Research in Veterinary Science 1991, 51, 344-346
Ostertagia ostertagi in neonatal calves: establishment of infection in ruminating and non-ruminating calves F. S A T R I J A , P. N A N S E N , C. M. C H R I S T E N S E N , Department of Veterinary Microbiology, The Royal Veterinary
and Agricultural University, 13 Bulowsvej, DK-1870 Frederiksberg C, Denmark
An experiment was carried out to study the role of the ruminal function in the establishment of Ostertagia ostertagi in neonatal calves. Three groups of calves were fed either milk only (groups A and C), or hay and concentrate in addition to milk (group B) from birth. At the time of infection, ruminal function was negligible in groups A and C, whereas it was well developed in group B. Calves of groups A and B were each given 25,000 normal ensheathed infective larvae of O ostertagi and those of group C were given 25,000 infective larvae exsheathed in vitro. Daily faecal egg output and post mortem worm counts 28 days after infection were higher in calves with well developed ruminal function than those having only negligible ruminal function. In the latter group, exsheathed larvae established at a lower rate than did ensheathed larvae. The results suggest that the degree of development of the ruminal function influences the establishment of O ostertagi. G E N E R A L L Y , calveS are more susceptible to Ostertagia ostertagi t h a n are heifers or cows (Michel et al 1979, Nansen et al 1990). However, neonatal calves which are milkfed seem more or less refractory to establishment of infection (Rohrbacher et al 1958; P. Steffan and P. Nansen, unpublished data). This p h e n o m e n o n indicates a possible role of either the diet or o f ruminal function in the establishment of parasites in neonatal calves. The present experiment was designed to study the role of ruminal function in the establishment of O ostertagi by comparing the course of infection in neonatal ruminating and non-ruminating calves. Thirteen neonatal Jersey bull calves, reared under conditions designed to exclude worm infection, were used. They were placed individually in pens with slatted or concrete floors. The calves were allocated to groups Of fiv e (group A) or four calves (groups B and C). Calves of groups A and C were fed only milk replacer (Kalvital) from birth in an attempt to prevent the development of the rumen, while calves in group B were fed milk replacer and, in addition, hay and concentrate ad libitum, allowing the development of ruminal function (Godfrey 1961, Puri et al 1983). After approximately seven weeks, the calves were given O ostertagi larvae per os. Calves of groups A and B each received 25,000 normal infective larvae and those of group C each received 25,000 exsheathed infective larvae. Exsheathment of the larvae was performed in vitro using a 0 . 2 per cent sodium hypochlorite solution according to a method described by Coles et al (1980). In order to test the infectivity o f these exsheathed larvae in a ruminating animal, an eightweek-old Jersey bull calf, fed with hay and concentrate, was
infected with 25,000 exsheathed larvae of the same batch. At post m o r t e m examination, 14-4 per cent exsheathed larvae had become established in the test calf. Every second day from day 14 to day 28 after infection, the total quantity o f faeces excreted per 24 hours was collected. The n u m b e r o f trichostrongyle eggs per gram of faeces (epg) was determined using a modified McMaster procedure and the total daily faecal egg output calculated. Ruminal function was evaluated around the time of infection by plasma ~-hydl'oxybutyrate and glucose assay (Purl et al 1983) and by recording ruminal movements. ~3hydroxybutyrate concentrations were determined according to the method described by McMurray et al (1984) while plasma glucose levels were measured using the Gluc-DH method (Merck System 14053). The calves were slaughtered 28 days after infection, reticuloruminal weight was estimated and morphological development and abomasal lesions given scores from + to + + + + . Post m o r t e m worm recoveries were made according to the method described by GrCnvold et al (1989). At the time of infection, three calves of group B showed a significant development of ruminal function in contrast to the calves of groups A and C. This was evidenced by the tendency to higher plasma B-hydroxybutyrate levels (group B, 0.17 ± 0"06 m m o l litre -1 versus group A, 0.10 4- 0.00 m m o l litre-1; group C, 0"10 ± 0.00 m m o l litre -1) and lower plasma glucose levels (group B, 5.66 ± 1.40 m m o l litre - l versus group A, 8" 27 ± 1.60 m m o l litre- 1; group C, 7.48 ± 0-99 m m o l litre-l). One calf in group B was excluded from the experiment because of a lack o f ruminal function development. The ruminating calves o f group B showed significant m o v e m e n t s of their forestomachs around the start of the infection, and had m u c h higher weights and greater morphological development of their reticulorumens than had calves of the other groups (Table 1). Calves of group B had significantly higher daily faecal egg outputs than those of groups A and C ( P < 0 " 0 5 to P < 0 . 