Notes on necropsy and herbage processing ... - Science Direct

Veterinary Parasitology, 46 (1993) 205-213 Elsevier Science Publishers B.V., Amsterdam

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Notes on necropsy and herbage processing techniques for gastrointestinal nematodes of ruminants M. Eysker and F.N.J. Kooyman Department of Parasitology, University of Utrecht, P.O. Box 80.165, 3508 TD Utrecht, Netherlands

ABSTRACT Eysker, M. and Kooyman, F.N.J., 1993. Notes on necropsy and herbage processing techniques for gastrointestinal nematodes of ruminants. Vet. Parasitol., 46:205-213. Necropsy techniques for the digestive tract of ruminants used at the University of Utrecht differ from those used elsewhere in three respects: ( 1 ) the abomasum is opened immediately after slaughter and the "contents" are treated separately from the "washings"; (2) the first 10 m of the small intestine are treated separately from the remainder of the intestine; (3) the aliquots are coloured with iodine before being examined for worms. The majority of the worms are found in the washings of the abomasum and the first part of the small intestine, whereas the contents of the abomasum and the remainder of the small intestine contain the bulk of the digesta. Because inhibited stages of Ostertagia, Haemonchus and, particularly, the very small third stage larvae ( L 3) of Trichostrongylus can be overlooked easily in digesta, these methods imply a more rapid and accurate enumeration of worms. This is more important in small ruminants than in cattle because a much higher proportion of the inhibited larvae will be washed out of the mucosa and because Trichostrongylus is more important in small ruminants. Herbage sampling methods for monitoring gastrointestinal nematode infections on cattle pastures in northwest Europe should also be suitable for lungworm. The agar-bile technique of Jorgensen is an elegant method, but disadvantages are that many gastrointestinal nematode larvae exsheath, resulting in identification difficulties, and recovery oflungworm larvae decreases as a result of ageing. A simple sucrose flotation method, based on the principle that a sucrose solution does not mix easily with water containing nematodes, has been tested at our laboratory. After centrifugation, a very clean suspension can be withdrawn from the interface. For lungworm larvae recovery is much better with this technique than with the agar-bile technique, but for gastrointestinal nematode larvae it is somewhat lower.

INTRODUCTION

Standardisation of veterinary helminthology techniques, such as faecal egg counts, pasture larval counts and worm counts is virtually non-existent. As a result, comparison of these important parameters between laboratories is Correspondence to: M. Eysker, Department of Parasitology, University of Utrecht, P.O. Box 80.165, 3508 T D Utrecht, Netherlands.

© 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4017/93/$06.00

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M. EYSKER AND F.N.J. KOOYMAN

problematic. An important reason for this lack of uniformity is the low level of accuracy inherent in the techniques of these parameters. Improvement of accuracy usually involves such increases in the amount of labour that they cannot be justified. Thus, investigators tend to be conservative in choice of methods used and comparability of their own results over many years becomes the norm. New methods are usually adopted only when they are easily accomplished and provide greater accuracy than existing methods. The objective of this paper is to present methods for necropsy and worm counts and for the processing of herbage samples for larval recovery used at the University of Utrecht and the advantages of these methods over those used in other laboratories. NECROPSY AND WORM COUNT TECHNIQUES

Methods Methods used for the processing of the abomasum and small intestine of ruminants often have the objective of minimising the amount of time spent in the slaughterhouse and in collecting aliquots. However, much more time is consumed in worm counts and identification, but preservation of samples allows more time for these tasks. The methods used at Utrecht involve somewhat more labour at necropsy; however, they allow for more rapid, and probably also more accurate, worm counts.

Abomasum The abomasum is separated from the small intestine immediately after slaughter and is then opened longitudinally along the greater curvature. The contents are collected in a polyethylene bag and the abomasum in another polyethylene bag for later processing in the laboratory. At the laboratory the abomasum is washed thoroughly in water, then soaked for 5 h in 1.51 of saline at 37°C and finally washed again and discarded. Each fraction (contents, water wash, saline wash) is treated separately. Each fraction is placed in a sampling bucket and water is added until the contents of the bucket represent 50)< or 100× the contents of a 55 ml sampling jar. After stirring for l0 min with the aid of a vibromixer, samples are taken with the sampling jar. Formalin is added to a concentration of 5%. Aliquots are poured through a sieve with a mesh size of 0.063 m m and coloured with a few drops of iodine. Aliquots are examined and worms counted and collected using a dissecting microscope at a magnification of approximately × 12-16 and identification of species and developmental stages is done with a c o m p o u n d microscope at a magnification of )< 40-400.

