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The pathogenicity of daily intakes of Cooperia oncophora larvae in growing calves
Veterinary Parasitology, 5 (1979) 261-269 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
261
T H E P A T H O G E N I C I T Y O F D A I L Y I N T A K E S O F COOPERIA ONCOPttORA LARVAE IN GROWING CALVES
R.L. COOP, A.R. SYKES* and K.W. ANGUS
Moredun Research Institute, Edinburgh EH17 7JH (Great Britain) *Present address: Department of Animal Science, Lincoln College, Canterbury (New Zealand) (Accepted 7 August 1978)
ABSTRACT Coop, R.L., Sykes, A.R. and Angus, K.W., 1979. The pathogenicity of daily intakes of Cooperia oncophora larvae in growing calves. Vet. Parasitol., 5: 261--269. Groups of three 4~4-month-old calves were offered a complete ruminant diet ad libitum and given, on 5 days each week for 20 weeks, either 0, 5000, 10 000 or 20 000 Cooper/a oncophora larvae. All calves were killed 2 weeks after receiving the last dose of larvae. Cooperia infection reduced liveweight gain by 13.5% but did not significantly affect voluntary dry matter intake or produce any obvious clinical signs. No relationship was evident between the rate of larval intake and worm burden or faecal egg count. It appeared that a degree of acquired resistance developed in the majority of infected calves by 8--10 weeks. At slaughter, early fourth stage larvae predominated in four calves and maximum worm recoveries from the intestine were only 1.5% of the total larval intake. The majority of Cooperia were located in the duodenum and anterior jejunum. Pathological changes were restricted to areas surrounding the worms and consisted only of compression and distortion of villi in contact with parasites. Slight hypoalbuminaemia occurred between weeks 7 and 13 post inoculation in the group which received 20 000 larvae/day. There were no significant effects on serum total protein, globulin, urea, calcium, magnesium or phosphorus concentrations, or skeletal composition,
INTRODUCTION T h e m o s t prevalent gastrointestinal parasites o f cattle in t h e U n i t e d Kingd o m are Ostertagia and Cooperia. Ostertagia ostertagi has b e e n f r e q u e n t l y studied, b u t t h e r e is little i n f o r m a t i o n o n t h e effects o f Cooperia oncophora o n a n i m a l p e r f o r m a n c e . E p i d e m i o l o g i c a l studies (Michel, 1 9 6 9 ; Michel et al., 1 9 7 0 ) have s h o w n a sharp increase in Cooperia oncophora larvae in J u l y / A u g u s t t o levels o f a b o u t 5 0 0 - - 2 0 0 0 / k g h e r b a g e d r y m a t t e r (DM), w h i c h are m a i n t a i n e d over t h e winter, t h e n decline in t h e spring. A n e x p e r i m e n t , using c o m p a r a b l e levels o f c o n t i n u o u s i n f e c t i o n , was c o n d u c t e d t o assess t h e e f f e c t o f Cooperia oncophora o n g r o w t h rate, f o o d intake, c o n c e n t r a t i o n o f s e r u m c o n s t i t u e n t s a n d intestinal m o r p h o l o g y .
262 MATERIALS A N D METHODS
Animals and experimental design Twelve Ayrshire bull calves, reared indoors under worm-free conditions, were allocated at 4'/~ months o f age to four groups of three and the treatments randomly assigned. The calves were offered ad libitum a complete pelleted ration (Ruminant A, 13.5% CP: Wainman et al., 1970). Three groups (1, 2 and 3) were given daiiy on 5 days each week for 20 weeks (July to Decmber) 5000, 10 000 or 20 000 Cooperla oncophora larvae, respectively. Control, non-infected (Group 0) and all infected calves were killed 22 weeks after the start of inoculation. Assessment of larval infectivity during the experiment was available from two yearling donor calves (359 and 854) given single doses of 50 000 and 108 000 Cooperia oncophora, respectively.
Infective larvae C. oncophora was isolated from a local field infection and passaged once through a sheep and then three times through calves. Faeces from d o n o r calves were mixed with sterile peat, cultured at 26 ° C for 7 days, and larvae extracted b y means of a Baermann apparatus. Larvae stored at 7 ° C were used within 4 weeks up to week 10 o f the experiment, and thereafter stored for a maximum of 10 weeks. Bodyweight and voluntary food intake All calves were weighed weekly. Individual daily food refusals were weighed weekly and dry matter contents o f diet and refusals calculated after drying at 105 ° C for 72 h.
Faecal DM and egg count Faeces were taken twice weekly from the rectum and the n u m b e r of worm eggs per gramme fresh faeces (e.p.g.) estimated b y the m e t h o d of Jackson (1974), using flexible cellulose tubes to isolate the egg layer. Faecal DM was calculated after drying at 105 ° C for 72 h.
