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Spread of lungworm (Dictyocaulus viviparus) infection by Pilobolus fungi among stabled calves
Preventive Veterinary Medicine, 5 (1987) 43-50
43
Elsevier Science Publishers B.V., AmsterdAm - - Printed in The Netherlands
Spread of Lungworm (Dictyocaulus viviparus) Infection by Pilobolus Fungi Among Stabled Calves J O R N G R ~ N V O L D iand R O L F JESS J ~ R G E N S E N s
~Institute o[ Hygiene and Microbiology and 2Institute of Internal Medicine, Royal Veterinary and Agricultural University, 13, Biilowsvej, DK-1870 Frederiksberg C (Denmark)
(Accepted for publication 10 March 1987)
ABSTRACT
Gr~nvold, J. and Jergensen, R.J., 1987. Spread of lungworm (Dictyocaulus viviparus) infection by Pilobolus fungi among stabled calves. Prey. Vet. Med., 5: 43-50.
Pilobolus kleinii spores isolated from cattle faeces were administered daily by the oral route to two donor calves excreting D&tyocaulus viviparus larvae in their faeces. Faeces were collected at daily intervals from the donor calves, and on each day a pooled sample of 500 g was placed outside a pen containing two parasite-free experimental calves. A wire screen prevented the experimental calves from coming into contact with the artificially deposited faecal portions. Pilobolus kleinii fruit bodies were observed on the faecal surface in large numbers 5-10 days after deposition. Sporangial discharge was observed to be directed against a window which served as the only light source. Sporangia were found up to 110 cm away from the faeces, and it was estimated that at least 29% of them had been transmitted to a feeding trough placed close to the wire screen. Infective D. viviparus larvae were observed on some of the discharged sporangia. The experimental calves started excreting low numbers of D. viviparus larvae in their faeces 40-49 days after the first portions were deposited. These results indicate that P. kleinii was reponsible for the infection of the experimental calves with D. viviparus, through the discharge of sporangia carrying infective lungworm larvae.
INTRODUCTION
The cattle lungworm Dictyocaulus viviparus causes clinical problems among grazing young susceptible calves. According to Jorgensen (1981), who reviewed the subject, there are few reports describing indoor-acquired infections. Enigk and Dfiwel (1962) working in the Federal Republic of Germany examined the prevalence of indoor-acquired infections. They found that 13% out of 398 2-4-month-old calves excreted low numbers of larvae in their faeces at the 0167-5877/87/$03.50
© 1987 Elsevier Science Publishers B.V.
44 time of turning out. The authors believed that the source of infection was contaminated straw. On a Rumanian farm, 36 calves acquired serious parasitic bronchitis during the housing period ( Simionescu et al., 1972 ). It was suggested that the source of infection was feeding and drinking troughs contaminated by older cattle which were carriers. On a Danish farm, six permanently stabled bull calves, which had never been fed fresh grass, died due to acute respiratory disease during the indoor period from November 1983 to January 1984. Infectious bovine rhinotracheitis (IBR) virus was believed to be the main cause of the outbreak, but mature specimens of D. viviparus were found in the lungs of four of the calves (Jorgensen et al., 1985 ). Faecal analyses performed in December 1983 revealed two larval excreters among 25 previously grazed heifers stabled in nine slatted-floor box units, and four larval excreters among bull calves. These four calves were distributed in four out of 21 slatted-floor boxes containing a total of 120 bull calves. Airborne transmission of infective larvae on the sporangia of Pilobolus fungi was suggested since Pilobolus fungi are known to play an important role in the translation of infective D. viviparus larvae on pastures (Jorgensen et al., 1982). The present investigation was designed to examine whether Pilobolus fungi can transmit infective D. viviparus larvae to indoor-reared susceptible calves. MATERIALSAND METHODS
Donor calves Two Jersey calves, 3-4 months old, were artificially infected orally with 4000 and 6000 D. viviparus 3rd stage larvae, respectively. Later, when the calves excreted D. viviparus larvae in their faeces, they were orally given 1.5 million spores of P. kleinii, daily, The spores of P. kleinii were isolated from cattle dung.