NECROPSY AND HERBAGE SAMPLING TECHNIQUES

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Small intestine The entire small intestine is stripped from the mesentery with scissors to facilitate separation of the first 10 m from the remainder. The anterior 10 m of the intestine and the remainder are treated separately. Each segment is emptied in the sampling bucket, slit open longitudinally with scissors and the mucosal surface is washed thoroughly. Procedures for preparation and treatment of aliquots, as well as worm counts and identification, are similar to those used for the abomasum.

Results and discussion Data for Ostertagia abomasal counts in 12 calves, Cooperia small intestinal counts in 12 calves, and Haemonchus and Ostertagia abomasal counts in 12 lambs, are shown in Tables 1,2 and 3, respectively. The results for sheep date back more than 10 years and peptic digestion of the washed mucosa of the abomasum was done instead of the saline wash (Eysker, 1978 ). These results are representative of those from several hundred calves and sheep necropsied at Utrecht. They indicate that in most animals the bulk of the adult and developing stages will be found in the washings of the abomasum and the first part of the small intestine. Moreover, Tables 1 and 3 show that large proportions of inhibited early fourth stage larvae (EL4) of Ostertagia and Haemonchus are found in the washings of the abomasum while the numbers of these stages in the contents are very low. The majority of the EL4 of Ostertagia are TABLE 1

Mean numbers and percentages o f Ostertagia populations recovered from three abomasal fractions o f 12 calves housed on 2 October and necropsied on 15 N o v e m b e r 1989 Washings

Contents

Saline

2033 500-6400

242 0-1200

1375 100-3650

57 33-89

5 0-21

38 11-67

208 0-2400

22567 1200-92500

Adults +juveniles Number Mean Range

Percentage Mean

Range

EL4 Number Mean Range Percentage Mean

Range

5225 300-26400 21 3-46

< 1 0-4

79 50-97

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TABLE2 Mean numbers and percentages of Cooperia populations recovered from two fractions of the small intestine of 12 tracer calves grazed from 17 to 21 August (calves 1-6) or from 29 September to 2 October (calves 7-12) 1989 First 10 m of small intestine

Remainder of small intestine

52425 3300-157600

157501 0-87400

75 12-100

25 0-88 l

9933 0-41600

15392 l 0-160500

64 16-100

36 0-841

Adults +juveniles Numbers Mean Range Percentage Mean Range

EL4 Number Mean Range Percentage Mean Range

lln one calf with extremely high worm burdens which was necropsied in extremis 88% of the 99600 adult and juveniles and 84% of the 190100 EL4 were in the rest of the small intestine. TABLE 3

Haemonchus and Ostertagia counts in washings, contents and digests I of the abomasa of 12 sheep necropsied in autumn-winter 1975-1976

Haemonchus Washings

Ostertagia Contents

Digests

Washings

Contents

Digests

8600 700-29500

1142 100-4000

158 0-900

83 64-95

14 5-36

3 0-8

Adults+juveniles Number Mean Range Percentage Mean Range

2908 0-26400

442 0-3000

8 0-100

79 33-100

20 0-67

10792 200-26500

83 0-300

6783 300-17600

1533 100-3900

67 0-300

3075 200-6800

57 40-85

1 0-3

39 15-60

40 11-90

1 0-6

59 10-89

1 0-9

EL4 Numbers Mean Range Percentage Mean Range

Ipeptic digestion of the abomasa was done according to Eysker ( 1978 ).


