Mucosal larval recovery techniques of cyathostomes: can they be standardized?

Veterinary Parasitology 85 (1999) 137–149

Mucosal larval recovery techniques of cyathostomes: can they be standardized? M. Eysker a,∗ , T.R. Klei b a

Department of Parasitology and Tropical Veterinary Medicine, Utrecht University, P.O. Box 80.165, 3508 TD Utrecht, The Netherlands b Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA

Abstract The methods used currently for enumeration of mucosal stages of cyathostomes include transmural illumination (TMI) and peptic digestion (DIG). Enumerating the inhibited early L3 (EL3) and differentiating between mucosal developing L3 (DL3) and L4 (ML4) is only possible when DIG is used. However, in some studies higher numbers of DL (DL3 + ML4) have been found when using TMI as opposed to DIG. This finding, however, is not consistent. Moreover, results are not consistent when DIG and TMI are compared for treated and control groups of horses in drug trials. Studies performed on the effect of freezing on DIG and TMI also show inconsistent results between laboratories. However, there is, evidence that digested samples can be preserved in 5–10% formalin or in 70% ethanol when used as a buffered isotonic solution. Based on these results it is premature to recommend standardized mucosal larval recovery techniques. Consequently, TMI and DIG should both be applied. Recommendations for further research for the development of standardized techniques are given. ©1999 Elsevier Science B.V. All rights reserved. Keywords: Cyathostomes; Equids; Mucosal larvae; Inhibited early L3

1. Introduction Simultaneous emergence of large numbers of cyathostome larvae from the mucosa can result in serious and often fatal disease in young horses in winter and spring (Chiejina and Mason, 1977; Mirck, 1977; Ogbourne, 1978; Mair et al., 1990; Love et al., 1992; van Loon et al., 1995). In the etiology of this disease development of inhibited larvae in the mucosa is thought to be very important. Inhibition of development has been considered a feature of the ∗ Corresponding author. Tel: +31-30-2531223; fax: +31-30-2540784 E-mail address: [email protected] (M. Eysker)

0304-4017/99/$ – see front matter ©1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 9 9 ) 0 0 0 9 4 - 1

138

M. Eysker, T.R. Klei / Veterinary Parasitology 85 (1999) 137–149

biology of cyathostomes since Gibson (1953) demonstrated in mature horses that mucosal larvae can still resume development after 3 years. Ogbourne (1975) found indications for a seasonal inhibition similar to that in trichostrongylids in ruminants of some of the most prevalent cyathostomes. Later it was determined that inhibited development occurs in the early third stage (EL3) and it became obvious in The Netherlands that a large majority of the cyathostome populations in young horses in autumn and winter consists of these EL3 (Eysker et al., 1984, 1997). Subsequent studies in Louisiana demonstrated in a very different climatic region that EL3 dominate the cyathostome populations in tracer foals year-round (Klei et al., 1993b). For research on the population dynamics of the cyathostomes it is obvious that suitable techniques have to be used to enumerate the mucosal stages, including the EL3, which measure approximately only 0.5 mm. Furthermore, such enumeration is vital in controlled efficacy trials for anthelmintics. Mucosal EL3, developing L3 (DL3) and L4 (ML4) are encapsulated within the lamina propria or the submucosa. This implies that techniques should be used which either make them visible within the mucosa or after removal from the mucosa. Therefore, the most widely used techniques for enumeration of the mucosal stages include transmural illumination (TMI) and peptic digestion (DIG). In this communication we will discuss the advantages and disadvantages of both methods and suggest techniques for standardizing enumeration of these stages. This is important because the WAAVP guidelines for evaluating the efficacy of equine anthelmintics do not dictate which techniques should be used for this purpose (Duncan et al., 1988).

2. Methods 2.1. Transmural illumination (TMI) TMI implies that the mucosa from each compartment of the large intestine (caecum, ventral colon and dorsal colon) will be examined using a dissecting microscope and a light source. Exact methods vary among laboratories. Usually, small pieces of mucosa are removed at regular intervals from the caecum, ventral colon and dorsal colon. These pieces are then processed according to the standard operating procedures of the laboratory. In one major laboratory (Klei et al., 1993a; Monahan et al., 1995) each piece is stretched and nailed, serosa down, on a wooden structure with a circular aperture of 32 cm2 . Larvae in the aperture will be counted under a dissecting microscope. In other laboratories (Reinemeyer and Aguilar, 1992; Eysker et al., 1997) the serosa is removed and the whole piece of mucosa (approximately 16–25 cm2 ) is pressed between two petri dishes, one of which contains a grid to facilitate counting. The combined weight of all examined pieces from each compartment (caecum, ventral colon, dorsal colon) is used to derive a % aliquot of the total weight of the compartment in order to calculate the total number of developing larvae (DL = DL3 + ML4). Variations of these methods are used by other laboratories; these variations may affect the results of enumeration. Reinemeyer and Herd (1986) reported that higher numbers of larvae were found by TMI than by peptic digestion. However, a shortcoming of TMI is that it can not be used to