Experimental calves Two indoor-reared 5-month-old Jersey calves (Calf 10 and Calf 46) were used. Weekly faecal samples taken over a period of 3 months before the start of the experiment were consistently negative for D. viviparus larvae.
Experimental stable The experimental calves were placed in a pen (Fig. 1 ) which was part of an isolated room free of insects and rats. A 60-cm-high table was placed just outside the pen level with the feeding trough. A 60-cm-high screen was placed
45 A
ooo|
coo.j
I
°°°l'° oool' B ooo;,
l
l
/ | o! lm
Fig. 1. Diagram showing the experimental setup. The two experimental calves were placed in the pen (A). Outside the stable on a table (B), faecal portions ( C ) containingDictyocaulus viviparus larvae and Pilobolus fungi were placed level with the feeding trough (D). Light came through a window (E) at a height of 2 m. Discharged P. kleinii sporangia were collected on a horizontal piece of white paper (F). Between the faecal portions and the feeding trough, small Petri dishes ( G ) coated with a layer of 3% agar were placed vertically at a height of 5 cm.
between the table and the feeding trough to prevent the calves from getting in touch with portions of faeces manually deposited on the table.
Experiment Faeces containing D. viviparus larvae and viable P. kleinii spores were collected from the donor calves. Portions of approximately 500 g of faeces were put into plastic trays ( diameter, 15 cm; height, 3 cm) and placed on the table (Fig. 1 ). Placement of faecal portions started on 20 February 1985. In the first 22 days, 16 portions of faeces from one donor calf were placed as described above. During the period 29-36 days after the start of the experiment, 13 portions of faeces from the other donor calf were deposited as above (Table I). The faeces were placed in three rows with a maximum distance of 45 cm from the feeding trough. Fructification of P. kleinii ceased after periods of 12-23 days, after which time the faeces portions were removed from the stable.
Concentration of Dictyocaulus viviparus larvae in faeces At intervals, fresh faeces were collected from the recta of donor and experimental calves, and the presence and concentration ofD. viviparus larvae in 10g samples was determined by a modified Baermann method (Jorgensen and Madsen, 1982 ).
46 TABLE I N u m b e r of faecal portions of 500 g a n d t h e c o n c e n t r a t i o n of Dictyocaulus viviparus larvae in t h e faeces placed in front of t h e feeding t r o u g h of t h e experimental calves w i t h i n a distance of 45 cm No. of days after t h e s t a r t of t h e e x p e r i m e n t
No. of faecal portions deposited No. of larvae in 10 g of faeces (double B a e r m a n n i z a t i o n )
0
1
2
5
6
7
8
9
13
14
15
1
1
1
1
1
1
1
1
1
1
1
0 0
0 3
3 4
0 1
0 0
1 2
0 4
---
0 0
---
0 0
No. of days after t h e s t a r t of t h e e x p e r i m e n t
No. of faecal portions deposited No. of larvae in 10 g of faeces (double Baermannization )
16
19
20
21
22
29
30
1
1
1
1
0 0
---
31
32
33
34
35
36
1
1
1
1
1
3
2
2
2
0 0
14 37
42 79
90 109
106 135
200 229
226 276
164 169
197 206
Distribution of Pilobolus kleinii sporangia in the stable A single 500-g portion of faeces was placed on the table. During the following week, fruit bodies of P. kleinii were observed on the faeces, and a piece of white paper ( 20 × 150 cm) was placed horizontally in front of the faeces in the direction of the window (Fig. 1). The next day the paper was examined for the presence of discharged sporangia sticking to the paper.
Qualitative observations of airborne Dictyocaulus viviparus larvae Small Petri dishes (diameter 5.5 cm) containing a bottom layer of 3% agar were placed vertically at a height of 5 cm between the portions of faeces and the feeding trough (Fig. 1 ) with the agar layer facing the faeces. Sporangia of P. kleinii were collected on the agar, facing the faeces portions, in the period Day 37-Day 42. The Petri dishes were examined daily for the presence of D. viviparus larvae.