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found in the saline or digest fractions. This seems more pronounced in cattle than in sheep, but results are not completely comparable, as in sheep peptic digestion of the mucosa was done instead of a saline wash. The small proportions of Haemonchus found in digest fractions of sheep abomasa indicated a more loose attachment of inhibited larvae of this species to the mucosa. Such small proportions of inhibited Haemonchus were also observed in saline washings of abomasa of cattle in Zimbabwe (M. Eysker and V.S. Pandey, unpublished results, 1988-1990). In those few cases in which large numbers of Cooperia, including many fourth stage larvae (L4), were found in the posterior part of the small intestine it was usually in calves with extremely large infections and necropsied in extremis. A distinct advantage of examining three fractions of the abomasum and two fractions of the small intestine, is that most worms, including almost all inhibited stages, will be separated from the large bulk of the digesta. When this is not done, and also when the materials are not coloured with iodine, small stages such as inhibited larvae may be easily overlooked. This may not be too great a problem with the relatively large and stout inhibited Cooperia and Nematodirus larvae in cattle. However, it is no coincidence that the very small inhibited early third stage larvae of Trichostrongylus in small ruminants were overlooked until these necropsy techniques were used (Eysker, 1978). Thus, examination of different abomasal, and intestinal fractions allows a more accurate and less time-consuming enumeration of small larval stages, particularly for the abomasum and small intestine of small ruminants, and for cattle abomasa in Haemonchus regions. Enumeration of the adult stages of small worms such as Ostertagia spp. and Trichostrongylus spp. are also less burdensome. As indicated earlier, a disadvantage of the described techniques is that they are slightly more labour intensive on the day of necropsy than conventional techniques. An unexpected advantage of these necropsy techniques was observed with cattle in Zimbabwe. Cooperia counts in the small intestine of four calves from each of two farms are shown in Table 4. On Farm 1, Cooperia pectinata was the dominant species and, together with Cooperiapunctata, occupied primarily the first 10 m of the small intestine. In contrast, Cooperia spatulata was mainly located in the posterior segment of the small intestine. On Farm 2, C. spatulata was virtually the only Cooperia sp. present and in the absence of both other species it is mainly located in the first part of the small intestine, though to a lesser extent than for C. pectinata at Farm 1. This p h e n o m e n o n appeared to be consistent in other calves which were examined from both farms and it suggested some level of competition between C. pectinata and C. spatulata for the anterior part of the small intestine.

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TABLE 4 Differential Cooperia counts of the first 10 m (SI 1 ) and the remainder (SI 2 ) of the small intestine of eight calves from two farms in Zimbabwe Farm 1

Males

Farm 2

Males

Calf no. Fraction Females pect. of small intestine

punc. spat.

Calf no. Fraction Females pect. punc. spat. of small intestine

1

200 0 600 100 300 300 200 0

5

2 3 4

SII S12 Sll SI2 SII SI2 SI1 S12

1300 100 4200 2300 3400 6600 3300 1300

1100 0 1300 0 1900 200 1800 100

200 600 700 800 0 2400 0 1500

6 7 8

SI1 SI2 SII S12 SI1 SI2 SII SI2

250 100 2500 1100 2450 800 150 200

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

200 0 1350 700 1650 300 50 0

Pect., C. pectinata; punc., C. punctata; spat., C. spatulata. GRASS S A M P L I N G T E C H N I Q U E S

Methods Grass sampling techniques for estimating numbers of larvae of gastrointestinal nematodes in northwest Europe should also be suitable for the small and lethargic infective larvae of Dictyocaulus viviparus. A very elegant method for this purpose is the agar-bile technique (Jorgensen, 1975). The bile stimulates larvae, in particular the extremely sluggish lungworm larvae, to migrate out of the debris which remains trapped in an agar slab. Consequently, extremely clean nematode suspensions result. However, a disadvantage of this method is that gastrointestinal nematode larvae often exsheath, making generic differentiation difficult. Another disadvantage at our laboratory was that results with the agar-bile method were not very consistent and this was reason enough to continue with a Z n S O 4 centrifugal flotation method (Kloosterman, 1971; Eysker et al., 1988). With this method, however, only 20% of the washings from herbage were examined for larvae and this still contained large amounts of debris. Thus, the enumeration of larvae was a slow and tedious job and the method was not very sensitive for lungworm larvae, which are usually present in small numbers. In 1990, a modification of the agar-bile technique was developed by one of us (FK) which gave better results. However, comparison of the modification with the Z n S O 4 flotation method, indicated that a substantial proportion of ageing lungworm larvae were not found with the agar-bile technique. No differences in the numbers of gastrointestinal nematode larvae were observed. When at-