Slaughter procedures Calves were killed b y intravenous sodium pentobarbitone and were n o t bled out. The small intestine was immediately removed and divided into four equal segments for worm recoveries, each being processed separately. For histology, short lengths (approximately 4 cm) of intestine were taken from the d u o d e n u m and at 1-m intervals along the remainder of the small intestine (total length
263 ranged from 16 to 21 m). Each length was trimmed to yield both transverse and longitudinal sections. The intestine was opened longitudinally and examirled for macroscopic pathological changes, washed thoroughly, and then left to be d i g e ~ d in physiological saline at 39 ° C for 6 h. Washings and digests were passed through a 300 mesh/in (approximately 118 mesh/cm) screen and the retained material fixed in 10% formalin. Ten to 20% aliquots from each segment were searched for worms and the total number and their stage of development determined. The fourth lumbar vertebra from each calf was taken for chemical evaluation and the volume, weight of ash and fat-free organic matter determined by the methods of Sykes et al. (1973). Intestinal histology Tissues were fixed in Baker's calcium-formol for 24 h and post-fixed in a modified Bouin's solution (190 parts saturated picric acid; 10 parts 40% formaldehyde; five parts glacial acetic acid). Following processing to paraffin-wax, sections 5 ~m thick were cut and stained by Mayer's haematoxylin and eosin (H & E). Serum constituents Calves were bled weekly and the serum separated and stored at - 2 0 ° C. Total serum protein, albumin, globulin and urea were determined by the methods used by Sykes and Field (1973), and calcium by that of Sykes and Field (1974). Serum phosphorus was estimated by the method of Robinson et al. (1971), and magnesium by atomic absorption spectrophotometry. Statistics Data were analysed by analysis of variance. Means are given with one SE. RESULTS Clinical observations No clinical signs of disease were seen in any of the calves over the 22 weeks. There was no significant difference in mean faecal DM between infected (mean 16.1, range 11.4--19.9%) and controls (metal 16.1, range 12.6--18.3%) although on a few occasions individual infected calves passed slightly soft faeces for 2--3 days, particularly during week 3.
264
Faecal egg count There was no relationship between faecal egg count and larval intake. Worm eggs appeared in the faeces of all calves by week 3, and in three calves (one from group 2 and t w o from group 3) the initial rapid increase in egg o u t p u t to peaks of 1900--3900 e.p.g, at weeks 5--6 was followed by a sharp decline. A further three calves (one from group 1 and two from group 2) showed a low response, m a x i m u m egg counts of 420, 230 and 280 e.p.g., respectively, occurring 5 weeks after initial infection. With the exception of one calf from group 3, faecal egg counts ranged from zero to 76 e.p.g, between weeks 15 and 22.
Worm burden There was large variation in worm recoveries within groups: maxima of 1.5, 0.5 and 0.6% of the total larvae given were recovered from individuals in groups 1, 2 and 3, respectively (Table I). The majority of Cooperia were located in the first quarter of the small intestine, except in one calf of group 1 where a greater proportion was present in the second quarter. Early fourthstage larvae predominated in four of the calves. Twenty-three and 18%, of the larvae given were recovered from the donor calves 359 and 854 at weeks 4 and 9 post infection, confirming the viability of the larvae used in the experiment.
Liveweight gain and food intake The mean cumulative gains in bodyweight for groups 0, 1, 2 and 3 are given in Fig.1. There was a significant group X time interaction (P < 0.05) attributable to the slightly lower rate of gain in the three infected groups. Weight gain TABLE I
Cooperia oncophora r e c o v e r e d f r o m t h e small i n t e s t i n e o f e x p e r i m e n t a l l y i n f e c t e d calves. Total worms
P e r c e n t a g e o f early f o u r t h stage larvae
Group 1
7600 1130 < 100
82 44
Group 2
5250 2100 < 100
77 70
Group 3
11750 6920 1580
87 31 49
265
200,
160
120
__....,o
/
......... ..... wP~"
80
,o/ 0
Week Fig.1. Cumulative liveweight gain. Group 0 (control), • o; group 1, o - - - o ; group 2 • ........•; group 3, • •. sed, SE of difference between means. Mean initial bodyweight: -- 106 ~ 13.0; 107 -+ 2.4; 104 -+ 7.3 and 108 +- 3.5 kg for groups 0, 1, 2 and 3, respectively.
was similar up to week 7, b u t from this period to week 21, the mean growth rates for groups 1, 2 and 3 were 1031, 1061 and 1041 g/day, respectively, compared with 1265 g/day for the control group. Voluntary dry matter intake increased from mean values of 4.30, 4.08, 4.38 and 4.41 kg DM/day at week 1 to values of 7.70, 6.29, 7.75 and 7.10 kg DM/day at week 22 for groups 1, 2, 3 and 0, respectively. There were no significant differences between groups.
