sporangia. However, it is possible that a more pronounced effect of Pilobolus translocation on the infection parameters is to be expected under field conditions where calves are grazing more selectively by avoiding the grass growing near faecal pats.
338 ACKNOWLEDGEMENTS
The technical assistance of N. Midtgaard and K. Madsen is acknowledged. The authors' sincere thanks are due to K. Sehested, Atomic Experimental Station, RisO, for his guidance and help in connection with the use of the electron accelerator for sterilization purposes, and to Allen and Hanburys Research Laboratories, Ware, England, for providing infective D. viviparus larvae. They are also indebted to H. Dissing, Institute of Thallophytes, University of Copenhagen, for fruitful discussions on the botanical aspects. This study was supported b y a grant from the Carlsberg Research Foundation.
REFERENCES Bizzel, W.E. and Ciordia, H., 1965. Dissemination of infective larvae of trichostrongylid parasites of ruminants from faeces to pastures by the fungus Pilobolus spp. J. Parasitol., 51: 184. Buller, A.H.R., 1934. Researches on Fungi. Vol. 6, Longmans, London, 513 pp. Doncaster, C.C., 1981. Observations on relationships between infective juveniles of bovine lungworm, Dictyocaulus viviparus (Nematoda: Strongylida) and the fungi, Pilobolus kleinii and Pilobolus crystaUinus (Zygomycotina: Zygomycetes). Parasitology, 82: 421--428. Duncan, J., Armour, J., Bairden, K., Urquhart, G.M. and J~brgensen, R.J., 1979. Studies on the epidemiology of bovine parasitic bronchitis. Vet. Rec., 104: 274--278. Inderbitzin, F., 1976. Experimentale erzeugte Entwicklungshemmung yon Dicty ocaulus viviparus des Rindes. Thesis, Institut fiir Parasitologie der Universit~t ZUrich, 45 pp. Ingold, C.T., 1971. Fungal spores. Their liberation and dispersal. Clarendon Press, Oxford, 302 pp. Ingold, C.T., 1978. The biology of Mucor and its allies. The Institute of Biology's Studies in Biology No. 88, London, 59 pp. Iskander, A.R. and J~brgensen, R.J., 1980. Identification of infective Dictyocaulus viviparus larvae isolated from herbage by the bile--agar technique. Acta Vet. Scand., 21 : 330----335. J~brgensen, R.J., 1975. Isolation of infective Dictyocaulus larvae from herbage. Vet. Parasitol., 1 : 61--67. Jc,brgensen, R.J., 1980a. Dictyocaulus viviparus: migration in agar of larvae subjected to a variety o f physico~chemical exposures. Exp. Parasitol., 49: 106--115. J~brgensen, R.J., 1980b. Epidemiology of bovine dictyocaulosis in Denmark. Vet. Parasitol., 7: 153--167. J~brgensen, R.J., 1980c. Bovine dictyocaulosis. Pattern of infection and the prevention of parasitic bronchitis. Acta Vet. Scand., 21: 658--676. J~brgensen, R.J., 1981. Recent Danish studies on the epidemiology of bovine parasitic bronchitis. In: P. Nansen, R.J. J~brgensen, E.J.L. Soulsby (Editors), Epidemiology and Control of Nematodiasis in Cattle. Current Topics in Veterinary Medicine and Animal Science, Vol. 9, Nijhoff, The Hague, Boston, London, pp. 215--239. Michel, J.F. and Ollerenshaw, C.B., 1963. Helminth diseases of grazing animals. In: A.N. Warden, K.C. Sellers and D.E. Tribe (Editors), Animal Health, Production and Pasture. Longmans, London, 786 pp. Michel, J.F. and Rose, J.H., 1954. Some observations on the free-living stages of the cattle lungworm in relation to their normal environment. J. Comp. Pathol., 64: 195. Nevenic, V., Jovanovic, M., Sokolic, A., Cuperlovic, K. and Movsesijan, M., 1962. Jednostavan postupak za ustanovljenje larvica Dictyocaulus filaria (In: Croatic with English abstract). Vet. Glas., 16: 203--209.
