Effect of concurrent infection with Ostertagia circumcincta and Trichostrongylus vitrinus on the performance of lambs

Research in Veterinary Science /986. 40. 24/-245

Effect of concurrent infection with Ostertagia circumcincta and Trichostrongylus vitrinus on the performance of lambs R. L. cOOP, A. C. FIELD, R. B. GRAHAM, K. W. ANGUS, F. JACKSON, Moredun Research Institute, 408 Gilmerton Road, Edinburgh EHI7 7JH

The interaction between Ostertagia circumcincta and Trichostrongylus vitrinus was investigated in lambs continuously infected over 12 weeks. Four groups of seven lambs were given either zero, 1000 T vitrinus larvae per day, 2500 0 circumcincta larvae per day or infected concurrently. Overall Iiveweight gain was reduced by 17, 20 and 30 per cent in T vitrinus, o circumcincta and concurrent infection, respectively. T vitrinus infection significantly lowered serum phosphorus concentrations by week 4. In the concurrent infection the decline in phosphorus concentration was more gradual and only differed significantly from the controls during the final four weeks of the trial. There were no significant differences between the total numbers of T vitrinus or o circumcincta recovered from single or combined infections. The lack of an additive effect on performance may be due to a delayed establishment of T vitrinus in the presence of 0 circumcinta.

males) which had been housed since birth under conditions designed to minimise worm infection were used. Three weeks before the start of the experiment the lambs received a dose of fenbendazole (5 mg kg - I) and were allocated to four equal groups to provide uniformity of sex, weight and conformation. The lambs were penned individually and allowed to acclimatise to the pelleted ruminant ration. Groups I and 2 were infected daily on five days each week for 12 weeks with 1000 T vitrinus larvae and 2500 0 circumcincta larvae respectively. Group 3 lambs similarly received a daily combined dose of 1000 T vitrinus and 2500 0 circumcincta larvae. Group 4 lambs remained uninfected as controls. Infected and control lambs were killed by an intravenous injection of sodium pentobarbitone after 14 weeks on experiment.

THE major nematodes responsible for outbreaks of parasitic gastroenteritis in lambs in the United Kingdom are Nematodirus bat/us in the spring and Ostertagia circumcincta and Trichostrongylus vitrinus in the summer and autumn. The impact of a daily infection with either 0 circumcincta (Sykes and Coop 1977, Coop et al 1982) or T vitrinus (Sykes et al 1979) larvae on the performance of growing lambs has been assessed. The present experiment investigated the interaction between these two parasites to ascertain whether the effect of concurrent infection on animal performance is additive, as has been reported for infections of o circumcincta and T colubriformis (Steel et al 1982). The rate of infection chosen for either T vitrinus or o circumcincta was such that only a moderate effect on productivity would occur with the single infection.

Lambs were offered a complete ruminant ration (ruminant A, Wainman et al 1970) at approximately 120 per cent of consumption. The average concentraof crude protein in the dry matter (DM) tion (g kg was 143. Individual food refusals were collected daily, pooled over seven days and a sample taken for DM determination. Lambs were weighed at weekly intervals before feeding.

Bodyweight andjood intake

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Injective larvae Faeces from donor lambs infected with either T vitrinus or 0 circumcincta were cultured at 22°C for 10 days and infective larvae extracted in water using a standard Baermann technique. Larvae were stored at 4 to 7°C and used within three weeks. Faecal egg count

Materials and methods Animals and experimental design Twenty-eight three-and-a-half-month-old Greyface cross Suffolk lambs (females and castrated

Faecal samples were taken from the rectum twice weekly and the number of eggs per gram (epg) estimated using the flotation method described by Christie and Jackson (1982).

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R. L. Coop, A. C. Field, R. B. Graham, K. W. Angus, F. Jackson 20

Blood analysis Blood samples were taken from a jugular vein at weekly intervals and serum stored at - 20°C. Serum pepsinogen concentration was determined by a modification of the method of Mylrea and Hotson (1969) using a glycine/hydrochloric acid buffer (pH 2'0) and bovine albumin as substrate. Pepsinogen activity was expressed as milli-units (mu) where I unit equals I fJmole of tyrosine released per litre serum per minute at 37°C. Serum phosphorus concentrations were determined using a commercial kit (Auto/Stat; Pierce and Warriner) based on phosphomolybdate complex formation (Henry 1964). Analyses were performed on a Multistat III microcentrifugal analyser (Instrumentation Laboratories).