Serum constituents There was a significant group × time interaction (P < 0.01) for serum albumin concentration, attributable to the slight hypoalbuminaemia in group 3 which reached significance (P < 0.05 to P < 0.001) between weeks 7 and 13 (Fig.2). There was a negligible interaction for serum globulin, although the concentration increased slightly in group 3 between weeks 5--12. There was no significant effect on total serum protein, urea, calcium, magnesium or phosphorus concentrations.
Bone chemical analyses There were no significant differences between any of the groups in mean bone volume, ash weight or in the following ratios: ash/volume , organic matter/volume, or ash/organic matter.
266
36
32
,/~,
~.7
7,
'Y'" V
=E 28
='~-~" " -, ..,-o-:.
='~::
"
" J
,"'" %,
"=
''.
"~ ~o.~ "'"
"'"'"~"~
2o/ Week • ; group 1, sed, SE of difference b e t w e e n means.
Fig. 2. M e a n s e r u m a l b u m i n c o n c e n t r a t i o n s . G r o u p 0 ( c o n t r o l ) , •
group 2, • ........ •; group 3, •
•.
o----o;
Intestinal pathology The only consistent pathological changes were present in areas occupied by worms. The changes were confined to the first quarter of the small intestine in one calf from group 2 and one from group 3, but extended over the proximal half of the small intestine in a second calf from group 3. They comprised limited compression or distortion of the lateral margins of the intestinal villi in contact with the bodies of the worms (Fig.3) but there was no blunting or other morphological alteration to villi. Worms with no associated changes were seen in the proximal intestine in a further three calves, one from each of the infected groups. In one other calf from group 1 more goblet cells were present in jejunal crypts and eosinophils were more numerous in the ileal mucosa than in Controls, but no worms were seen. Post-mortem artefacts, viz: -- sloughing of surface epithelium of villi, or adhesion and fragmentation of groups of adjacent villi, were absent from the first quarter of the small intestine in both control and infected calves. These artefacts became increasingly obvious through the second to the third quarter, but t h e y occurred relatively infrequently in the ileum. DISCUSSION
There are no detailed descriptions of pathological lesions caused by C. oncophora in cattle, though Herlich (1965) described some macroscopic changes in the intestines of experimentally infected calves. Although data are available
Fig. 3. Cooperia oncophora in the small intestine of a calf from group 3. Left: local distortion of otherwise normal villi can be seen (H & E, × 100). Right: higher magnification, showing compression of adjacent villi and minor flattening of the epithelium (H & E, × 250).
t~ O~
268
for other Cooperia species precise comparisons are difficult because of differences in inoculation regimes and, probably, between methods of slaughter and handling of specimens at necropsy. Our results showed that there was no relationship between the size of worm burden and the extent and severity of intestinal lesions. Furthermore, there was no evidence of penetration by the parasite into the mucosa or sub-mucosa as reported for C. punctata or C. pectinata (Hung, 1926; Bailey, 1949; Herlich, 1967). Our necropsy procedures provided excellent histology and opportunity to study the site of greatest worm activity while more distally, artefacts attributed to post-mortem changes became numerous. This observation emphasises the need for multiple site selection and adequate controls as well as appropriate slaughter techniques. The lack of pathogenicity is probably explained by the rapid acquisition of resistance , indicated by the sharp decline in faecal egg count, and the presence of small numbers of worms at slaughter with minimal lesions in the small intestine. This is similar to the rapidly developing refractory state experienced by cattle grazing pastures contamined with Cooperia spp. (Smith and Archibald, 1968). The high proportion of early fourth-stage larvae in some of the calves may also reflect an acquired resistance since Michel (1963) indicated that C. oncophora was inhibited at the early fourth stage after repeated infections. In addition, some inhibition of larvae in later infections may have resulted from prolonged storage at 7 ° C from week 10. These conditions have been shown to induce inhibition (Michel et al., 1974, 1975). Our results appear to confirm field observations and other experimental studies (Alicata and Lynd, 1961; Herlich, 1965; Keith, 1967) which have suggested that C. oncophora is less pathogenic for cattle than either C. pectinata or C. punctata. In the present study, larval intakes of up to 100 000 per week produced no clinical signs and only slightly impaired performance. However, this does not exclude the possibility that sudden large intakes of larvae in less well-fed calves may be pathogenic. The experimental findings of Herlich (1967) in which trickle infections of C. pectinata were less pathogenic than comparable large doses, offer indirect support for this viewpoint. The lack of any significant effect on serum calcium or phosphorus concentrations or on the skeleton suggests lack of interference with intestinal absorption. The slight hypoalbuminaemia may, however, indicate macromolecular leakage into the gastrointestinal tract. Although food intake was unimpaired in the infected groups, and there was only a small effect on food efficiency, the latter may be important in mixed infections of C. oncophora and Ostertagia ostertagi, especially if the effects are multiplicative rather than additive.