0 0 1 ) . Groups A and C did not differ significantly in this respect. At post m o r t e m examination, the abomasal mucosae of the animals showed typical lesions o f ostertagiasis, ie, haem0rrhage, oedema and hyperplasia with formation o f small nodules. Group B had significantly higher total worm counts than group A ( P < 0 . 0 5 ) . Both group A and group B had significantly higher worm counts than group C which had been given in vitro exsheathed larvae ( P < 0 . 0 1 ) . Coles et al (1980) f o u n d that in vitro exsheathment with sodium hypochlorite can be somewhat toxic to third stage larvae, at least if they are exposed for a
344
345
O s t e r t a g i a o s t e r t a g i e s t a b l i s h m e n t in n e o n a t a l calves TABLE 1: Morphological development and weight of the reticulorumen, mean daily faecal egg output and total worm burden of ruminating and non-ruminating calves given 2 5 , 0 0 0 ensheathed or exsheathed Ostertagia ostertagi infective larvae (groups A and C -- non-ruminating; B -- ruminating)
Group A Ensheathed larvae
Reticulorumen Weight Morphological (g/kg development Iiveweight) + + + + + + + +
Mean (SD) /3 Ensheathed larvae
Mean (SD)
+ + + + + +
+ + + + +
+ + + + +
8.2 (1 ' 7 0 ) + + + + + + + + + + +
Mean (SD) C Exsheathed larvae
10,8 7.6 7,4 6.3 8.7
Abomasal lesion
20"0 18.3 16.3
7.1 8.6 6-3 7.7
+ + + + + + + + + + + +
+ + + + + +
7 •4 (0" 97)
long time. However, the high establishment of the exsheathed O ostertagi larvae in the ruminating control calf suggests that this agent produced no deleterious effect in the present experiment. In all three groups only a few fourth stage larvae were recorded (less than 3 per cent of the total worm counts). W h e n worm n u m b e r s and egg outputs are considered (Table 1), it m a y be concluded that worm fecundity, as reflected by the n u m b e r s of eggs per individual worm, was markedly higher in the animals with ruminal function. There are two main possibilities to be considered in accounting for higher worm establishment in ruminating calves. First, the provision of ruminal carbon dioxide which m a y act as a m a j o r c o m p o n e n t for triggering exsheathment of third stage larvae in the r u m e n (Rogers and Sommerville 1968). However, the infection with in vitro exsheathed larvae did not bring the establishment rate o f group C up to the level of group B, although such exsheathed larvae became well established in the test calf. A n o t h e r consideration is that the difference in the diets offered to the calves m a y cause differences in both ruminal and abomasal p H as well as other physiological parameters, which in turn m a y influence the development and establishment of the parasites. At least for Haemonchus conlortus in sheep, the p H of the r u m e n fluid m a y affect the rate of larval exsheathment (Silverman and Podger 1964). Unfortunately, information about ruminal and abomasal p H at the time o f infection o f the present calves was not available. However, Godfrey (1961) records that the ruminal p H of milk fed calves is within the range of 5" 15 to 5.75, while the ruminal p H of calves fed with concentrate and roughage in addition to milk, is within the range of 5.7 to 6.6. The slightly higher p H m a y cause better conditions for the exsheathment and development of the larvae in the rumen.
14,135 31,335 32,746 81,576 58,593 43,667 ( 2 4 , 4 7 4 " 1)
18.2 (1 - 85) ++ + + +
Mean daily faecal egg output
Total worm count 3984 2659 3864 2318 4771 3519 (1010"5)
271,291 325,196 291,258
4759 4843 4937
295,915 (27,252.6)
4846 (89.0)
0 5532 36,702 33,446 18,920 ( 1 8 , 8 3 6 ' 2)
15 1850 1395 413 9t 8 (849" 8)
This study has shown a higher establishment rate of O ostertagi in calves with significant ruminal function than in animals having only a negligible ruminal function. It clearly points to a role o f the ruminal function but the actual mechanism(s) that enhances establishment awaits further study.
Acknowledgements The authors wish to thank the staff of the section for animal reproduction, department of clinical studies, the Royal Veterinary and Agricultural University, Copenhagen, for technical assistance. We are grateful to our colleagues, Dr Rolf Jess JCrgensen, Dr Jesper Monrad and Dr T h o m a s Krogh for advice and comments. The experiment was supported by grants from the Danish Agricultural and Veterinary Research Council (grant n u m b e r 13-4292-2) and from the Danida Fellowship P r o g r a m m e .