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Worm populations At slaughter the abomasum and small intestine were ligated and immediately removed. A sample of abomasal fluid was taken for determination of pH. For recovery of 0 circumcincta the abomasum was opened along its greater curvature, then washed and digested using the procedures described by Coop et al (1977). The material remaining on a 300 mesh sieve (aperture 53 fJm) was made up to 2 litres with 5 per cent formalin and I to IO per cent aliquots searched for the estimation of total worm populations. The small intestine was opened longitudinally, washed to remove the digesta and small lengths allowed to autolyse in physiological saline at 39°C for four hours. The surface of the intestine was then rubbed to remove the digested mucosa. Intestinal washings and digests were processed separately as above, and total worm burdens estimated from I to 10 per cent aliquots.

Topography of small intestine The surface topography of fixed pieces of small intestine (approximately 16 crn-) taken at O' 5 m (site A), I· 5 m (site B) and 2· 5 m (site C) from the pylorus was examined under a dissecting microscope and categorised numerically (Coop et al 1979) using criteria suggested by Pout (1970) and Barker (1974).

Histological procedures An abomasal fold was removed from each lamb by cutting along its fixed margin, fixed in IO per cent formol saline for 24 hours and after routine processing several portions were embedded in paraffin wax according to the technique described by Christie et al (1975). Sections 5 fJm thick were cut and stained by Mayer's haematoxylin and eosin. A qualitative

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FIG 1: Cumulative liveweight gain, Group 1 ... - - .... group 2 0- -0. group and group

assessment of ostertagia damage was based on the criteria described by Coop et al (1977). Three pieces of small intestine (approximately 3 to 4 cm long) were taken from sites A, Band C, fixed in IOper cent Baker's calcium formol saline and cut and stained as above. To enable a comparison of intraepithelial globule leucocyte (IGL) numbers in lambs with varying degrees of villous atrophy only those IGLS in crypts were estimated.

Statistical analyses Data were compared using a 2 x 2 factorial analysis with a split-plot for comparison of overall effects. Abomasal pH and total worm burden data were analysed by Student's t test. Means in the text are given ±I SD. Results

Bodyweight and food intake The cumulative weight gain for groups I to 4 is shown in Fig I. Reductions in growth rate were apparent in the T vitrinus infected lambs (group I) by week 8 and were significant from week IO(P
Concurrent ostertagia and trichostrongylus infection 1·8

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FIG 2: Mean daily food dry matter intake. Group 1 A- -A, group 2 0--0, group 3 . - . and group

(group 3 vs groups I or 2, not significant at any week). There was no significant difference in the overall DM intake between any of the four groups (Fig 2). There was, however, a slight reduction in DM intake (9' 6 to 12· 8 per cent) between weeks 2 to 6 in the lambs receiving ostertagia alone (P<0·05) compared with the controls.

Serum pepsinogen The mean serum pepsinogen concentrations for groups 2, 3 and 4 are shown in Fig 3. The rate of increase and magnitude of the response were similar in the two groups receiving ostertagia larvae. Concentrations were significantly higher than controls at week 2 and at succeeding weeks to week 13(P
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FIG 4: Mean serum inorganic phosphorus concentrations, Group 1 . - - A , group 2 0--0, group 3 . - . and group 4

Serum inorganic phosphorus There were no consistently significant differences in serum phosphorus concentration between group 2 lambs and the controls although the values in the former group were slightly lower between weeks 4 to 8 (Fig 4). In group I (1000 T vitrinus) there was a sharp decline in serum phosphorus concentration from week 3, the values being significantly lower than controls.(P
Faecal egg count Mean egg counts in groups I and 3 were similar, reaching a peak 4 to 5 weeks after initial infection (Table I). Faecal egg counts of lambs receiving a circumcincta alone (group 2) were low throughout the trial, the highest individual value being 255 epg.

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Worm populations The individual worm populations and their stage of development are shown in Fig 5. There were no significant differences between the total numbers of T vitrinus or a circumcincta recovered from single or combined infections (mean number of a circumcincta: group 2, 6727, group 3, 7469; mean number of T vitrinus: group 1,6656, group 3, 5099). Over 91 per cent of the T vitrinus populations consisted of adult worms.