269
ACKNOWLEDGMENTS W e a r e g r a t e f u l t o M r J. G i b s o n a n d M r R. H o p e f o r m a i n t a i n i n g t h e c a l v e s a n d t o M r R. B r o w n , M r A . D i n g w a l l , M r R. G r a h a m , M r F . J a c k s o n , M r J . E . P a t t e r s o n a n d Miss C. K e r r f o r s k i l l e d t e c h n i c a l a s s i s t a n c e . W e a r e i n d e b t e d t o M r M . G . C h r i s t i e f o r o r i g i n a l l y i s o l a t i n g t h e Cooperia oncophora a n d t o M r M. M c L a u c h l a n f o r a d v i c e w i t h s t a t i s t i c a l a n a l y s e s .
REFERENCES Alicata, J.E. and Lynd, F.T., 1961. Growth rate and other signs of infection in calves experimentally infected with Cooperia punctata. Am. J. Vet. Res., 22: 704--707. Bailey, W.S., 1949. Studies on calves experimentally infected with Cooperia punctata (v. Linstow, 1907) Ransom, 1907. Am. J. Vet. Res., 10: 119--129. Herlich, H., 1965. The effects of the intestinal worms, Cooperia pectinata and Cooperia oncophora, on experimentally infected calves. Am. J. Vet. Res., 26: 1032--1036. Herlich, I-L, 1967. Effects of Cooperia pectinata on calves: two levels of repeated oral inoculation. Am. J. Vet. Res., 28: 71--77. Hung, See-Lu., 1926. The pathology of Cooperia punctata infestation in calves. North Am. Vet., 73: 33--36. Jackson, F., 1974. New technique for obtaining nematode ova from sheep faeces. Lab. Pract., 23: 65--66. Keith, R.K., 1967. The pathogenicity of experimental infections of Cooperia pectinata, Ransom, 1907 in calves. Aust. J. Agric. Res., 18: 861--864. Michel, J.F., 1963. The phenomenon of host resistance and the course of infection of Ostertagia ostertagi in calves. Parasitology, 53: 63--84. Michel, J.F., 1969. Observations on the epidemiology of parasitic gastro-enteritis in calves. J. Helminthol., 43: 111--133. Michel, J.F., Lancaster, M.B. and Hong, C., 1970. Field observations on the epidemiology of parasitic gastro-enteritis in calves. Res. Vet. Sci., 11: 255--259. Michel, J.F., Lancaster, M.B. and Hong, C., 1974. Studies on arrested development of Ostertagia ostertagi and Cooperia oncophora. J. Comp. Pathoh, 84: 539--554. Michel, J.F., Lancaster, M.B. and Hong, C., 1975. Arrested development of Ostertagia ostertagi and Cooperia oncophora. Effect of temperature at the free-living third stage. J. Comp. Pathol., 85: 133--138. Robinson, R., Roughan, M.E. and Wagstaff, D.F., 1971. Measuring inorganic phosphate without using a reducing agent. Ann. Clin. Biochem., 8: 168--170. Smith, H.J. and Archibald, R.McG., 1968. Experimental helminthiasis in parasite-free calves on marshland pastures. Can. Vet. J., 9: 46--55. Sykes, A.R. and Field, A.C., 1973. Effects of dietary deficiencies of energy, protein and calcium on the pregnant ewe. IV Serum total protein, albumin, globulin, transferrin and plasma urea levels. J. Agric. Sci., 80: 29--36. Sykes, A.R. and Field, A.C., 1974. Seasonal changes in plasma concentrations of proteins, urea, glucose, calcium and phosphorus in sheep grazing a hill pasture and their relationship to changes in body composition. J. Agric. Sci., 83: 161--169. Sykes, A.R., Nisbet, D.I. and Field, A.C., 1973. Effects o f dietary deficiencies of energy, protein and calcium on the pregnant ewe. V Chemical analyses and histological examination of some individual bones. J. Agric. Sei., 81: 433--440. Wainman, F.W., Blaxter, K.L. and Pullar, J.D., 1970. The nutritive value for ruminants of a complete processed diet based on barley straw. J. Agric. Sci., 74: 311--314.