References COLES, G. C., SIMPKIN, K. G. & BRISCOE, M. G. (1980)Routine cryopreservation of ruminant nematode larvae. Research in Veterinary Science 28, 391-392 GODFREY, N. W. (1961) The functional development of the calf II. Development of rumen function in the calf. Journal of Agricultural Science 57, 177-183 GRONVOLD, J., HENRIKSEN, S. Aa., NANSEN, P., WOLSTRUP, J. & THYLIN, J. (1989) Attempts to control infection with Ostertagia ostertagi (Trichostrongylidae) in grazing calves by adding mycelium of the nematode-trapping fungus Arthrobotrys oligospora (Hyphomycetales) to cow pats. Journal of Helminthology 63, 115- 126 McMURRAY, C. H., BLANCHFLOWER, W. J. & RICE, A. D.
346
F. Satrija, P. Nansen, C. M. Christensen
(1984) Automated kinetic method for D-3-hydroxybutyrate in plasma or serum. Clinical Chemistry 30, 421-425 MICHEL, J. F., LANCASTER, M. B. & HONG, C. (1979) The effect of age, acquired resistance, pregnancy and lactation on some reactions of cattle to infection with Ostertagia ostertagi. Parasitology 79, 157-168 NANSEN, P., STEFFAN, P., MONRAD, J., GRONVOLD, J. & HENRIKSEN, S. Aa. (1990) Effects of separate mixed grazing on trhicostrongylosis in first- and second-season grazing calves. Veterinary Parasitology 36, 265-276 PURl, J. P., NANGIA, O. P., GARG, S. L., PUNIA, J. S. & SINGH, N. (1983) Early development of rumen function in buffalo-calves, nitrogen metabolism and blood metabolism as influenced by age and diet. Indian Journal of Animal Science 28, 391-392
ROGERS, W. P. & SOMMERVILLE, R. I. (1968) The infectious process and its relationship to development of eariy parasitic stages of nematodes. Advances in Parasitology 6, 327-348 ROHRBACHER, G. H., PORTER, D. A. & HERLICH, H. (1958) The effects of milk in the diet of calves and rabbits upon the development of trichostrongylid nematodes. American Journal of Veterinary Research 19, 625 -631 SILVERMAN, P . H . & PODGER, K. R. (1964) In vitro exsheathment of some nematode infective larvae. Experimental Parasitology 15, 314-324
Received May 7, 1991 Accepted July 24, 1991
Ostertagia ostertagi in neonatal calves: establishment of infection in ruminating and non-ruminating calves F. S A T R I J A , P. N A N S E N , C. M. C H R I S T E N S E N , Department of Veterinary Microbiology, The Royal Veterinary
and Agricultural University, 13 Bulowsvej, DK-1870 Frederiksberg C, Denmark
An experiment was carried out to study the role of the ruminal function in the establishment of Ostertagia ostertagi in neonatal calves. Three groups of calves were fed either milk only (groups A and C), or hay and concentrate in addition to milk (group B) from birth. At the time of infection, ruminal function was negligible in groups A and C, whereas it was well developed in group B. Calves of groups A and B were each given 25,000 normal ensheathed infective larvae of O ostertagi and those of group C were given 25,000 infective larvae exsheathed in vitro. Daily faecal egg output and post mortem worm counts 28 days after infection were higher in calves with well developed ruminal function than those having only negligible ruminal function. In the latter group, exsheathed larvae established at a lower rate than did ensheathed larvae. The results suggest that the degree of development of the ruminal function influences the establishment of O ostertagi. G E N E R A L L Y , calveS are more susceptible to Ostertagia ostertagi t h a n are heifers or cows (Michel et al 1979, Nansen et al 1990). However, neonatal calves which are milkfed seem more or less refractory to establishment of infection (Rohrbacher et al 1958; P. Steffan and P. Nansen, unpublished data). This p h e n o m e n o n indicates a possible role of either the diet or o f ruminal function in the establishment of parasites in neonatal calves. The present experiment was designed to study the role of ruminal function in the establishment of O ostertagi by comparing the course of infection in neonatal ruminating and non-ruminating calves. Thirteen neonatal Jersey bull calves, reared under conditions designed to exclude worm infection, were used. They were placed individually in pens with slatted or concrete floors. The calves were allocated to groups Of fiv e (group A) or four calves (groups B and C). Calves of groups A and C were fed only milk replacer (Kalvital) from birth in an attempt to prevent the development of the rumen, while calves in group B were fed milk replacer and, in addition, hay and concentrate ad libitum, allowing the development of ruminal function (Godfrey 1961, Puri et al 1983). After approximately seven weeks, the calves were given O ostertagi larvae per os. Calves of groups A and B each received 25,000 normal infective larvae and those of group C each received 25,000 exsheathed infective larvae. Exsheathment of the larvae was performed in vitro using a 0 . 2 per cent sodium hypochlorite solution according to a method described by Coles et al (1980). In order to test the infectivity o f these exsheathed larvae in a ruminating animal, an eightweek-old Jersey bull calf, fed with hay and concentrate, was