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Pathological findings Focally severe lesions characteristic of chronic T vitrinus infection (Coop et al 1979), including

244

R. L. Coop, A. C. Field, R. B. Graham, K. W. Angus, F. Jackson TABLE 1: Mean faecal egg count of lambs infected with Tvitrinus (group 1), circumcincta {group 21and both parasites (group 3)

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Group 1 Group 2 Group 3

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4

5

6

7

Weeks 8

9

10

11

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120 32 208

716 85 469

345 186 499

374 58 326

254 62 347

384 86 489

381 45 392

227 9 306

207 24 214

120 8 287

81 65 264

extensive villous atrophy with mucosal flattening, were present in the anterior small intestines of all the lambs from groups I and 3. The severity of the lesions was similar in both groups. However, all group I lambs had numerous IGLS in their intestinal mucosa, whereas no IGLS were seen in four of the seven group 3 lambs (Fig 5). Typical lesions of ostertagiasis, namely hyperplasia of the fundic mucosa and loss of cell differentiation in the gastric glands, were present in the abomasa of both group 2 and group 3 lambs. There were no obvious differences in the severity of the abomasal lesions between the two groups. Abomasal pH was significantly higher in groups 2 (4'83±1'21, P
T colubrijormis as opposed to T vitrinus and a diet with a higher protein content (3' 3 per cent nitrogen). However, the higher protein diet would tend to reduce the pathogenic effects, not increase them. There are insufficient data available to speculate on the host response to T colubrijormis and T vitrinus although there is some evidence that resistance to T vitrinus tends to develop more rapidly than to T colubrijormis (Jackson et aI1983). Further studies on the interaction between ostertagia and T vitrinus have confirmed the present findings and have also shown that the effects are not additive over a range of larval intakes (R. L. Coop and A. C. Field unpublished data). One possible explanation for the apparent lack of an additive effect is that the presence of 0 circumcincta interferes with the establishment and accumulation of T vitrinus. This view is supported by the

Discussion The effect of either 0 circumcincta or T vitrinus infection alone on the rate of liveweight gain (20 per cent and 17 per cent reduction respectively) and on DM intake was similar to previous results with moderate levels of continuous infection given over a similar length of time (Sykes et a11979, Coop et aI1982). The timing of the check in growth rate from around week 2 to week 4 for 0 circumcincta and week 7 to week 8 for T vitrinus also agrees with these studies and those of Steel et al (1982) for lambs infected with 0 circumcincta and T colubrijormis. However, the lack of an additive or multiplicative effect on performance contrasted with the results of Steel et al (1982). They found that infection rates of 38,000 0 circumcincta per week concurrent with 3000 T colubrijormis per week depressed growth rate by 73 per cent over 16 weeks compared to reductions of 30 per cent and 20 per cent respectively for 0 circumcincta and T colubrijormis when given alone. The greater loss of productivity in the concurrent infection was primarily due to lowered food intake but this was further exacerbated by a reduction in the efficiency of retention of apparently digested nitrogen. The reason for the marked difference in the two sets of results is unknown and at present impossible to resolve as the experimental conditions differed considerably. For example, Steel et al (1982) used Merino cross lambs, a higher intake of ostertagia larvae,

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FIG 5: Total number and stage of development of 0 circumcinct» and Tvitrinusrecovered from groups 1, 2and3. Adult worms and developing stages 0, early fourth stage larvae 0 circumcincta and third stage T vitrinus •. *IGLs absent from intestinal mucosa