261
T H E P A T H O G E N I C I T Y O F D A I L Y I N T A K E S O F COOPERIA ONCOPttORA LARVAE IN GROWING CALVES
R.L. COOP, A.R. SYKES* and K.W. ANGUS
Moredun Research Institute, Edinburgh EH17 7JH (Great Britain) *Present address: Department of Animal Science, Lincoln College, Canterbury (New Zealand) (Accepted 7 August 1978)
ABSTRACT Coop, R.L., Sykes, A.R. and Angus, K.W., 1979. The pathogenicity of daily intakes of Cooperia oncophora larvae in growing calves. Vet. Parasitol., 5: 261--269. Groups of three 4~4-month-old calves were offered a complete ruminant diet ad libitum and given, on 5 days each week for 20 weeks, either 0, 5000, 10 000 or 20 000 Cooper/a oncophora larvae. All calves were killed 2 weeks after receiving the last dose of larvae. Cooperia infection reduced liveweight gain by 13.5% but did not significantly affect voluntary dry matter intake or produce any obvious clinical signs. No relationship was evident between the rate of larval intake and worm burden or faecal egg count. It appeared that a degree of acquired resistance developed in the majority of infected calves by 8--10 weeks. At slaughter, early fourth stage larvae predominated in four calves and maximum worm recoveries from the intestine were only 1.5% of the total larval intake. The majority of Cooperia were located in the duodenum and anterior jejunum. Pathological changes were restricted to areas surrounding the worms and consisted only of compression and distortion of villi in contact with parasites. Slight hypoalbuminaemia occurred between weeks 7 and 13 post inoculation in the group which received 20 000 larvae/day. There were no significant effects on serum total protein, globulin, urea, calcium, magnesium or phosphorus concentrations, or skeletal composition,
INTRODUCTION T h e m o s t prevalent gastrointestinal parasites o f cattle in t h e U n i t e d Kingd o m are Ostertagia and Cooperia. Ostertagia ostertagi has b e e n f r e q u e n t l y studied, b u t t h e r e is little i n f o r m a t i o n o n t h e effects o f Cooperia oncophora o n a n i m a l p e r f o r m a n c e . E p i d e m i o l o g i c a l studies (Michel, 1 9 6 9 ; Michel et al., 1 9 7 0 ) have s h o w n a sharp increase in Cooperia oncophora larvae in J u l y / A u g u s t t o levels o f a b o u t 5 0 0 - - 2 0 0 0 / k g h e r b a g e d r y m a t t e r (DM), w h i c h are m a i n t a i n e d over t h e winter, t h e n decline in t h e spring. A n e x p e r i m e n t , using c o m p a r a b l e levels o f c o n t i n u o u s i n f e c t i o n , was c o n d u c t e d t o assess t h e e f f e c t o f Cooperia oncophora o n g r o w t h rate, f o o d intake, c o n c e n t r a t i o n o f s e r u m c o n s t i t u e n t s a n d intestinal m o r p h o l o g y .
262 MATERIALS A N D METHODS
Animals and experimental design Twelve Ayrshire bull calves, reared indoors under worm-free conditions, were allocated at 4'/~ months o f age to four groups of three and the treatments randomly assigned. The calves were offered ad libitum a complete pelleted ration (Ruminant A, 13.5% CP: Wainman et al., 1970). Three groups (1, 2 and 3) were given daiiy on 5 days each week for 20 weeks (July to Decmber) 5000, 10 000 or 20 000 Cooperla oncophora larvae, respectively. Control, non-infected (Group 0) and all infected calves were killed 22 weeks after the start of inoculation. Assessment of larval infectivity during the experiment was available from two yearling donor calves (359 and 854) given single doses of 50 000 and 108 000 Cooperia oncophora, respectively.
Infective larvae C. oncophora was isolated from a local field infection and passaged once through a sheep and then three times through calves. Faeces from d o n o r calves were mixed with sterile peat, cultured at 26 ° C for 7 days, and larvae extracted b y means of a Baermann apparatus. Larvae stored at 7 ° C were used within 4 weeks up to week 10 o f the experiment, and thereafter stored for a maximum of 10 weeks. Bodyweight and voluntary food intake All calves were weighed weekly. Individual daily food refusals were weighed weekly and dry matter contents o f diet and refusals calculated after drying at 105 ° C for 72 h.
Faecal DM and egg count Faeces were taken twice weekly from the rectum and the n u m b e r of worm eggs per gramme fresh faeces (e.p.g.) estimated b y the m e t h o d of Jackson (1974), using flexible cellulose tubes to isolate the egg layer. Faecal DM was calculated after drying at 105 ° C for 72 h.