Temperature, relative humidity and light intensity in the stable Recordings of temperature and relative humidity were obtained from a termohygrograph placed on the table (Fig. 1). At noon the light intensity was registered by a luxmeter.
47
m
12. 10. I--
6'
4.
2
10 20 30 40 50 60 70 80 90 100 110 Distance (cm)
Fig. 2. The horizontal distribution of discharged Pilobolus kleinii sporangia. RESULTS
Temperature, relative humidity and light intensity in the stable The mean temperature was 11.3 ° C, with a minimum and maximum of 8 and 15 ° C, respectively. Fluctuations in the relative humidity were between 65 and 100%, with a mean value of 85.9%. Peak light intensities at noon were within the range 250-350 Lux.
Observations on Pilobolus kleinii fructification and sporangial discharge Large numbers ofP. kleinii fruit bodies (5-10 fruit bodies/cm 2) were observed after 5-10 days on the surface of all the faecal portions. The fruitbodies pointed directly at the window in the other end of the stable with an angle of 20 °-30 ° from the horizontal plane. The horizontal distribution of discharged sporangia from a single portion of faeces in the direction of the window is shown in Fig. 2. Approximately 29% of the sporangia were transmitted to distances of between 45 and 95 cm. As the faecal portions were placed 0-45 cm from the feeding trough, the results in Fig. 2 indicate that at least 29% of the discharged sporangia were transmitted to the feeding trough ( 50 cm in breadth).
48 TABLE II Concentration of Dictyocaulus viviparus larvae in portions of rectal faecal samples collected from the experimental calves at intervals. Each time, 2 or 4 samples of 10 g of faeces were examined Calf No.
No. of days after the s t a r t of the experiment 40
41
49
54
57
61
64
68
70
76
79
82
89
10
0 0
0 0
1 0
0 0 0 0
0 0 0 0
0 0 0 0
1 0 0 0
0 0 0 0
0 0 0 0
0 0
0 0 0 0
0 0 0 0
0 0 0 0
46
7 6
4 3
0 0
0 0 0 0
0 0 0 0
0 0 0 0
1 0 0 0
2 1 1 0
1 0 0 0
3 2
1 1 1 0
1 0 0 0
0 0 0 0
98
103
110
117
4 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
Qualitative observations of airborne Dictyocaulus viviparus larvae Small numbers ofD. uiviparus larvae appeared in the Petri dishes during the period Day 37-Day 42, indicating that the larvae were transmitted together with sporangia of P. kleinii caught on the agar surface during the same period. This result also indicates that some D. viviparus larvae may have been transmitted to the feeding trough on Pilobolus sporangia.
Findings of Dictyocaulus viviparus in faeces of the experimental calves The results of the faecal analysis are shown in Table II. On Day 49 and Day 64, Calf 10 was positive at a very low level. Calf 46 excreted D. viviparus larvae in the faeces at a higher level on Day 40 and Day 41 and in the period Day 64-Day 98. No significant elevation in the respiratory rates appeared but coughing was heard occasionally from both calves during the experiment. DISCUSSION
The present results indicate that P. kleinii can grow and discharge its sporangia in the indoor environment at a mean temperature of 11.3 °C and a mean relative humidity of 85.9%. The fruit bodies of P. kleinii grew directly against the source of light, which was a window in the opposite end of the stable, admitting 250-350 Lux at noon. With an angle of 20-30 ° from the horizontal plane, the fruit bodies discharged their sporangia up to a distance of 110 cm horizontally. Boon et al. (1983} found that sporangia of P. kleinii discharged at an angle of 45 ° had a horizontal distribution in accordance with our results. They also found that sporangia harbouring infective D. viviparus larvae were deposited approximately 20 cm further away than sporangia free of larvae.