Concurrent ostertagia and trichostrongylus injection change in serum phosphorus concentrations. It is well known that intakes of T vitrinus or T colubrijormis larvae markedly depress blood phosphorus levels about 3 to 4 weeks after the start of continuous infection (Coop et al 1976, Sykes et al 1979) and this reduction occurred in group 1 lambs. In the concurrent infection (group 3) this rapid depression of phosphorus concentration was absent in the initial stages of infection, although phosphorus levels tended to be lower than for ostertagia alone and similar to group 1 during the last three weeks of the trial. This pattern is consistent with a delay in the accumulation of the T vitrinus population. In addition, although the severity of the topographical and histological lesions in the intestine were fairly similar in groups 1 and 3 at slaughter, the apparent absence of an IGL response, which is an index of developing resistance in the ruminant, in four of the group 3 lambs suggests that the development of resistance was not as far advanced in the concurrently infected group. Some interaction between 0 circumcincta and T vitrinus would seem likely as other workers have shown specific and unspecific responses between various species of nematodes . inhabiting the gastrointestinal tract in ruminants (Turner et a11962, Mapes and Coop 1970, Dash 1981, Kloosterman et aI1984). It must be emphasised that the results were obtained over a three month trial and that the interaction may differ over an extended period. Previous studies (Gibson and Parfitt 1976, Symons et a11981, Coop et al 1982) have shown that lambs continuously exposed to 0 circumcincta larvae develop resistance to infection after about eight to 12 weeks. In the present study plasma pepsinogen concentrations had decreased by week 10, indicating that abomasal damage was becoming less severe. It may well be that in the presence of a declining ostertagia population T vitrinus is able to establish more readily. In the concurrent group there was a tendency for the reduction in growth rate to be more marked during the final three weeks of the trial. Delayed accumulation of T vitrinus in the presence of an 0 circumcincta infection might in part account for the observation that intestinal Trichostrongylus species infections predominantly occur in lambs in the autumn and early winter (Reid and Armour 1972, 1975, Boag and Thomas 1977) at a time when they would have acquired some resistance to ostertagia infection.

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Acknowledgements We are grateful to Mr M. McLauchlan for advice and assistance with statistical analyses and to Mrs E. Jackson, Mr S. Wright and Miss G. Hutchison for skilled technical assistance. References BARKER, I. K. (1974) International Journal jor Parasitology 4, 153-163 BOAG, B. & THOMAS, R. J. (1977) Research in Veterinary Science 22,62-67 CHRISTIE, M. G. & JACKSON, F. (1982) Research in Veterinary Science 32, 113-117 CHRISTIE, M. G., ANGUS, K. W. & HOTSON, I. K. (1975) International Journal jor Parasitology,S, 193-198 COOP, R. L., ANGUS, K. W. & SYKES, A. R. (1979) Research in Veterinary Science 26, 363-371 COOP, R. L., SYKES, A. R. & ANGUS, K. W. (1976) Research in Veterinary Science 21, 253-258 COOP, R. L., SYKES, A. R. & ANGUS, K. W. (1977) Research in Veterinary Science 23, 76-83 COOP, R. L., SYKES, A. R. & ANGUS, K. W. (1982) Journal of Agricultural Science, Cambridge 98, 247-255 DASH, K. M. (1981) International Journal jor Parasitology II, 201-207 GIBSON, T. E. & PARFITT, J. W. (1976) Journal oj Comparative Pathology 86, 547-555 HENR Y, R. 1. (1964) Clinical Chemistry: Principles and Technics. Hoeber Medical Division, New York, Harper and Row. pp411-416 JACKSON, F., ANGUS, K. W. & COOP, R. L. (1983) Research in Veterinary Science 34, 301-304 KLOOSTERMAN, A., ALBERS, G. A. A. & VAN DEN BRINK, R. (1984) Veterinary Parasitology IS, 135-150 MAPES, C. J. & COOP, R. L. (1970) Journal of Comparative Pathology 80, 123-136 MYLREA, P. J. & HOTSON, I. K. (1969) British Veterinary Journal 125, 379-388 POUT, D. (1970) British Veterinary Journal 126, 357-363 REID, J. F. S. & ARMOUR, J. (1972) Research in Veterinary Science 13, 225-229 REID, J. F. S. & ARMOUR, J. (1975) Research in Veterinary Science 18, 307-313 STEEL, J. W., JONES, W. O. & SYMONS, L. E. A. (1982) Australian Journal of Agricultural Research 33, 131-140 SYKES, A. R. & COOP, R. L. (1977) Journal of Agricultural Science, Cambridge 88, 671-677 SYKES, A. R., COOP, R. L. & ANGUS, K. W. (1979) Research in Veterinary Science 26, 372-377 SYMONS, L. E. A., STEEL, J. W. & JONES, W. O. (1981) Australian Journal of Agricultural Research 32,139-148 TURNER, J. H., KATES, K. C. & WILSON, G. I. (1962) Proceedings of the Helminthological Society oj Washington 29, 210-216 WAINMAN, F. W., BLAXTER, K. L. & PULLAR, J. D. (1970) Journal oj Agricultural Science. Cambridge 74, 311-314

Accepted April 29, 1985