339 Robinson, J., 1962a. Pilobolus spp. and the translation of the infective larvae of Dictyocaulus viviparus from faeces to pasture. Nature, 193: 353-I-354. Robinson, J., 1962b. The role o f the fungus Pilobolus in the spread of the infective larvae of Dic~yocaulus viviparus. Parasitology, 52: 17. Rose, J.H., 1956. The bionomics of the free-living larvae of Dictyocaulus viviparus. J. Comp. Pathol. Ther., 66: 228--240. Soliman, K.N., 1952. Observations on the survival on pasture of preparasitic stages of Dictyocaulus viviparus in Southern England. II. Br. Vet. J., 108: 204--213. Taylor, E.L., 1951. Parasitic bronchitis in cattle. Vet. Rec., 6 3 : 8 5 9 873.
331
S P R E A D O F I N F E C T I V E D I C T Y O C A U L U S V I V I P A R US L A R V A E I N PASTURE AND TO GRAZING CATTLE: EXPERIMENTAL EVIDENCE OF THE ROLE OF PILOBOLUS FUNGI
R.J. J O R G E N S E N *
Institute o f Veterinary Microbiology and Hygiene, Royal Veterinary and Agricultural University, Bu'lowsvej 13, DK 1870 Copenhagen V (Denmark) H. RO NNE and C. HELSTED
Zoological Laboratory, University of Copenhagen, Universitetsparken 15, DK-2100, Copenhagen 0 (Denmark) A.R. I S K A N D E R
Animal Health Research Institute, Dokki, Cairo (Egypt) (Accepted for publication 17 February 1982)
ABSTRACT J~brgensen, R.J., R~bnne, H., Helsted, C. and Iskander, A.R., 1982. Spread of infective Dictyocaulus viviparus larvae in pasture and to grazing cattle: experimental evidence of the role of Pilobolus fungi. Vet. Parasitol., 10: 331--339. Four calves experimentally infected with Dictyocaulus viviparus were made Pilobolusfree by hygienic measures and by feeding them irradiation sterilized feed. Two of the calves were orally administered laboratory cultured Pilobolus sporangia daily. As a result, the faeces from one pair contained D. viviparus larvae and Pilobolus spores, and the faeces from the other pair contained D. viviparus larvae, but no Pilobolus spores. T w o identical plots were used for deposition of the two kinds of faeces, and one of them remained free o f Pilo bolus fructification. Herbage sampling and the use of tracer calves revealed that on this plot the larval contamination and the infectivity of the pasture were greatly reduced. A mean larval count of 1321 near the faecal pats (0--5 cm) in the plot where Pilobolus was observed was reduced to 69 per kg of herbage on the Pilobolus-free plot. At a distance o f 100 cm from the pats, a reduction from 99 to 3 larvae per kg herbage was found. Each plot was grazed by four parasite-free tracer calves for 3 days. During the subsequent stabling period o f these calves, the lungworm larval excretion of those from the Pilobolusfree plot was reduced by 90% and the clinical symptoms were milder than those which grazed the plot which contained the fungus. The mean post m o r t e m worm counts after 4 weeks o f stabling showed a reduction from 167 to 25 worms. A more marked effect of Pilobolus fungi on the transmission of D. viviparus infection is to be expected under field conditions where calves are grazing more selectively than in the present study.
*Present address: Institute of Internal Medicine, Royal Veterinary and Agricultural University, Biilowsvej 13, DK-1870 Copenhagen V, Denmark.