M. Eysker, T.R. Klei / Veterinary Parasitology 85 (1999) 137–149

139

enumerate the smaller inhibited EL3 (Reinemeyer and Aguilar, 1992; Klei et al., 1993a; Xiao et al., 1994; Monahan et al., 1995; Eysker et al., 1997). Moreover, small DL3 are also missed by TMI (Eysker et al., 1997). Another disadvantage of TMI is that it will not allow differentiation between DL3 and ML4, and larvae can only be classified as mucosal DL.

2.2. Peptic digestion Different laboratories also use a variety of techniques for peptic digestion (DIG). A consensus seems to be that 10 g pepsin (1 : 10 000) and 15 ml of HCl 36–38% per liter water is used as digestion fluid, and the mucosal and submucosal tissues must be removed prior to digestion (Eysker et al., 1988; Xiao et al., 1994; Monahan et al., 1995; Murphy and Love, 1997). Depending on the laboratory the incubation period may vary from 2 h (Monahan et al., 1995) to 4 h (Eysker et al., 1988; Xiao et al., 1994) and incubation temperatures varied from 40◦ C (Eysker et al., 1988; Xiao et al., 1994; Murphy and Love, 1997) to 37◦ C (Eysker et al., 1997; Monahan et al., 1995). Another very important aspect of DIG is that samples must be shaken constantly during digestion. Murphy and Love (1997) used a ’milking’ technique. After 3 h of incubation the digest solution was removed from each sample and fixed with 10% formalin. The undigested tissue was reincubated with fresh digest solution for a further 3 h. This process was repeated several times until the intestinal tissue was fully digested. The preserved fluids from each tissue sample were then combined. Many variations of the digest method persist. Eysker et al. (1984, 1988) performed only peptic digestion using 10% by weight of the mucosa of each compartment of the large intestine. The numbers of DL, refered to as ‘L4’were counted from these 10% samples. Enumeration of the EL3 was done by counting 1% aliquots of these samples, thus each EL3 identified represented 1000 larvae. Chapman and Klei (unpublished) cut the caecum and ventral colon in half to compare TMI (one half) with DIG (other half). In total, 10% of the mucosal scrapings of the DIG half was digested for enumeration of mucosal stages. Actual counts were done on aliquots. Chapman and Klei (unpubl.) standardised digestion methods by using screw cap 250 ml Erlemeyer flasks with 200 ml of HCl/pepsin for tissue samples of a maximum 25 g weight. Before counting the larvae, digested materials were usually washed over a screen with an aperture of approximately 0.063 mm (Reinemeyer and Aguilar, 1992; Xiao et al., 1994; Monahan et al., 1995; Eysker et al., 1984, 1988, 1997; Chapman and Klei, unpubl.). With the exception of Xiao et al. (1994), the above authors used iodine colouration to allow more contrast between the larvae and debris. Murphy and Love (1997) did not sieve their materials. After sedimentation of their materials, they reduced the total volume of each digest to 200 ml. Subsequently, counting was performed on 4 ml aliquots. Advantages of DIG over TMI are that EL3 can be enumerated, and that DL3 and ML4 can be differentiated. The latter information is of particular importance when the efficacy of drugs with limited efficacy against mucosal DL are being evaluated. Anthelmintic effect may be restricted to ML4 or a well defined portion there of (Eysker et al., 1997). In a study on the persistent effect of moxidectin against cyathostomes, the ability to differentiate between DL3 and ML4 was essential to estimate the duration of this effect (Vercruysse et al., 1998).

140

M. Eysker, T.R. Klei / Veterinary Parasitology 85 (1999) 137–149

Table 1 Comparison of mucosal developing stages of caythostomes counted in each sample after DIG or TMIa Moxidectin group

Caecum Ventral colon Dorsal colon Total Total-dorsal colon

Control group

+

=

–

0

+

=

–

0

11 11 5 27 22

2 4 4 10 6

5 3 3 11 8

0 0 6 6 0

6 6 2 14 12

0 0 2 2 0

12 11 6 29 23

0 1 8 9 1

a

More larvae after DIG (+); Equal numbers of larvae in TMI and DIG (=); Less larvae after Dig (−); No larvae found in TMI and DIG (0). (Eysker et al., 1997).