49 In our experiment, the environmental conditions were obviously favourable for the transmission of sporangia to the fodder, and it was estimated that at least 29% of the sporangia were transmitted to the feeding trough. In addition, it appeared that the conditions were also favourable for the translation of infective D. viviparus larvae. Our findings therefore offer an alternative explanation to the mode of infection in previous reports (e.g. Enigk and Diiwel, 1962; Simionescu et al., 1972) on indoor-acquired lungworm infections. We were not able to establish an experiment with control calves, as it is very difficult to get hold of cattle faeces free of Pilobolus fungi. But the demonstration of D. viviparus larvae transmitted together with sporangia of P. kleinii strongly indicates that the source of infection among the experimental calves were airborne D. viviparus larvae spread by Pilobolus fungi. Faecal analysis of the experimental calves confirmed that translation of D. viviparus had taken place because they excreted larvae in two waves. The first wave started 40 and 49 days after placement of the first 16 faeces portions. The second wave started 35 days after placement of the last 13 faecal portions containing the highest concentration of D. viviparus larvae. As the fructification of P. kleinii commenced 5-10 days after placement of the faecal portions, the infection of the experimental calves could have taken place as early as 25-39 days before they began excreting D. viviparus larvae, which is in accordance with a prepatence period of 3-4 weeks. However, Pilobolus translocation of infective D. viviparus larvae is only possible when infected faeces are left approximately 1 week in the stable, allowing the development of both the fungi and the parasite larvae. Moreover, the growth of P. kleinii is inhibited by admixture of more than 10% of cattle urine, so translation of infective D. viviparus larvae by Pilobolus fungi is only possible in parts of the stable where the admixture of urine is minimal (Gronvold and Jorgensen, 1987). In an indoor experiment, Boon et al. (1984) showed that continuous exposure of calves to infective D. viviparus larvae resulted in prolonged larval excretion over a period of at least 10 weeks, and the worm burden increased linearly with the level of infection. These results support the view that, under favourable indoor conditions, Pilobolus transmission of infective D. viviparus larvae from faeces to fodder might contribute to the overwintering of bovine lungworm infection. ACKNOWLEDGEMENTS We wish to express our thanks to A. Korner and N. Midtgaard for their technical assistance. We are also grateful to Dr. H.J. Bos, Intervet International B.V., Boxmeer, Holland, for providing the infective Dictyocaulus viviparus larvae. This study was supported by the Danish Agricultural and Veterinary
5O
Research Council and the Danish Natural Science Research Council, Grant No. 13-3463.
REFERENCES Boon, J.H., Kloosterman, A. and Breukink, M., 1984. Parasitological, serological and clinical effects of continuous graded levels of Dictyocaulus viviparus inoculations in calves. Vet. Parasitol., 16: 261-272. Boon, J.H., Cremers, H.W.J.M., Hendriks, J. and van Vliet, G., 1983. Longworminfecties bij rundvee, sen blijvend probleem? Tijdschr. Diergeneeskd., 108: 435-438. Enigk, K. and Diiwel, D., 1962. Beitrag zur Epizootologie der Dictyocaulose des Rindes. Dtsch. Tier~irztl. Wochenschr., 69: 72-78. Gronvold, J. and Jorgensen, R.J., 1987. The occurrence of Pilobolus fungi in Danish stables. Prev. Vet. Med., 5: 37-41. Jorgensen, R.J., 1981. Studies on the lungworm Dictyocaulus viviparus (Bloch, 1782) and its epidemiology in young cattle with a description of an attempt to prevent parasitic bronchitis. Ph.D. Thesis, Copenhagen. Acta Vet. Scand. Suppl., 76: 1-77. Jorgensen, R.J. and Madsen, K., 1982. Undersogelse af faecesprover for lungeormlarver. Dansk Vet. Tidsskr., 65: 517-518. Jorgensen, R.J., Bisgaard Madsen, E., Gronvold, J. and Heje, N.-I., 1985. Akut respirationsvejslidelse blandt permanent opstaldede ungtyre. Dansk Vet. Tidsskr., 68: 358-363. Jorgensen, R.J., Ronne, H., Heisted, C. and Iskander, A.R., 1982. Spread of infective Dictyocaulus v iviparus larvae in pasture and to grazing cattle: experimental evidence of the role of Pilobolus fungi. Vet. Parsitol., 10: 331-339. Simionescu, E., Orbulescu, D., Boloanta, T. and Talos, V., 1972. Consideratii asupra strongilozei pulmonare la bovine, evoluta in stabulatie. Ser. Med. Vet., 13: 181-188.