Slaughter procedures Calves were killed b y intravenous sodium pentobarbitone and were n o t bled out. The small intestine was immediately removed and divided into four equal segments for worm recoveries, each being processed separately. For histology, short lengths (approximately 4 cm) of intestine were taken from the d u o d e n u m and at 1-m intervals along the remainder of the small intestine (total length
263 ranged from 16 to 21 m). Each length was trimmed to yield both transverse and longitudinal sections. The intestine was opened longitudinally and examirled for macroscopic pathological changes, washed thoroughly, and then left to be d i g e ~ d in physiological saline at 39 ° C for 6 h. Washings and digests were passed through a 300 mesh/in (approximately 118 mesh/cm) screen and the retained material fixed in 10% formalin. Ten to 20% aliquots from each segment were searched for worms and the total number and their stage of development determined. The fourth lumbar vertebra from each calf was taken for chemical evaluation and the volume, weight of ash and fat-free organic matter determined by the methods of Sykes et al. (1973). Intestinal histology Tissues were fixed in Baker's calcium-formol for 24 h and post-fixed in a modified Bouin's solution (190 parts saturated picric acid; 10 parts 40% formaldehyde; five parts glacial acetic acid). Following processing to paraffin-wax, sections 5 ~m thick were cut and stained by Mayer's haematoxylin and eosin (H & E). Serum constituents Calves were bled weekly and the serum separated and stored at - 2 0 ° C. Total serum protein, albumin, globulin and urea were determined by the methods used by Sykes and Field (1973), and calcium by that of Sykes and Field (1974). Serum phosphorus was estimated by the method of Robinson et al. (1971), and magnesium by atomic absorption spectrophotometry. Statistics Data were analysed by analysis of variance. Means are given with one SE. RESULTS Clinical observations No clinical signs of disease were seen in any of the calves over the 22 weeks. There was no significant difference in mean faecal DM between infected (mean 16.1, range 11.4--19.9%) and controls (metal 16.1, range 12.6--18.3%) although on a few occasions individual infected calves passed slightly soft faeces for 2--3 days, particularly during week 3.
264
Faecal egg count There was no relationship between faecal egg count and larval intake. Worm eggs appeared in the faeces of all calves by week 3, and in three calves (one from group 2 and t w o from group 3) the initial rapid increase in egg o u t p u t to peaks of 1900--3900 e.p.g, at weeks 5--6 was followed by a sharp decline. A further three calves (one from group 1 and two from group 2) showed a low response, m a x i m u m egg counts of 420, 230 and 280 e.p.g., respectively, occurring 5 weeks after initial infection. With the exception of one calf from group 3, faecal egg counts ranged from zero to 76 e.p.g, between weeks 15 and 22.
Worm burden There was large variation in worm recoveries within groups: maxima of 1.5, 0.5 and 0.6% of the total larvae given were recovered from individuals in groups 1, 2 and 3, respectively (Table I). The majority of Cooperia were located in the first quarter of the small intestine, except in one calf of group 1 where a greater proportion was present in the second quarter. Early fourthstage larvae predominated in four of the calves. Twenty-three and 18%, of the larvae given were recovered from the donor calves 359 and 854 at weeks 4 and 9 post infection, confirming the viability of the larvae used in the experiment.
Liveweight gain and food intake The mean cumulative gains in bodyweight for groups 0, 1, 2 and 3 are given in Fig.1. There was a significant group X time interaction (P < 0.05) attributable to the slightly lower rate of gain in the three infected groups. Weight gain TABLE I
Cooperia oncophora r e c o v e r e d f r o m t h e small i n t e s t i n e o f e x p e r i m e n t a l l y i n f e c t e d calves. Total worms
P e r c e n t a g e o f early f o u r t h stage larvae
Group 1
7600 1130 < 100
82 44
Group 2
5250 2100 < 100
77 70
Group 3
11750 6920 1580
87 31 49
265
200,
160
120
__....,o
/
......... ..... wP~"
80
,o/ 0
Week Fig.1. Cumulative liveweight gain. Group 0 (control), • o; group 1, o - - - o ; group 2 • ........•; group 3, • •. sed, SE of difference between means. Mean initial bodyweight: -- 106 ~ 13.0; 107 -+ 2.4; 104 -+ 7.3 and 108 +- 3.5 kg for groups 0, 1, 2 and 3, respectively.
was similar up to week 7, b u t from this period to week 21, the mean growth rates for groups 1, 2 and 3 were 1031, 1061 and 1041 g/day, respectively, compared with 1265 g/day for the control group. Voluntary dry matter intake increased from mean values of 4.30, 4.08, 4.38 and 4.41 kg DM/day at week 1 to values of 7.70, 6.29, 7.75 and 7.10 kg DM/day at week 22 for groups 1, 2, 3 and 0, respectively. There were no significant differences between groups.