A disadvantage of DIG in comparison with TMI is that in some studies the numbers of DL were lower (Reinemeyer and Herd, 1986; Eysker et al., 1997; Vercruysse et al., 1998). 2.3. Comparison of TMI and DIG In several studies the same samples examined by TMI were subsequently used for DIG (Reinemeyer and Aguilar, 1992; Xiao et al., 1994; Monahan et al., 1995; Eysker et al., 1997; Murphy and Love, 1997; Vercruysse et al., 1998) and results of both techniques could be compared for each sample. However, Xiao et al. (1994) only used TMI for DL and DIG for EL3. In some studies numbers of DL were higher using TMI than using DIG (Reinemeyer and Herd, 1986; Eysker et al., 1997; Vercruysse et al., 1998). Closer examination of the results of Eysker et al. (1997) demonstrates that more DL were found after TMI in six control ponies, but in six ponies, treated 5 weeks earlier with moxidectin, more DL were found after DIG (Table 1). The explanation presented by the authors was that the larger mucosal stages, which were highly prevalent in control ponies, were not efficiently recovered by DIG. In contrast, very small DL3, which had resumed development during the 5 weeks between treatment and necropsy, were not large enough to be enumerated by TMI (Eysker et al., 1997). Thus, disappearance of DL during the process of digestion seems to be more prominent for the large DL than for the small DL. The reason for this observation may be that large larvae can be easily damaged in the process of scraping and subsequently be more vulnerable to disintegration during digestion. Lower recovery of DL by DIG compared to TMI, however, was not observed in other studies (Monahan et al., 1995). Klei et al. (1997) even found lower numbers numbers of DL in treated groups with DIG than with TMI; in contrast to the observations of Eysker et al. (1997). This may imply that DL which have been damaged or killed by treatment can still be found by TMI, but not by DIG. It is obvious that more studies need to be performed to determine what can be achieved with both techniques. 2.4. Freezing materials before TMI and/or DIG Necropsy procedures for horses are very time consuming, and techniques permitting enumeration of mucosal stages to be delayed would be helpful. A useful option would be freezing of the materials prior to being processed. Klei et al. (1997) indicate that freezing

M. Eysker, T.R. Klei / Veterinary Parasitology 85 (1999) 137–149

141

does not affect the results for TMI and EL3 by DIG. However, lower numbers of DL were found by DIG after the samples were frozen and the DL were more difficult to visualize. In contrast Gamboa et al. (1997) found higher numbers of EL3 and DL in frozen than in fresh samples after DIG. 2.5. Fixation after digestion Eysker (unpubl. results) observed that DL and EL3 from digested mucosa eventually disintegrate when they are stored in 5% formalin, suggesting that samples should be examined as soon as possible after digestion. However, Klei et al. (1997) demonstrated that counts were not affected by time when digested materials when fixed in 5 or 10% buffered formalin or in 70% ethanol/glycerin. Thus, fixation is possible when care is taken that an isotonic solution is used. 2.6. Variation within intestinal compartment Within the caecum and ventral colon, the numbers of mucosal stages vary between sites (Table 2). Because numbers of mucosal stages are usually low, this is of little concern regarding the dorsal colon. A consequence of this large variation is that counts based on a limited number of samples from each compartment may be very inaccurate. This problem can be solved for DIG when the entire mucosa is scraped off and digested. Considering the amount of digestion fluid and the ’shaking’ capacity needed, this is hardly a feasible option. Aliquots of digests by weight (up to 10%) should preferably be digested. The problem is then to compile representative samples, since large sheets of mucosa may be present after scraping. Chapman and Klei (unpubl.) solved this problem by mincing the scrapings. A possible disadvantage of mincing, though not apparent in their data, is that large DL may be physically damaged. An alternative may be to initially take a longitudinal strip of the organs (which approaches 2.5% to scrape and digest), since most of the variation can be expected in the length (Chapman and Klei, unpubl.). The reproducibility of counts from different tissue samples from the same scrapings, or different longitudinal strips, should be evaluated. 2.7. Time between anthelmintic treatment and necropsy In controlled anthelmintic trials, different time intervals have been used between treatment and necropsy. In most trials, where appropriate techniques were used to enumerate EL3, an interval of 2 weeks was used (Reinemeyer and Aguilar, 1992; Eysker et al., 1992; Xiao et al., 1994; Monahan et al., 1995). An important question that remains is whether larvae which have been killed by the anthelmintic can still be found using TMI or DIG. This was illustrated by Klei et al. (1997), who observed higher DL burdens by TMI than by DIG in ponies treated with an anthelmintic; whereas this was not the case in their non-treated controls. Therefore, Eysker et al. (1997) used a 5-week interval between treatment and necropsy. However, their study demonstrated a serious disadvantage associated with this long interval, in that there was indication of resumption of development by part of the EL3