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tempts were made to improve separation of larvae from debris by using sucrose as a flotation medium, results were greatly improved. Details of this modification are given below. Grass samples (200-500 g) are soaked for 3 h in 5 1 of water and 10 ml of a detergent (teepol). The grass is then removed and washed three times. All soaking and washing water plus detergent is sieved through a screen of 1 m m mesh to remove large particles and a screen of 0.020 m m mesh to remove fine debris. The latter material contains the recovered larvae and is collected in a wide-mouth centrifuge tube of 125 ml. The larval suspension is centrifuged for 5 min at 1800×g, the supernatant is removed and the pellet is resuspended in approximately 60 ml of water to which at the bottom of the tube 60 ml of a sucrose solution ( 1.17 density) is added. Without mixing the larval suspension and the sucrose, the whole is centrifuged again for 5 min at 1800Xg. The interface containing larvae is removed with a syringe. A few drops of iodine are added to the interface material. Counting and differentiation can be done 1 h later after decolorisation with sodium thiosulphate. Results and discussion Samples treated with the sucrose interface method were almost as clean as those treated with the agar-bile technique. The main difference was that additional organisms, such as Rotifera, were present in interface samples. Table 5 presents a comparison of the recovery of larvae of Haemonchus contortus or gastrointestinal nematodes of cattle and Dictyocaulus viviparus which were added to larvae-free grass samples processed with the agar-bile method and the sucrose interface method. It shows that recovery of fresh lungworm larvae is high and equal with both methods. However, aged larvae ( 1 year) were found with the sucrose interface method but not with the agarbile technique. This is in contrast to results of Jorgensen ( 1975 ) who claimed equal recovery of fresh and aged lungworm larvae with this technique. However, the results are consistent with our earlier field data. The proportions of H. contortus larvae or gastrointestinal nematode larvae of cattle were higher with the agar-bile technique but still quite high with the interface method. Table 6 gives the results from field samples processed with both techniques. To assess these the debris from herbage samples was split into two equal portions before being processed with either method. A much higher recovery of lungworm larvae was observed with the sucrose interface method, but recovery of gastrointestinal nematode larvae was lower. Overall the sucrose interface method appears to be more sensitive and efficient for lungworm larvae and somewhat less efficient for gastrointestinal nematode larvae than the modified agar-bile technique and it is also less laborious. A c o m m e n t which could be made is the possibility that lungworm larvae, which are not found with the agar-bile technique, may not be viable

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TABLE 5 Recovery (%) of infective larvae ofHaemonchus contortus, gastrointestinal nematodes of cattle (GI) and Dictyocaulus viviparus larvae with the modified agar-bile (AB) method and the sucrose interface (SI) method Method

H. contortus

GI ( n = 159)

(n=638)

D. viviparus 2-3 weeks (n=212)

AB Before washing 1

After washing 2

67 60 70 69 80 85

1 year (n=130)

63 55 70 67 96 85 64 73

3 6

SI Before washing

After washing

28 29 37 34 55 38

68 75 77 68 83 94 71 53

87 97

ILarvae added before the washing procedure. 2Larvae added after the washing procedure.

TABLE 6 Numbers (kg-~ dry grass) of infective Dictyocaulus viviparus and gastrointestinal nematode larvae recovered from pasture samples with the modified agar-bile method and the sucrose interface method Date

22/8/1991 29/8/1991 5/9/1991 12/9/1991 19/9/1991 26/9/1991

Gastrointestinal nematodes

D. viviparus

Agar-bile

Sucrose

Agar-bile

Sucrose

5040 1670 3370 842 41500 110000

3070 1030 2960 669 21700 79700

3520 893 0 0 0 0

7710 12710 1317 0 2521 0


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enough to be infective. Thus, application of the sucrose interface method might result in an overestimation of pasture infectivity. However, as viability cannot be assured there is no reason to reject a method which is more sensitive in detecting lungworm larvae in the field. Therefore, it will be continued as the method of choice at Utrecht for processing grass samples from cattle and horse pastures. In the case of horse pastures the agar-bile technique has been proven to be inadequate. Comparison between the Z n S O 4 flotation method and the sucrose interface method showed no differences in efficiency for horse pastures. Where lungworm is not a widespread and critical health problem, such as in most of the USA, a method which is more efficient for gastrointestinal nematodes than the sucrose interface method may be a better choice. However, the interface principle can also be used in other instances when parasitic stages have to be collected from faeces in as clean a condition as possible. In Utrecht, for example, it is used for mass collection of nematode eggs from sheep and cattle faeces and for the detection of low numbers of lungworm larvae in large quantities (500 g) of cattle faeces.

REFERENCES Eysker, M., 1978. Inhibition of the development of Trichostrongylus spp. as third stage larvae in sheep. Vet. Parasitol., 4: 29-33. Eysker, M., Boersema, J.H., Kooyman, F.N.J. and Berghen, P., 1988. Possible resistance of small strongyles from female Shetland ponies in The Netherlands against albendazole. Am. J. Vet. Res., 49: 995-999. Jorgensen, R.J., 1975. Isolation of infective Dictyocaulus larvae from herbage. Vet. Parasitol., 1: 61-67. Kloosterman, A., 1971. Observations of the epidemiology of trichostrongylosis of calves. Ph.D. Thesis, Wageningen, 114 pp.