0304-4017/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing Company
332 INTRODUCTION The free-living stages of the cattle lungworm Dictyocaulus viviparus are known to possess a relatively poor migratory ability, and their motility is correspondingly low (Taylor, 1951; Soliman, 1952; Michel and Rose, 1954; Roses 1956) and of a relatively short duration in the absence of host stimuli (J¢rgensen, 1980a). However, outbreaks of verminous bronchitis among grazing cattle m a y result from the sudden availability of large numbers of infective stages on the herbage (Michel and Ollerenshaw, 1963; Duncan et al., 1979; J~rgensen, 1980b, c, 1981). A possible explanation of this apparent discrepancy may be sought in the report of Robinson (1962a) who showed that D. viviparus larvae may be transmitted b y the violent discharge of the sporangia of the coprophilous Pilobolus spp. fungi, the so-called Pilobolus gun. Robinson believed that this interesting p h e n o m e n o n represents a significant factor in the epidemiology of parasitic bronchitis, and he reported on the presence of Pilobolus spores in 95% of bovine faeces f r o m British cattle (Robinson, 1962b). Since then no original observations have appeared on this relationship, except the recent laboratory studies b y Doncaster (1981). Danish field studies concerned with the epidemiology of D. viviparus have shown that during the grazing season the translocation of larvae takes place in approximately one week (JCrgensen, 1980b, c, 1981). In one of these studies an increase in the pasture larval contamination was observed around noon (J~brgensen, 1980c), concomitant with the circadian periodicity of Pilobolus sporanglal discharge (Buller, 1934; Ingold, 1971, 1978). As a result of these observations it was decided to determine the role of Pilobolus spp. in the epidemiology of D. viviparus under field conditions. MATERIALS AND METHODS
Calves Four Jersey calves weighing 150--170 kg each were artificially infected orally with 1000 D. viviparus 3rd stage larvae each. The faeces produced b y these donor calves were monitored and collected for field use. Eight one-year-old black pied Danish Friesian bull calves with a mean b o d y weight o f 323 kg (range 262--421 kg) were used as tracers. They were bought from one farm where t h e y had been intensively fed indoors since birth. The calves were equally divided according to b o d y weight into t w o groups of 4 calves each (Groups 1 and 2).
Stabling and feeding o f donor calves After the first week following artificial infection with D. viviparus, the donor calves were thoroughly washed and transferred to individual, steam
333 sterilized stable compartments. They were fed sterilized lucerne pellets and sterilized calf concentrate in order to clean t h e m f r o m Pilobolus spores.
Sterilization o f feed and faeces Feed for the four d o n o r calves was placed in polyvinylchloride (PVC) bags in portions o f 2--4 kg. The bags were flattened to a m a x i m u m height of 4 cm and exposed to ionizing irradiation on b o t h sides at a dose of 4 Mrad, which is k n o w n to be lethal to microorganisms (K. Sehested, 1979, personal communication). A total of 600 kg of feed was sterilized. Fifty kg of fresh bovine faeces were similarly sterilized.
Cu Ituring o f Pilo bolus sporangia Fifty grams of faeces f r o m parasite-free cattle were kept in non-transparent commercial plastic flower pots on wet mineral wool. Each pot was provided with a transparent plastic lid, kept at r o o m temperature n o t exeeding 25°C, and exposed to artificial illumination during day-time. Sporangia were harvested f r o m the lids and mixed with 0.5% liquid agar at 40°C. The mixtures were allowed to set to a semi-liquid consistency in syringes. The syringes were stored at 4°C until use. Two of the d o n o r calves were later orally administered these sporangia in agar in order to contaminate their faeces with Pilobolus spores.
Hots A two-year<~ld ungrazed rye grass pasture was cut one week before the c o m m e n c e m e n t o f the field experiment, leaving a rather coarse and open 15cm high pasture. Two square plots (Plots 1 and 2) of 750 m 2 were each fenced off, leaving a distance of 12 m between them.
Pilobolus contamination o f the faeces The faeces produced by the four donor calves were collected twice daily after the first week of infection, and the weight recorded. Each batch of faeces was checked for the presence of Pilobolus as described above, and for the presence, and concentration of D. viviparus larvae by the m e t h o d of Nevenic et al. (1962). After one week of feeding sterilized feed, the faeces of the four calves were free of Pilobolus spores. Three weeks after artificial infection, two of the donors (Pair 1) received 1000 Pilobolus sporangia per os daily while the remaining two calves (Pair 2) were not given the fungi. During this period, the daily faecal production f r o m each pair was stored separately overnight in closed PVC bags. During the following day, the a m o u n t of faeces and the total number o f D. viviparus larvae in the two portions were standardized by (1) transferring Pilobolus-free faeces with a relatively high concentration of larvae to the
334 other portions, (2) adding sterilized faeces and/or (3) disposal of surplus faeces. Thus the two portions of faeces were similar, except for the presence (Batch 1) or absence (Batch 2) of Pilobolus spores.