142

M. Eysker, T.R. Klei / Veterinary Parasitology 85 (1999) 137–149

Table 2 Numbers of mucosal developing stages of cyathostomes counted in each replicate sample after TMI and DIG (Eysker et al., 1997) Control group Animal number 33 TMI

Dig

37 TMI

Dig

41 TMI

Dig

31 TMI

Dig

35 TMI

Dig

39 TMI

Dig

89 103 49

40 84 42

14 20 18

6 5 19

43 59 48

21 16 7

8 21 19

9 24 31

20 15 15

17 7 7

31 41 35

41 49 31

Ventral colon I 35 II 54 III 35

13 24 32

9 22 13

3 8 11

22 21 12

19 6 16

9 8 0

12 15 0

11 5 4

9 9 0

5 39 9

45 42 10

Dorsal Colon I 0 II 0 III 0

0 0 0

1 0 0

1 0 0

1 0 1

0 0 1

2 0 0

0 0 0

2 4 2

1 2 1

5 0 5

4 1 6

Dig

34 TMI

Dig

36 TMi

Dig

38 TMi

Dig

40 TMi

Dig

42 TMi

Dig

31 10 32

30 23 31

21 41 36

65 38 59

50 15 57

9 6 8

27 20 9

23 18 11

25 14 20

7 4 6

7 5 13

47 3 48

18 22 3

32 65 9

38 25 10

65 53 8

10 9 1

25 12 9

6 15 32

2 15 24

1 1 1

1 1 1

0 0 0

1 11 4

1 4 16

3 1 1

0 1 1

4 0 1

5 1 1

3 0 0

5 0 0

0 0 6

0 1 2

I II III

Moxidectin-treated group 32 TMI Caecum I 12 II 10 III 13 Ventral colon I 9 II 2 III 14 Dorsal colon I 0 II 0 III 0

population remaining in the treated group following treatment. Therefore, a post-treatment period of 5 weeks prior to necropsy appears too long. However, when a period of 2 weeks is used, the effect of treatment may be underestimated, particularly when TMI is used. 3. Discussion As mentoned previously a wide variety of TMI and DIG techniques are used in different laboratories. This indicates that standardization of procedures would be most useful. Currently, it is unacceptable to use TMI as the only method for enumeration of mucosal stages, as EL3 and very small DL3 will be overlooked. When DIG techniques that yield consistent and reproducible results within and between different laboratories, are developed TMI might then be deleted as an additional technique. However, the present inconsistency

M. Eysker, T.R. Klei / Veterinary Parasitology 85 (1999) 137–149

143

of results is so great that TMI and DIG should both be applied to enumerate DL. The results of both the techniques should then be given in publications. The results of studies involving freezing materials prior to TMI and digestion are also inconsistent. In particular the observation that more larvae are found after freezing than in fresh samples (Gamboa et al., 1997) is confusing. More studies, preferably from different laboratories, should be performed to address this dilemma. Fixation of materials prior to counting can be done (Klei et al., 1997), however, it is imperative that buffered isotonic fixatives are used. Enumeration of mucosal stages by TMI and DIG is not very accurate, except perhaps for DIG when the whole mucosa is scraped. This, however, is very laborious. At this time it seems premature to make definitive recommendations for standardized techniques for enumeration of mucosal stages. 4. Current recommendations 1. TMI and DIG should be used together in studies where the mucosal stages of cyathostomes are to be enumerated. When a reproducible DIG technique is developed, TMI will no longer be necessary. 2. Incubation temperature, incubation period, volume of digestion fluid and incubation flask, and sample weight should be further evaluated for DIG. At the present time a promising technique for DIG is to constantly mix a maximum of 25 g of mucosa with 200 ml of digestion fluid in 250 ml Erlemeyer flaks at 37◦ C for 2 h (Chapman and Klei, unpubl.). However, additional studies should be performed in other laboratories to substantiate and standardize the methods. These studies should also include a comparsion of DIG with TMI. 3. The effect of freezing should be evaluated further in different laboratories, particularly on DIG. 4. Digested materials can be stored, but the fixative (5 or 10% formalin; glycerin/ethonol 70%) must be isotonic. 5. The reproducibility of DIG should be examined for different samples from the same compartment. These studies should concentrate on the ventral colon because most variation can be expected in this compartment (Table 2). Different 5 or 10% replicates, or different narrow longitudinal strips, should be taken from scraped mucosa of the ventral colon and counts should be compared after digestion, sieving through a 0.063 mm sieve, and colouring with iodine. Preferably these results should also be compared with enumeration done after digestion of several small pieces of mucosa from the same ventral colon. If similar results are obtained from these small pieces and from the 5–10% replicates, the laborious exercize of performing total scrapings can be avoided. 6. The optimal interval between treatment and necropsy should be determined for drug trials. Acknowledgements Prof A.W.C.A. Cornelissen is thanked for his critical remarks on the manuscript.