43
Elsevier Science Publishers B.V., AmsterdAm - - Printed in The Netherlands
Spread of Lungworm (Dictyocaulus viviparus) Infection by Pilobolus Fungi Among Stabled Calves J O R N G R ~ N V O L D iand R O L F JESS J ~ R G E N S E N s
~Institute o[ Hygiene and Microbiology and 2Institute of Internal Medicine, Royal Veterinary and Agricultural University, 13, Biilowsvej, DK-1870 Frederiksberg C (Denmark)
(Accepted for publication 10 March 1987)
ABSTRACT
Gr~nvold, J. and Jergensen, R.J., 1987. Spread of lungworm (Dictyocaulus viviparus) infection by Pilobolus fungi among stabled calves. Prey. Vet. Med., 5: 43-50.
Pilobolus kleinii spores isolated from cattle faeces were administered daily by the oral route to two donor calves excreting D&tyocaulus viviparus larvae in their faeces. Faeces were collected at daily intervals from the donor calves, and on each day a pooled sample of 500 g was placed outside a pen containing two parasite-free experimental calves. A wire screen prevented the experimental calves from coming into contact with the artificially deposited faecal portions. Pilobolus kleinii fruit bodies were observed on the faecal surface in large numbers 5-10 days after deposition. Sporangial discharge was observed to be directed against a window which served as the only light source. Sporangia were found up to 110 cm away from the faeces, and it was estimated that at least 29% of them had been transmitted to a feeding trough placed close to the wire screen. Infective D. viviparus larvae were observed on some of the discharged sporangia. The experimental calves started excreting low numbers of D. viviparus larvae in their faeces 40-49 days after the first portions were deposited. These results indicate that P. kleinii was reponsible for the infection of the experimental calves with D. viviparus, through the discharge of sporangia carrying infective lungworm larvae.
INTRODUCTION
The cattle lungworm Dictyocaulus viviparus causes clinical problems among grazing young susceptible calves. According to Jorgensen (1981), who reviewed the subject, there are few reports describing indoor-acquired infections. Enigk and Dfiwel (1962) working in the Federal Republic of Germany examined the prevalence of indoor-acquired infections. They found that 13% out of 398 2-4-month-old calves excreted low numbers of larvae in their faeces at the 0167-5877/87/$03.50
© 1987 Elsevier Science Publishers B.V.
44 time of turning out. The authors believed that the source of infection was contaminated straw. On a Rumanian farm, 36 calves acquired serious parasitic bronchitis during the housing period ( Simionescu et al., 1972 ). It was suggested that the source of infection was feeding and drinking troughs contaminated by older cattle which were carriers. On a Danish farm, six permanently stabled bull calves, which had never been fed fresh grass, died due to acute respiratory disease during the indoor period from November 1983 to January 1984. Infectious bovine rhinotracheitis (IBR) virus was believed to be the main cause of the outbreak, but mature specimens of D. viviparus were found in the lungs of four of the calves (Jorgensen et al., 1985 ). Faecal analyses performed in December 1983 revealed two larval excreters among 25 previously grazed heifers stabled in nine slatted-floor box units, and four larval excreters among bull calves. These four calves were distributed in four out of 21 slatted-floor boxes containing a total of 120 bull calves. Airborne transmission of infective larvae on the sporangia of Pilobolus fungi was suggested since Pilobolus fungi are known to play an important role in the translation of infective D. viviparus larvae on pastures (Jorgensen et al., 1982). The present investigation was designed to examine whether Pilobolus fungi can transmit infective D. viviparus larvae to indoor-reared susceptible calves. MATERIALSAND METHODS
Donor calves Two Jersey calves, 3-4 months old, were artificially infected orally with 4000 and 6000 D. viviparus 3rd stage larvae, respectively. Later, when the calves excreted D. viviparus larvae in their faeces, they were orally given 1.5 million spores of P. kleinii, daily, The spores of P. kleinii were isolated from cattle dung.