Deposition o f faeces on plots Placement on the plots of the faeces standardized as above t o o k place from 8--24 June, 1979. It was carried out manually by dropping portions of approximately 500 g f r o m a height of l m . On the 24th of June, a total of 104 dung pats or 72 kg o f faeces containing 2.2 million D. viviparus larvae had been deposited on Plot 1. Faeces of equal a m o u n t s from Pair 2 (Batch 2) were similarly deposited on Plot 2.
Herbage examination for infective D. viviparus larvae Herbage samples were collected daily from areas near the faecal pats (0--5 cm) as well as at a distance of 100 cm, and at a distance of more than 100 cm on both plots. The samples were processed separately and examined for D. viviparus larvae by t h e bile--agar technique described by J¢rgensen (1975). Infective Dictyocaulus larvae were identified using Iskander and J~brgensen (1980) criteria, and t h e n u m b e r of larvae were expressed per kg of fresh herbage.
Grazing by tracer calves Based on the larval counts obtained from Plot 1, it was decided to graze the two groups o f tracer calves on their respective plots for 3 days only. The calves were t u r n e d o u t on the 25th of June, 1979, and during the following 3 days, the calves were tethered individually. They were moved once or twice daily, and their behaviour was observed.
Stabling and post mortem worm counts of tracer calves The tracer calves were moved indoors on 29th June, 1979, and stabled conventionally for four weeks before slaughter. The faecal larval excretion was determined f r o m 19th July to 26th July. At post mortem, the lungs were perfused with water according to the m e t h o d of Inderbitzin (1976), and the bronchial tree was opened. Worms present in the washings and worms observed in t h e bronchial tree were collected and counted. RESULTS
Observations on Pilobolus fructification Observations carried out daily on the experimental plots during the afternoons revealed no Pilobolus fruitbodies on faeces deposited on Plot 2. On
335
Plot 1, fruitbodies were observed for the first time on the 20th of June, 1979, on faeces deposited 8, 10, and 12 days previously. During the following week, growth and fructification was observed to occur daily on Plot 1. Pasture larval counts The results o f the examination o f herbage samples for presence of infective D. viviparus larvae are shown in Table I. It appears that the larval concentration close to faecal pats was greatly reduced on Plot 2 (average 94.8%). At a distance o f 100 cm or more, the reduction reached 97% and 91.5%, respectively. TABLE I
Dictyocaulus viviparus larvae o n h e r b a g e f r o m p l o t s i n f e c t e d with, and free f r o m , Pilobolus spp. f u n g i
Distance P l o t
N u m b e r of l a r v a e / k g of fresh h e r b a g e
from faecal pats
June
(cm)
24
Total July
25
26
27
28
29
30
1
2
3 4
<5
1 2
423 9
489 28
217 20
192 12
1321 69
100
1 2
53 0
25 0
8 3
13 0
99 3
>100
1 2
2 0
0 0
8 0
29 0
9 0
6 0
5 5
0 0
0 0
0 0 0 0
59 5
Behaviour o f tracer calves During t h e first day on pasture, the calves were excited. The faeces previously deposited b y hand was trampled and therefore partly spread into the surrounding pasture. During the next t w o days, the calves started to graze, apparently at random. The a m o u n t o f herbage ingested appeared to be limited. Clinical and post mortem findings Coughing was n o t e d among 3 of the calves of Group 1 during the 3rd and 4th week o f stabling, whereas only one calf which had grazed the Pilobolusfree field coughed during the same period. The mean respiratory rates are shown in Fig. 1, from which a slight b u t consistent difference in respiratory rates appears. The difference was most pronounced during relatively warm weather conditions during the middle of July.
336
RESP./MIN.