144

M. Eysker, T.R. Klei / Veterinary Parasitology 85 (1999) 137–149

References Chiejina, S.N., Mason, J.A., 1977. Immature stages of Trichonema spp. as a cause of diarrhoe in adult horses in spring. Vet. Rec. 100, 360–361. Duncan, J.L., Arundel, J.H., Drudge, J.H., Malczewski, A., Slocombe, J.O.D., 1988. World association for the advancement of veterinary parasitology (WAAVP) guidelines for evaluating the efficacy of equine anthelmintics. Vet. Parasitol. 30, 57–72. Eysker, M., Jansen, J., Mirck, M.H., 1984. Inhibited development of Cyathostominae in the horse in the early third stage. Res. Vet. Sci. 37, 355–356. Eysker, M.J.H., Kooyman, F.N.J., Berghen, P., 1988. Possible resistance of small strongyles from female ponies in The Netherlands against albendazole. Am. J. Vet. Res. 49, 995–999. Eysker, M., Boersema, J.H., Kooyman, F.N.J., 1992. The effect of ivermectin treatment against inhibited early third stage, late third stage, late third stage and fourth stage larvae and adult stages of the cyathostomes in Shetland ponies and spontaneous expulsion of these helminths. Vet. Parasitol. 42, 295–302. Eysker, M., Boersema, J.H., Grinwis, G.C.M., Kooyman, F.N.J., Poot, J., 1997. Controlled dose confirmation study of a 2% moxidectin equine gel against equine internal parasites in The Netherlands. Vet. Parasitol. 70, 165–173. Gamboa, R., Dipietro, J.A., Paul, A.J., 1997. Comparison of the recovery of cyathostome larvae from fresh and frozen equine mucosa. In: Proc. 42nd Annual Meeting AAVP, Reno, p. 89. Gibson, T.E., 1953. The effect of repeated anthelmintic treatment with phenothiazine on the faecal egg counts of housed with some observations on the life-cycle of Trichonema spp. in the horse. J. Helminthol. 27, 29–40. Klei, T.R., Chapman, M.R., French, D.D., Taylor, H.W., 1993a. Evaluation of ivermectin at an elevated dose against encysted equine cyathostome larvae. Vet. Parasitol. 47, 99–106. Klei, T.R., French, D.D., Monahan, C.M., Chapman, M.R., 1993b. Epidemiology of equine gastrointestinal parasites in southern Louisiana. In: Proc. 39th Annual Meeting AAVP, San Francisco, p. 36. Klei, T.R., Chapman, M.R., French, D.D., 1997. Experimental reevalution of methods for the enumeration of mucosal cyathostome larvae. In: Proc. 16th Int. Conf. AAVP, Sun City, South Africa, p. 48. van Loon, G., Deprez, P., Muylle, E., Sustronck, B., 1995. Larval Cyasthostominosis as a cause of death in two regularly dewormed horses. J. Vet. Med. Series A 42, 301–306. Love, S., Mair, T.S., Hillyer, M.H., 1992. Chronic diarrhoea in adult horses: a review of 51 referred cases. Vet. Rec. 130, 217–219. Mair, T.S., de Westerlaken, L.V., Cripps, P.J., Love, S., 1990. Diarrhoea in adult horses: a survey of clinical cases and assessment of some prognostic indices. Vet. Rec. 126, 479–481. Mirck, M.H., 1977. Cyathostominose: een vorm van ernstige strongylidose. Tijdschr. Diergeneeskd. 102, 932–934. Monahan, C.M., Chapman, M.R., French, D.D., Taylor, H.W., Klei, T.R., 1995. Dose titration of moxidectin oral gel against gastrointestinal parasites of ponies. Vet. Parasitol. 59, 241–248. Murphy, D., Love, S., 1997. The pathogenic effects of experimental cyathostome infections in ponies. Vet. Parasitol. 70, 99–110. Ogbourne, C.P., 1975. Epidemiological studies on horses infected with nematodes of the family Trichonematidae (Witenberg, 1925). Int. J. Parasitol. 5, 667–672. Ogbourne, C.P., 1978. Pathogenesis of cyathostome (Trichonema) infections of the horse: its biology and veterinary importance. Commonwealth Institute of Helminthology, Miscellaneous Publication No. 5, p. 25. Reinemeyer, C.R., Herd, R.P., 1986. Comparison of two techniques for quantification of encysted larvae in the horse. Am. J. Vet. Res. 47, 507–509. Reinemeyer, C.R., Aguilar, R., 1992. Dose titration study of moxidectin gel as an oral equine antehlmintic. In: Proc. 37th Annual Meeting AAVP, Boston, p. 50. Xiao, L., Herd, R.P., Majefski, G.A., 1994. Comparative efficacy of moxidectin and ivermectin against hypobiotic and encysted cyathostomes and other equine parasites. Vet. Parasitol. 53, 83–90. Vercruysse, J., Eysker, M., Demeulenaere, D., Smets, K., Dorny, P., 1998. Persistent efficacy of a 2 equine moxidectin gel on establishment of Cyathostominae in horses. Vet. Rec., in press.