Experimental calves Two indoor-reared 5-month-old Jersey calves (Calf 10 and Calf 46) were used. Weekly faecal samples taken over a period of 3 months before the start of the experiment were consistently negative for D. viviparus larvae.
Experimental stable The experimental calves were placed in a pen (Fig. 1 ) which was part of an isolated room free of insects and rats. A 60-cm-high table was placed just outside the pen level with the feeding trough. A 60-cm-high screen was placed
45 A
ooo|
coo.j
I
°°°l'° oool' B ooo;,
l
l
/ | o! lm
Fig. 1. Diagram showing the experimental setup. The two experimental calves were placed in the pen (A). Outside the stable on a table (B), faecal portions ( C ) containingDictyocaulus viviparus larvae and Pilobolus fungi were placed level with the feeding trough (D). Light came through a window (E) at a height of 2 m. Discharged P. kleinii sporangia were collected on a horizontal piece of white paper (F). Between the faecal portions and the feeding trough, small Petri dishes ( G ) coated with a layer of 3% agar were placed vertically at a height of 5 cm.
between the table and the feeding trough to prevent the calves from getting in touch with portions of faeces manually deposited on the table.
Experiment Faeces containing D. viviparus larvae and viable P. kleinii spores were collected from the donor calves. Portions of approximately 500 g of faeces were put into plastic trays ( diameter, 15 cm; height, 3 cm) and placed on the table (Fig. 1 ). Placement of faecal portions started on 20 February 1985. In the first 22 days, 16 portions of faeces from one donor calf were placed as described above. During the period 29-36 days after the start of the experiment, 13 portions of faeces from the other donor calf were deposited as above (Table I). The faeces were placed in three rows with a maximum distance of 45 cm from the feeding trough. Fructification of P. kleinii ceased after periods of 12-23 days, after which time the faeces portions were removed from the stable.
Concentration of Dictyocaulus viviparus larvae in faeces At intervals, fresh faeces were collected from the recta of donor and experimental calves, and the presence and concentration ofD. viviparus larvae in 10g samples was determined by a modified Baermann method (Jorgensen and Madsen, 1982 ).
46 TABLE I N u m b e r of faecal portions of 500 g a n d t h e c o n c e n t r a t i o n of Dictyocaulus viviparus larvae in t h e faeces placed in front of t h e feeding t r o u g h of t h e experimental calves w i t h i n a distance of 45 cm No. of days after t h e s t a r t of t h e e x p e r i m e n t
No. of faecal portions deposited No. of larvae in 10 g of faeces (double B a e r m a n n i z a t i o n )
0
1
2
5
6
7
8
9
13
14
15
1
1
1
1
1
1
1
1
1
1
1
0 0
0 3
3 4
0 1
0 0
1 2
0 4
---
0 0
---
0 0
No. of days after t h e s t a r t of t h e e x p e r i m e n t
No. of faecal portions deposited No. of larvae in 10 g of faeces (double Baermannization )
16
19
20
21
22
29
30
1
1
1
1
0 0
---
31
32
33
34
35
36
1
1
1
1
1
3
2
2
2
0 0
14 37
42 79
90 109
106 135
200 229
226 276
164 169
197 206
Distribution of Pilobolus kleinii sporangia in the stable A single 500-g portion of faeces was placed on the table. During the following week, fruit bodies of P. kleinii were observed on the faeces, and a piece of white paper ( 20 × 150 cm) was placed horizontally in front of the faeces in the direction of the window (Fig. 1). The next day the paper was examined for the presence of discharged sporangia sticking to the paper.
Qualitative observations of airborne Dictyocaulus viviparus larvae Small Petri dishes (diameter 5.5 cm) containing a bottom layer of 3% agar were placed vertically at a height of 5 cm between the portions of faeces and the feeding trough (Fig. 1 ) with the agar layer facing the faeces. Sporangia of P. kleinii were collected on the agar, facing the faeces portions, in the period Day 37-Day 42. The Petri dishes were examined daily for the presence of D. viviparus larvae.
Temperature, relative humidity and light intensity in the stable Recordings of temperature and relative humidity were obtained from a termohygrograph placed on the table (Fig. 1). At noon the light intensity was registered by a luxmeter.