/\
40
•
•
\
/
/
rl
0
'
• 4
~T
,
[] 6
,
i 8
.
, I0
o
. 12
.
\
G~lq
, 14
,
, 16
.
.
. 18
.
. 20
.
.
.
.
.
22
24
26
JULY
Fig. 1. Mean respiratory rates (resp./min) of the two groups of tracers and difference (Group 1-2) in mean respiratory rates between 2; = - - c , G r o u p 1 - - 2 .
t h e g r o u p s . • - - =, G r o u p 1; • - - - • , G r o u p
LARVAEIO / g 400
300
200
1
I00
0
18
20
22
24
26 JULY
Fig. 2. M e a n l a r v a l c o u n t s p e r 1 0 g o f f a e c e s ( l a r v a e / 1 0 g) o f t h e t w o t r a c e r g r o u p s . V e r t i c a l l i n e s i n d i c a t e r a n g e i n c o u n t s o f i n d i v i d u a l c a l v e s . • - - • , G r o u p 1; • - - - • , G r o u p 2.
Results o f faecal analysis for presence of D. viviparus larvae are shown in Fig. 2. It appears that both groups had patent infections, but that the number of larvae excreted by Group 2 was only approximately 10% of the larval excretion of Group 1.
337 During t h e period f r o m turning out until slaughter, Group 2 gained an average o f 26 kg compared with an average loss of 0.25 kg among the calves of Group 1. Post m o r t e m determinations of the number of worms in the tracer calves revealed seven times higher worm counts in Group I compared with Group 2. Individual worm counts are presented in Table II. TABLE II Numbers of worms recovered at necropsy from the tracer calves Calf no.
Group 1 1
Worm
counts
2
Group 2 3
4
146 77 102 343
Mean
167
5
6
7
26 11 4
8 58
25
DISCUSSION This experiment shows t h a t the pasture contamination with infective D. vivi. parus larvae as well as the infectivity of the pasture was greatly reduced in the absence o f Pilobolus fungi. It thus appears t h a t in the presence of Pilobolus fungi, the bulk o f infective larvae which were dispersed f r o m the faecal pats and which became available to grazing calves were Pilobolus translocated. Trichostrongyle species were n o t included in the present investigation. Whether Pilobolus spp. play a role in the translocation of such larvae under grazing conditions is u n k n o w n , although Bizzell and Ciordia (1965) observed Pilobolus translocation of Cooperia punctata and Trichostrongylus colubriformis larvae f r o m faecal cultures. In t h e present experiment, the lungworm larvae f o u n d in some pasture samples picked on t h e Pilobolus-free field as well as the infections acquired on that field could n o t have been dispersed by Pilobolus fungi since no fruit bodies were observed on the field despite careful daily observations of all deposited faecal pats. It is most likely t h a t the larval translocation on Plot 2 resulted f r o m mechanical spread of larval contaminated faecal material to the surrounding herbage by the bodies and hooves of the tethered and excited tracer calves. It is also likely t h a t the apparently non-selective grazing resulted in a relatively high intake of larvae spread in this manner. Therefore, the results o f the present experiment do not indicate that the spread of bovine dictyocaulosis is totally dependent on the presence and dispersal of Pilobolus sporangia. However, it is possible that a more pronounced effect of Pilobolus translocation on the infection parameters is to be expected under field conditions where calves are grazing more selectively by avoiding the grass growing near faecal pats.
338 ACKNOWLEDGEMENTS
The technical assistance of N. Midtgaard and K. Madsen is acknowledged. The authors' sincere thanks are due to K. Sehested, Atomic Experimental Station, RisO, for his guidance and help in connection with the use of the electron accelerator for sterilization purposes, and to Allen and Hanburys Research Laboratories, Ware, England, for providing infective D. viviparus larvae. They are also indebted to H. Dissing, Institute of Thallophytes, University of Copenhagen, for fruitful discussions on the botanical aspects. This study was supported b y a grant from the Carlsberg Research Foundation.