M. Eysker, T.R. Klei / Veterinary Parasitology 85 (1999) 137–149

145

Discussion (Klei — USA) We saw a difference in freezing after digestion, but the differences weren’t statistically significant. So if you freeze or don’t freeze, you find there is no significant difference between the populations of worms you get back. However, in our hands when you freeze and digest you get what Melanie (Chapman) describes as a floculant material which you don’t see if you don’t freeze for some reason. So it’s easier for us to look at the material digested fresh than it is digested after freezing, but we didn’t see a difference in numbers. (DiPietro — USA) Our experience was similar with frozen and fresh tissue. The differences in numbers weren’t statistically significant. And I would ask, so long as the lab knows you don’t get a false negative - i.e. a zero when there are really larvae there - and you treat all the samples the same way and freeze them, what difference does it make? The relative number may go up or down, but in drug studies you’re going to get your number. The only problem would be getting false negatives on EL3s because freezing precludes you from ever recovering any of them or the numbers are so small that it skews the data. (Klei — USA) If you use the same technique in the same study the numbers are not different. But if we want to describe the total populations of worms from one study to another, then we need to get closer to a real number somehow. Also, to clarify what you were saying about our work comparing digestion vs. transmural illumination (TMI) after treatment, what we saw was a decrease in the number of developing larvae we found in the treated worms after digestion versus TMI. So we’re seeing more worms in the TMI group than we were in the digested group and I think it’s because of some of those black worms. If we saw a worm we counted it - we didn’t try to differentiate dead versus alive. But if we digest them and they break up they aren’t counted. You get more disruption of some of those dead worms. In other words, the number we get back using the digestion technique is going to look like more of a real number. (Abbott — UK) At Glasgow, they have never continuously shaken mucosa from cattle, sheep, or horses when digesting tissues, and I wonder what sort of physical impact the actual shaking has. Do you think that could be contributing to the destruction of those larger larvae? (Eysker — The Netherlands) When I look at our experiences with material from ruminants, it is very obvious that by shaking the material you speed up the whole process tremendously. Whenever you don’t shake I expect at some stage larvae will come free from the mucosa. The whole process will take longer when you don’t shake it, and whenever these larvae come loose in the digestion period earlier, that the chance that they will be digested is greater than when you shake for shorter. (Abbott — UK) Coming back to this in particular, that is probably why Ken ( Bairden) started to do his “milking” procedure during digestion of equine mucosa. (Eysker — The Netherlands) Yes - and that’s possibly a reason why milking makes sense in his efforts. And why it didn’t in Tom’s (Klei). Because it was only time consuming and not giving you anything.