47
m
12. 10. I--
6'
4.
2
10 20 30 40 50 60 70 80 90 100 110 Distance (cm)
Fig. 2. The horizontal distribution of discharged Pilobolus kleinii sporangia. RESULTS
Temperature, relative humidity and light intensity in the stable The mean temperature was 11.3 ° C, with a minimum and maximum of 8 and 15 ° C, respectively. Fluctuations in the relative humidity were between 65 and 100%, with a mean value of 85.9%. Peak light intensities at noon were within the range 250-350 Lux.
Observations on Pilobolus kleinii fructification and sporangial discharge Large numbers ofP. kleinii fruit bodies (5-10 fruit bodies/cm 2) were observed after 5-10 days on the surface of all the faecal portions. The fruitbodies pointed directly at the window in the other end of the stable with an angle of 20 °-30 ° from the horizontal plane. The horizontal distribution of discharged sporangia from a single portion of faeces in the direction of the window is shown in Fig. 2. Approximately 29% of the sporangia were transmitted to distances of between 45 and 95 cm. As the faecal portions were placed 0-45 cm from the feeding trough, the results in Fig. 2 indicate that at least 29% of the discharged sporangia were transmitted to the feeding trough ( 50 cm in breadth).
48 TABLE II Concentration of Dictyocaulus viviparus larvae in portions of rectal faecal samples collected from the experimental calves at intervals. Each time, 2 or 4 samples of 10 g of faeces were examined Calf No.
No. of days after the s t a r t of the experiment 40
41
49
54
57
61
64
68
70
76
79
82
89
10
0 0
0 0
1 0
0 0 0 0
0 0 0 0
0 0 0 0
1 0 0 0
0 0 0 0
0 0 0 0
0 0
0 0 0 0
0 0 0 0
0 0 0 0
46
7 6
4 3
0 0
0 0 0 0
0 0 0 0
0 0 0 0
1 0 0 0
2 1 1 0
1 0 0 0
3 2
1 1 1 0
1 0 0 0
0 0 0 0
98
103
110
117
4 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
Qualitative observations of airborne Dictyocaulus viviparus larvae Small numbers ofD. uiviparus larvae appeared in the Petri dishes during the period Day 37-Day 42, indicating that the larvae were transmitted together with sporangia of P. kleinii caught on the agar surface during the same period. This result also indicates that some D. viviparus larvae may have been transmitted to the feeding trough on Pilobolus sporangia.
Findings of Dictyocaulus viviparus in faeces of the experimental calves The results of the faecal analysis are shown in Table II. On Day 49 and Day 64, Calf 10 was positive at a very low level. Calf 46 excreted D. viviparus larvae in the faeces at a higher level on Day 40 and Day 41 and in the period Day 64-Day 98. No significant elevation in the respiratory rates appeared but coughing was heard occasionally from both calves during the experiment. DISCUSSION
The present results indicate that P. kleinii can grow and discharge its sporangia in the indoor environment at a mean temperature of 11.3 °C and a mean relative humidity of 85.9%. The fruit bodies of P. kleinii grew directly against the source of light, which was a window in the opposite end of the stable, admitting 250-350 Lux at noon. With an angle of 20-30 ° from the horizontal plane, the fruit bodies discharged their sporangia up to a distance of 110 cm horizontally. Boon et al. (1983} found that sporangia of P. kleinii discharged at an angle of 45 ° had a horizontal distribution in accordance with our results. They also found that sporangia harbouring infective D. viviparus larvae were deposited approximately 20 cm further away than sporangia free of larvae.