infected with 25,000 exsheathed larvae of the same batch. At post m o r t e m examination, 14-4 per cent exsheathed larvae had become established in the test calf. Every second day from day 14 to day 28 after infection, the total quantity o f faeces excreted per 24 hours was collected. The n u m b e r o f trichostrongyle eggs per gram of faeces (epg) was determined using a modified McMaster procedure and the total daily faecal egg output calculated. Ruminal function was evaluated around the time of infection by plasma ~-hydl'oxybutyrate and glucose assay (Purl et al 1983) and by recording ruminal movements. ~3hydroxybutyrate concentrations were determined according to the method described by McMurray et al (1984) while plasma glucose levels were measured using the Gluc-DH method (Merck System 14053). The calves were slaughtered 28 days after infection, reticuloruminal weight was estimated and morphological development and abomasal lesions given scores from + to + + + + . Post m o r t e m worm recoveries were made according to the method described by GrCnvold et al (1989). At the time of infection, three calves of group B showed a significant development of ruminal function in contrast to the calves of groups A and C. This was evidenced by the tendency to higher plasma B-hydroxybutyrate levels (group B, 0.17 ± 0"06 m m o l litre -1 versus group A, 0.10 4- 0.00 m m o l litre-1; group C, 0"10 ± 0.00 m m o l litre -1) and lower plasma glucose levels (group B, 5.66 ± 1.40 m m o l litre - l versus group A, 8" 27 ± 1.60 m m o l litre- 1; group C, 7.48 ± 0-99 m m o l litre-l). One calf in group B was excluded from the experiment because of a lack o f ruminal function development. The ruminating calves o f group B showed significant m o v e m e n t s of their forestomachs around the start of the infection, and had m u c h higher weights and greater morphological development of their reticulorumens than had calves of the other groups (Table 1). Calves of group B had significantly higher daily faecal egg outputs than those of groups A and C ( P < 0 " 0 5 to P < 0 . 0 0 1 ) . Groups A and C did not differ significantly in this respect. At post m o r t e m examination, the abomasal mucosae of the animals showed typical lesions o f ostertagiasis, ie, haem0rrhage, oedema and hyperplasia with formation o f small nodules. Group B had significantly higher total worm counts than group A ( P < 0 . 0 5 ) . Both group A and group B had significantly higher worm counts than group C which had been given in vitro exsheathed larvae ( P < 0 . 0 1 ) . Coles et al (1980) f o u n d that in vitro exsheathment with sodium hypochlorite can be somewhat toxic to third stage larvae, at least if they are exposed for a
344
345
O s t e r t a g i a o s t e r t a g i e s t a b l i s h m e n t in n e o n a t a l calves TABLE 1: Morphological development and weight of the reticulorumen, mean daily faecal egg output and total worm burden of ruminating and non-ruminating calves given 2 5 , 0 0 0 ensheathed or exsheathed Ostertagia ostertagi infective larvae (groups A and C -- non-ruminating; B -- ruminating)
Group A Ensheathed larvae
Reticulorumen Weight Morphological (g/kg development Iiveweight) + + + + + + + +
Mean (SD) /3 Ensheathed larvae
Mean (SD)
+ + + + + +
+ + + + +
+ + + + +
8.2 (1 ' 7 0 ) + + + + + + + + + + +
Mean (SD) C Exsheathed larvae
10,8 7.6 7,4 6.3 8.7
Abomasal lesion
20"0 18.3 16.3
7.1 8.6 6-3 7.7
+ + + + + + + + + + + +
+ + + + + +
7 •4 (0" 97)
long time. However, the high establishment of the exsheathed O ostertagi larvae in the ruminating control calf suggests that this agent produced no deleterious effect in the present experiment. In all three groups only a few fourth stage larvae were recorded (less than 3 per cent of the total worm counts). W h e n worm n u m b e r s and egg outputs are considered (Table 1), it m a y be concluded that worm fecundity, as reflected by the n u m b e r s of eggs per individual worm, was markedly higher in the animals with ruminal function. There are two main possibilities to be considered in accounting for higher worm establishment in ruminating calves. First, the provision of ruminal carbon dioxide which m a y act as a m a j o r c o m p o n e n t for triggering exsheathment of third stage larvae in the r u m e n (Rogers and Sommerville 1968). However, the infection with in vitro exsheathed larvae did not bring the establishment rate o f group C up to the level of group B, although such exsheathed larvae became well established in the test calf. A n o t h e r consideration is that the difference in the diets offered to the calves m a y cause differences in both ruminal and abomasal p H as well as other physiological parameters, which in turn m a y influence the development and establishment of the parasites. At least for Haemonchus conlortus in sheep, the p H of the r u m e n fluid m a y affect the rate of larval exsheathment (Silverman and Podger 1964). Unfortunately, information about ruminal and abomasal p H at the time o f infection o f the present calves was not available. However, Godfrey (1961) records that the ruminal p H of milk fed calves is within the range of 5" 15 to 5.75, while the ruminal p H of calves fed with concentrate and roughage in addition to milk, is within the range of 5.7 to 6.6. The slightly higher p H m a y cause better conditions for the exsheathment and development of the larvae in the rumen.