Serum constituents There was a significant group × time interaction (P < 0.01) for serum albumin concentration, attributable to the slight hypoalbuminaemia in group 3 which reached significance (P < 0.05 to P < 0.001) between weeks 7 and 13 (Fig.2). There was a negligible interaction for serum globulin, although the concentration increased slightly in group 3 between weeks 5--12. There was no significant effect on total serum protein, urea, calcium, magnesium or phosphorus concentrations.
Bone chemical analyses There were no significant differences between any of the groups in mean bone volume, ash weight or in the following ratios: ash/volume , organic matter/volume, or ash/organic matter.
266
36
32
,/~,
~.7
7,
'Y'" V
=E 28
='~-~" " -, ..,-o-:.
='~::
"
" J
,"'" %,
"=
''.
"~ ~o.~ "'"
"'"'"~"~
2o/ Week • ; group 1, sed, SE of difference b e t w e e n means.
Fig. 2. M e a n s e r u m a l b u m i n c o n c e n t r a t i o n s . G r o u p 0 ( c o n t r o l ) , •
group 2, • ........ •; group 3, •
•.
o----o;
Intestinal pathology The only consistent pathological changes were present in areas occupied by worms. The changes were confined to the first quarter of the small intestine in one calf from group 2 and one from group 3, but extended over the proximal half of the small intestine in a second calf from group 3. They comprised limited compression or distortion of the lateral margins of the intestinal villi in contact with the bodies of the worms (Fig.3) but there was no blunting or other morphological alteration to villi. Worms with no associated changes were seen in the proximal intestine in a further three calves, one from each of the infected groups. In one other calf from group 1 more goblet cells were present in jejunal crypts and eosinophils were more numerous in the ileal mucosa than in Controls, but no worms were seen. Post-mortem artefacts, viz: -- sloughing of surface epithelium of villi, or adhesion and fragmentation of groups of adjacent villi, were absent from the first quarter of the small intestine in both control and infected calves. These artefacts became increasingly obvious through the second to the third quarter, but t h e y occurred relatively infrequently in the ileum. DISCUSSION
There are no detailed descriptions of pathological lesions caused by C. oncophora in cattle, though Herlich (1965) described some macroscopic changes in the intestines of experimentally infected calves. Although data are available
Fig. 3. Cooperia oncophora in the small intestine of a calf from group 3. Left: local distortion of otherwise normal villi can be seen (H & E, × 100). Right: higher magnification, showing compression of adjacent villi and minor flattening of the epithelium (H & E, × 250).
t~ O~
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for other Cooperia species precise comparisons are difficult because of differences in inoculation regimes and, probably, between methods of slaughter and handling of specimens at necropsy. Our results showed that there was no relationship between the size of worm burden and the extent and severity of intestinal lesions. Furthermore, there was no evidence of penetration by the parasite into the mucosa or sub-mucosa as reported for C. punctata or C. pectinata (Hung, 1926; Bailey, 1949; Herlich, 1967). Our necropsy procedures provided excellent histology and opportunity to study the site of greatest worm activity while more distally, artefacts attributed to post-mortem changes became numerous. This observation emphasises the need for multiple site selection and adequate controls as well as appropriate slaughter techniques. The lack of pathogenicity is probably explained by the rapid acquisition of resistance , indicated by the sharp decline in faecal egg count, and the presence of small numbers of worms at slaughter with minimal lesions in the small intestine. This is similar to the rapidly developing refractory state experienced by cattle grazing pastures contamined with Cooperia spp. (Smith and Archibald, 1968). The high proportion of early fourth-stage larvae in some of the calves may also reflect an acquired resistance since Michel (1963) indicated that C. oncophora was inhibited at the early fourth stage after repeated infections. In addition, some inhibition of larvae in later infections may have resulted from prolonged storage at 7 ° C from week 10. These conditions have been shown to induce inhibition (Michel et al., 1974, 1975). Our results appear to confirm field observations and other experimental studies (Alicata and Lynd, 1961; Herlich, 1965; Keith, 1967) which have suggested that C. oncophora is less pathogenic for cattle than either C. pectinata or C. punctata. In the present study, larval intakes of up to 100 000 per week produced no clinical signs and only slightly impaired performance. However, this does not exclude the possibility that sudden large intakes of larvae in less well-fed calves may be pathogenic. The experimental findings of Herlich (1967) in which trickle infections of C. pectinata were less pathogenic than comparable large doses, offer indirect support for this viewpoint. The lack of any significant effect on serum calcium or phosphorus concentrations or on the skeleton suggests lack of interference with intestinal absorption. The slight hypoalbuminaemia may, however, indicate macromolecular leakage into the gastrointestinal tract. Although food intake was unimpaired in the infected groups, and there was only a small effect on food efficiency, the latter may be important in mixed infections of C. oncophora and Ostertagia ostertagi, especially if the effects are multiplicative rather than additive.