49 In our experiment, the environmental conditions were obviously favourable for the transmission of sporangia to the fodder, and it was estimated that at least 29% of the sporangia were transmitted to the feeding trough. In addition, it appeared that the conditions were also favourable for the translation of infective D. viviparus larvae. Our findings therefore offer an alternative explanation to the mode of infection in previous reports (e.g. Enigk and Diiwel, 1962; Simionescu et al., 1972) on indoor-acquired lungworm infections. We were not able to establish an experiment with control calves, as it is very difficult to get hold of cattle faeces free of Pilobolus fungi. But the demonstration of D. viviparus larvae transmitted together with sporangia of P. kleinii strongly indicates that the source of infection among the experimental calves were airborne D. viviparus larvae spread by Pilobolus fungi. Faecal analysis of the experimental calves confirmed that translation of D. viviparus had taken place because they excreted larvae in two waves. The first wave started 40 and 49 days after placement of the first 16 faeces portions. The second wave started 35 days after placement of the last 13 faecal portions containing the highest concentration of D. viviparus larvae. As the fructification of P. kleinii commenced 5-10 days after placement of the faecal portions, the infection of the experimental calves could have taken place as early as 25-39 days before they began excreting D. viviparus larvae, which is in accordance with a prepatence period of 3-4 weeks. However, Pilobolus translocation of infective D. viviparus larvae is only possible when infected faeces are left approximately 1 week in the stable, allowing the development of both the fungi and the parasite larvae. Moreover, the growth of P. kleinii is inhibited by admixture of more than 10% of cattle urine, so translation of infective D. viviparus larvae by Pilobolus fungi is only possible in parts of the stable where the admixture of urine is minimal (Gronvold and Jorgensen, 1987). In an indoor experiment, Boon et al. (1984) showed that continuous exposure of calves to infective D. viviparus larvae resulted in prolonged larval excretion over a period of at least 10 weeks, and the worm burden increased linearly with the level of infection. These results support the view that, under favourable indoor conditions, Pilobolus transmission of infective D. viviparus larvae from faeces to fodder might contribute to the overwintering of bovine lungworm infection. ACKNOWLEDGEMENTS We wish to express our thanks to A. Korner and N. Midtgaard for their technical assistance. We are also grateful to Dr. H.J. Bos, Intervet International B.V., Boxmeer, Holland, for providing the infective Dictyocaulus viviparus larvae. This study was supported by the Danish Agricultural and Veterinary
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Research Council and the Danish Natural Science Research Council, Grant No. 13-3463.
REFERENCES Boon, J.H., Kloosterman, A. and Breukink, M., 1984. Parasitological, serological and clinical effects of continuous graded levels of Dictyocaulus viviparus inoculations in calves. Vet. Parasitol., 16: 261-272. Boon, J.H., Cremers, H.W.J.M., Hendriks, J. and van Vliet, G., 1983. Longworminfecties bij rundvee, sen blijvend probleem? Tijdschr. Diergeneeskd., 108: 435-438. Enigk, K. and Diiwel, D., 1962. Beitrag zur Epizootologie der Dictyocaulose des Rindes. Dtsch. Tier~irztl. Wochenschr., 69: 72-78. Gronvold, J. and Jorgensen, R.J., 1987. The occurrence of Pilobolus fungi in Danish stables. Prev. Vet. Med., 5: 37-41. Jorgensen, R.J., 1981. Studies on the lungworm Dictyocaulus viviparus (Bloch, 1782) and its epidemiology in young cattle with a description of an attempt to prevent parasitic bronchitis. Ph.D. Thesis, Copenhagen. Acta Vet. Scand. Suppl., 76: 1-77. Jorgensen, R.J. and Madsen, K., 1982. Undersogelse af faecesprover for lungeormlarver. Dansk Vet. Tidsskr., 65: 517-518. Jorgensen, R.J., Bisgaard Madsen, E., Gronvold, J. and Heje, N.-I., 1985. Akut respirationsvejslidelse blandt permanent opstaldede ungtyre. Dansk Vet. Tidsskr., 68: 358-363. Jorgensen, R.J., Ronne, H., Heisted, C. and Iskander, A.R., 1982. Spread of infective Dictyocaulus v iviparus larvae in pasture and to grazing cattle: experimental evidence of the role of Pilobolus fungi. Vet. Parsitol., 10: 331-339. Simionescu, E., Orbulescu, D., Boloanta, T. and Talos, V., 1972. Consideratii asupra strongilozei pulmonare la bovine, evoluta in stabulatie. Ser. Med. Vet., 13: 181-188.