146

M. Eysker, T.R. Klei / Veterinary Parasitology 85 (1999) 137–149

(Klei — USA) We did it differently - we didn’t use the whole piece of intestine. We in fact did the milking by scraping off the mucosa. So we started the milking process the same time we started our normal process, and what Melanie (Chapman) found is we ended up with liters and liters of fluid and it wasn’t any faster. What we’ve done to keep the time lower is what Maarten (Eysker) has stressed - 25 grams of tissue per 200 mL. Sometimes that may mean you have 3 or 4 flasks per animal. That’s going to increase what you have to look at but decrease the time you have to digest. The only cautionary thing is, in animals that have small numbers of developing larvae, - the sample size you look at is so much smaller for the digestion than it is for the TMI, so in really low infections you might not get much of a count. (Reinemeyer — USA) The only thing I would add is that we’ve had excellent success with the digestion technique. We use a lot of the same criteria that Tom (Klei) and Maarten (Eysker) have suggested. For temperature, I think 40C seems to work better than 37C. It’s definitely faster. One of the little tricks we’ve learned is to make the pepsin up very fresh right before you use it. One of the most important things is for the pepsin solution to be at the target temperature when you add it to the tissue. What we generally do is get a shaking environmental incubator, mix the pepsin with warm water, and start shaking it in the incubator. It’s up to 40 C when we put the tissues in. The only difference is, because we do horses instead of ponies and frequently have a lot more tissue, we set our upper limit at 40 g in 200 mL in a 250 mL flask and we get excellent digestion 19 times out of 20. Once in a while you get one that frustrates you a little bit but I think we might be able to go a little higher than 25 because we go up to 40 routinely. We acidify the pepsin solution just before we pour it in there, too, but I don’t know if that makes any difference. (DiPietro — USA) Frankly, I don’t know how far we’ve come in 3 years on this topic. (Abbott — UK) We still haven’t reached agreement. (DiPietro — USA) We’ve learned a lot more but whether we’ve defined what the universal process ought to be I don’t know, and we may never get there. (Anderson — USA) I’d like to add one observation on Craig (Reinemeyer)’s work. The fact that you have a higher amount of tissue but you also have a higher temperature speeding up the reaction. I would imagine for that amount of tissue if you used 25 grams at 40C you may destroy some of the larvae. (Reinemeyer — USA) It could be. The end point is still subjective. I don’t think you can stick them in there for 118 minutes and then pull them out, because you just have to keep monitoring things. Occasionally you’ll get one that in an hour and 10 minutes is ready to come out and then some others take 2 and a half hours. So you have to really kind of watch them. (DiPietro — USA) So what’s your descriptor of an end point? (Reinemeyer — USA) Well - that’s kind of hard to say. Basically, we consider the endpoint to be when the supernatant gets cloudy and there are no large particles of mucosa left on

M. Eysker, T.R. Klei / Veterinary Parasitology 85 (1999) 137–149

147

the bottom of the digestion container. If the mucosal pieces are the size of oatmeal, further digestion is required. Normally, mucosal pieces can be reduced to a granular consistency; and the only identifiable items should be nematodes or pieces of ingesta that weren’t washed off prior to processing. Heavily infected tissues often contain a lot of scar tissue and obviously take longer to digest, and some of these never get completely reduced. The occasional sample must be harvested even if larger mucosal particles are still present. (Klei — USA) Craig (Reinemeyer) is right on - that’s exactly what done is. (Abbott — UK) There’s still an element of art in it. (Klei — USA) There’s a subjective level about when it’s finished. (Eysker — The Netherlands) And did you compare in those trials to TMI? (Reinemeyer — USA) If we compare counts with TMI versus counts of the larger developing stages with digestion, 39 times out of 40 I will get fewer larvae with digestion of the more advanced stages. My personal feeling is that digestion destroys some of the larger larvae. (Abbott — UK) Controls or treated? (Reinemeyer — USA) Overall (Eysker — The Netherlands) That’s my experience. We find lower numbers of developing stages in digestion. The only exception was in this trial where we waited for 5 weeks after anthelmintic treatment because the small ones had time to be recruited into the developmental stages. (Monahan — USA) I was just curious. I know you already mentioned it’s a statistical nightmare as it exists. But would there be any utility in using the difference between the larger larvae visualized on transmural versus digestion as an indicator of the percent that were killed? If the larger larvae were more fragile or refractory than a greater decrease in the treatment would show effect, would it not? (Eysker — The Netherlands) Killed by the digestion or killed by the anthelmintic. (Monahan — USA) If they were killed by the anthelmintic and then dying in situ. (Eysker — The Netherlands) I think you should be very careful because inevitably there will be differences between labs in the results of the digestion. It will be quite hazardous to use this as parameter of the effect of your treatment difference between transillumination and digestion, because it can be different as a result of different labs. (Scholl — USA): When you have a drug that does in fact kill EL3s, how many days after treatment do you start to see the effect of those hypobiotic larvae that may in fact be somewhat removed from the normal processes that may kill later stages that are out of hypobiosis. The reason I ask is, is even 2 weeks long enough to see the effect of