14,135 31,335 32,746 81,576 58,593 43,667 ( 2 4 , 4 7 4 " 1)
18.2 (1 - 85) ++ + + +
Mean daily faecal egg output
Total worm count 3984 2659 3864 2318 4771 3519 (1010"5)
271,291 325,196 291,258
4759 4843 4937
295,915 (27,252.6)
4846 (89.0)
0 5532 36,702 33,446 18,920 ( 1 8 , 8 3 6 ' 2)
15 1850 1395 413 9t 8 (849" 8)
This study has shown a higher establishment rate of O ostertagi in calves with significant ruminal function than in animals having only a negligible ruminal function. It clearly points to a role o f the ruminal function but the actual mechanism(s) that enhances establishment awaits further study.
Acknowledgements The authors wish to thank the staff of the section for animal reproduction, department of clinical studies, the Royal Veterinary and Agricultural University, Copenhagen, for technical assistance. We are grateful to our colleagues, Dr Rolf Jess JCrgensen, Dr Jesper Monrad and Dr T h o m a s Krogh for advice and comments. The experiment was supported by grants from the Danish Agricultural and Veterinary Research Council (grant n u m b e r 13-4292-2) and from the Danida Fellowship P r o g r a m m e .
References COLES, G. C., SIMPKIN, K. G. & BRISCOE, M. G. (1980)Routine cryopreservation of ruminant nematode larvae. Research in Veterinary Science 28, 391-392 GODFREY, N. W. (1961) The functional development of the calf II. Development of rumen function in the calf. Journal of Agricultural Science 57, 177-183 GRONVOLD, J., HENRIKSEN, S. Aa., NANSEN, P., WOLSTRUP, J. & THYLIN, J. (1989) Attempts to control infection with Ostertagia ostertagi (Trichostrongylidae) in grazing calves by adding mycelium of the nematode-trapping fungus Arthrobotrys oligospora (Hyphomycetales) to cow pats. Journal of Helminthology 63, 115- 126 McMURRAY, C. H., BLANCHFLOWER, W. J. & RICE, A. D.
346
F. Satrija, P. Nansen, C. M. Christensen
(1984) Automated kinetic method for D-3-hydroxybutyrate in plasma or serum. Clinical Chemistry 30, 421-425 MICHEL, J. F., LANCASTER, M. B. & HONG, C. (1979) The effect of age, acquired resistance, pregnancy and lactation on some reactions of cattle to infection with Ostertagia ostertagi. Parasitology 79, 157-168 NANSEN, P., STEFFAN, P., MONRAD, J., GRONVOLD, J. & HENRIKSEN, S. Aa. (1990) Effects of separate mixed grazing on trhicostrongylosis in first- and second-season grazing calves. Veterinary Parasitology 36, 265-276 PURl, J. P., NANGIA, O. P., GARG, S. L., PUNIA, J. S. & SINGH, N. (1983) Early development of rumen function in buffalo-calves, nitrogen metabolism and blood metabolism as influenced by age and diet. Indian Journal of Animal Science 28, 391-392
ROGERS, W. P. & SOMMERVILLE, R. I. (1968) The infectious process and its relationship to development of eariy parasitic stages of nematodes. Advances in Parasitology 6, 327-348 ROHRBACHER, G. H., PORTER, D. A. & HERLICH, H. (1958) The effects of milk in the diet of calves and rabbits upon the development of trichostrongylid nematodes. American Journal of Veterinary Research 19, 625 -631 SILVERMAN, P . H . & PODGER, K. R. (1964) In vitro exsheathment of some nematode infective larvae. Experimental Parasitology 15, 314-324
Received May 7, 1991 Accepted July 24, 1991