REFERENCES Bizzel, W.E. and Ciordia, H., 1965. Dissemination of infective larvae of trichostrongylid parasites of ruminants from faeces to pastures by the fungus Pilobolus spp. J. Parasitol., 51: 184. Buller, A.H.R., 1934. Researches on Fungi. Vol. 6, Longmans, London, 513 pp. Doncaster, C.C., 1981. Observations on relationships between infective juveniles of bovine lungworm, Dictyocaulus viviparus (Nematoda: Strongylida) and the fungi, Pilobolus kleinii and Pilobolus crystaUinus (Zygomycotina: Zygomycetes). Parasitology, 82: 421--428. Duncan, J., Armour, J., Bairden, K., Urquhart, G.M. and J~brgensen, R.J., 1979. Studies on the epidemiology of bovine parasitic bronchitis. Vet. Rec., 104: 274--278. Inderbitzin, F., 1976. Experimentale erzeugte Entwicklungshemmung yon Dicty ocaulus viviparus des Rindes. Thesis, Institut fiir Parasitologie der Universit~t ZUrich, 45 pp. Ingold, C.T., 1971. Fungal spores. Their liberation and dispersal. Clarendon Press, Oxford, 302 pp. Ingold, C.T., 1978. The biology of Mucor and its allies. The Institute of Biology's Studies in Biology No. 88, London, 59 pp. Iskander, A.R. and J~brgensen, R.J., 1980. Identification of infective Dictyocaulus viviparus larvae isolated from herbage by the bile--agar technique. Acta Vet. Scand., 21 : 330----335. J~brgensen, R.J., 1975. Isolation of infective Dictyocaulus larvae from herbage. Vet. Parasitol., 1 : 61--67. Jc,brgensen, R.J., 1980a. Dictyocaulus viviparus: migration in agar of larvae subjected to a variety o f physico~chemical exposures. Exp. Parasitol., 49: 106--115. J~brgensen, R.J., 1980b. Epidemiology of bovine dictyocaulosis in Denmark. Vet. Parasitol., 7: 153--167. J~brgensen, R.J., 1980c. Bovine dictyocaulosis. Pattern of infection and the prevention of parasitic bronchitis. Acta Vet. Scand., 21: 658--676. J~brgensen, R.J., 1981. Recent Danish studies on the epidemiology of bovine parasitic bronchitis. In: P. Nansen, R.J. J~brgensen, E.J.L. Soulsby (Editors), Epidemiology and Control of Nematodiasis in Cattle. Current Topics in Veterinary Medicine and Animal Science, Vol. 9, Nijhoff, The Hague, Boston, London, pp. 215--239. Michel, J.F. and Ollerenshaw, C.B., 1963. Helminth diseases of grazing animals. In: A.N. Warden, K.C. Sellers and D.E. Tribe (Editors), Animal Health, Production and Pasture. Longmans, London, 786 pp. Michel, J.F. and Rose, J.H., 1954. Some observations on the free-living stages of the cattle lungworm in relation to their normal environment. J. Comp. Pathol., 64: 195. Nevenic, V., Jovanovic, M., Sokolic, A., Cuperlovic, K. and Movsesijan, M., 1962. Jednostavan postupak za ustanovljenje larvica Dictyocaulus filaria (In: Croatic with English abstract). Vet. Glas., 16: 203--209.
339 Robinson, J., 1962a. Pilobolus spp. and the translation of the infective larvae of Dictyocaulus viviparus from faeces to pasture. Nature, 193: 353-I-354. Robinson, J., 1962b. The role o f the fungus Pilobolus in the spread of the infective larvae of Dic~yocaulus viviparus. Parasitology, 52: 17. Rose, J.H., 1956. The bionomics of the free-living larvae of Dictyocaulus viviparus. J. Comp. Pathol. Ther., 66: 228--240. Soliman, K.N., 1952. Observations on the survival on pasture of preparasitic stages of Dictyocaulus viviparus in Southern England. II. Br. Vet. J., 108: 204--213. Taylor, E.L., 1951. Parasitic bronchitis in cattle. Vet. Rec., 6 3 : 8 5 9 873.