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ACKNOWLEDGMENTS W e a r e g r a t e f u l t o M r J. G i b s o n a n d M r R. H o p e f o r m a i n t a i n i n g t h e c a l v e s a n d t o M r R. B r o w n , M r A . D i n g w a l l , M r R. G r a h a m , M r F . J a c k s o n , M r J . E . P a t t e r s o n a n d Miss C. K e r r f o r s k i l l e d t e c h n i c a l a s s i s t a n c e . W e a r e i n d e b t e d t o M r M . G . C h r i s t i e f o r o r i g i n a l l y i s o l a t i n g t h e Cooperia oncophora a n d t o M r M. M c L a u c h l a n f o r a d v i c e w i t h s t a t i s t i c a l a n a l y s e s .
REFERENCES Alicata, J.E. and Lynd, F.T., 1961. Growth rate and other signs of infection in calves experimentally infected with Cooperia punctata. Am. J. Vet. Res., 22: 704--707. Bailey, W.S., 1949. Studies on calves experimentally infected with Cooperia punctata (v. Linstow, 1907) Ransom, 1907. Am. J. Vet. Res., 10: 119--129. Herlich, H., 1965. The effects of the intestinal worms, Cooperia pectinata and Cooperia oncophora, on experimentally infected calves. Am. J. Vet. Res., 26: 1032--1036. Herlich, I-L, 1967. Effects of Cooperia pectinata on calves: two levels of repeated oral inoculation. Am. J. Vet. Res., 28: 71--77. Hung, See-Lu., 1926. The pathology of Cooperia punctata infestation in calves. North Am. Vet., 73: 33--36. Jackson, F., 1974. New technique for obtaining nematode ova from sheep faeces. Lab. Pract., 23: 65--66. Keith, R.K., 1967. The pathogenicity of experimental infections of Cooperia pectinata, Ransom, 1907 in calves. Aust. J. Agric. Res., 18: 861--864. Michel, J.F., 1963. The phenomenon of host resistance and the course of infection of Ostertagia ostertagi in calves. Parasitology, 53: 63--84. Michel, J.F., 1969. Observations on the epidemiology of parasitic gastro-enteritis in calves. J. Helminthol., 43: 111--133. Michel, J.F., Lancaster, M.B. and Hong, C., 1970. Field observations on the epidemiology of parasitic gastro-enteritis in calves. Res. Vet. Sci., 11: 255--259. Michel, J.F., Lancaster, M.B. and Hong, C., 1974. Studies on arrested development of Ostertagia ostertagi and Cooperia oncophora. J. Comp. Pathoh, 84: 539--554. Michel, J.F., Lancaster, M.B. and Hong, C., 1975. Arrested development of Ostertagia ostertagi and Cooperia oncophora. Effect of temperature at the free-living third stage. J. Comp. Pathol., 85: 133--138. Robinson, R., Roughan, M.E. and Wagstaff, D.F., 1971. Measuring inorganic phosphate without using a reducing agent. Ann. Clin. Biochem., 8: 168--170. Smith, H.J. and Archibald, R.McG., 1968. Experimental helminthiasis in parasite-free calves on marshland pastures. Can. Vet. J., 9: 46--55. Sykes, A.R. and Field, A.C., 1973. Effects of dietary deficiencies of energy, protein and calcium on the pregnant ewe. IV Serum total protein, albumin, globulin, transferrin and plasma urea levels. J. Agric. Sci., 80: 29--36. Sykes, A.R. and Field, A.C., 1974. Seasonal changes in plasma concentrations of proteins, urea, glucose, calcium and phosphorus in sheep grazing a hill pasture and their relationship to changes in body composition. J. Agric. Sci., 83: 161--169. Sykes, A.R., Nisbet, D.I. and Field, A.C., 1973. Effects o f dietary deficiencies of energy, protein and calcium on the pregnant ewe. V Chemical analyses and histological examination of some individual bones. J. Agric. Sei., 81: 433--440. Wainman, F.W., Blaxter, K.L. and Pullar, J.D., 1970. The nutritive value for ruminants of a complete processed diet based on barley straw. J. Agric. Sci., 74: 311--314.