148

M. Eysker, T.R. Klei / Veterinary Parasitology 85 (1999) 137–149

some drugs on EL3s, so that we may be underestimating the effect, or is 5 weeks more reasonable? (Eysker — The Netherlands) Well - one thing is for certain. After 5 weeks when you use, for example, moxidectin you see no effect at all on the early third stage larvae. The people who can answer these questions best of whether two weeks are enough are probably those people who did the trials with fenbendazole or oxibendazole. In some of these trials the worm counts have been done after 2 weeks, right, Liz (Abbott)? (Abbott — UK) Yes. In Jim Duncan’s 1980 study, there was a two-week interval between treatment and necropsy and in a later (1993) unpublished study also done by Jim, the interval was three weeks. Efficacy against undifferentiated mucosal larvae was >95% for the larvicidal treatment. Perhaps the kinetics and metabolism of the drug needs to be considered. We know with fenbendazole that plasma drug levels are below the LOQ of the assay within two days of the last treatment. Fecal drug concentrations three days after the last treatment are less than 10% of those on the last treatment day and were below or near the LOQ of the assay 15 days after the last treatment. However, is moxidectin exerting its effect because of residual activity? Will it be possible to calculate the ideal treatment to necropsy interval when there are large differences between drug groups? One could sacrifice groups of horses at different time intervals after treatment to try to determine the optimum time interval, but that would be very expensive. (Eysker — The Netherlands) Perhaps drug companies could combine their efforts and finance studies like that. (Abbott — UK) That would be the only way to do it because you know the kinds of animal numbers it would take. (Lyons — USA) Regarding your larvicidal dose of fenbendazole on the EL3s, were they resistant? (Abbott — UK) Tom (Klei) had resistant strains present, and this was confirmed by poor efficacy against lumen adults. He can probably expand and explain his findings. (Klei — USA) In that group we had a population of animals with resistant worms and a population with completely susceptible worms, and the drug was effective against (EL3s of) both. The data were published jointly with Joe (DiPietro) and Craig (Reinemeyer). In ours we documented prior to treatment where the populations came from and the drug was effective against (EL3s of) both, but it was a little less effective against those that were resistant. (Eysker — The Netherlands) All mucosal stages? (Klei — USA) Total worm burdens, and mucosal stages. (Abbott — UK) Overall, the larvicidal dose was 90% effective against EL3s in that trial. (Klei — USA) There again, you are necropsying at 6 weeks and it is difficult to say whether you are killing all the worms in the lumen and getting some development. What you can do

M. Eysker, T.R. Klei / Veterinary Parasitology 85 (1999) 137–149

149

is look at total worms at the end of 6 weeks and if you do the counts the way we are now doing them, where we know how many EL3s are in the whole animal, not just a sample, how many developing larvae, how many adults, and how many L4s - now we can get a total worm burden as far as efficacy and adults to larvae. (Eysker — The Netherlands) How resistant was this benzimidazole resistant population? (Klei — USA) In some of the ponies in that herd the egg counts would stay the same or go up post treatment. (Abbott — UK) Fecal egg counts were done on a fortnightly basis through to necropsy. The treated group counts fell temporarily but then rose again. (Klei — USA) Interestingly, in that same trial we took fecal samples every week but we collected the whole rectal sample and we actually looked at that sample for worms. If you look at the controls, worms were just passing out all the time. It had nothing to do with drug treatment, it was just turnover and passage of worms. In the treated animals the worms were found in the first week and then we didn’t find any more. (Eysker — The Netherlands) Yes, well that’s the obvious thing you must be aware of. (Klei — USA) But I haven’t seen any data on it. (Eysker — The Netherlands) We did a trial where we used ivermectin at the end in a controlled as well as clinical trial and we also followed the number of worms excreted by the control ponies. There were three controls, three treated, but the effect, considering the clinical trial, was that non-treatment was almost 100% effective in some species in some of the control ponies because they shed worms in the feces as well after not having been treated. (Lyons — USA) I think Colglazier (AJVR: 40: 384-386) had a paper years ago on turnover of populations of cyathostomes. (Sangster — Australia) A quick comment about these benzimidazole resistant worms. In sheep there appear to be at least two steps in the development of benzimidazole resistance. In the first step where single doses are no longer effective if you give benzimidazole daily for 5 days, the drugs are still effective against those worms. In the second levels of resistance the drug delivered over 5 days is not effective. So I suspect the cyathostomes are now at that first level of resistance development, and so fenbendazole, daily for 5 days, is effective. How long it remains active is another question. I suspect that cyathostomes will develop to that second stage. (Abbott — UK) I think that’s why it’s important that these drugs are not over used. The larvicidal dose of fenbendazole should be used strategically and not every time the horse is dewormed.