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Shedding consistency of strongyle-type eggs in dutch boarding horses
Veterinary Parasitology 124 (2004) 249–258 www.elsevier.com/locate/vetpar
Shedding consistency of strongyle-type eggs in dutch boarding horses D. Do¨pfer*, C.M. Kerssens, Y.G.M. Meijer, J.H. Boersema, M. Eysker Division of Parasitology and Tropical Veterinary Diseases, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.165, 3508 TD, Utrecht, The Netherlands Received 6 February 2004; received in revised form 18 June 2004; accepted 26 June 2004
Abstract Faeces of 484 horses were sampled twice with an interval of 6 weeks while anthelmintic therapy was halted. Faecal eggs counts revealed that 267 (55.2%) horses had consistently low numbers of eggs per gram faeces (EPG) (EPG < 100 or = 100), 155 (32.0%) horses had consistently high EPGs (EPG > 100). Horses with consistently high EPGs were more often mares with access to pasture, aged less than 6 or more than 23 years, that were dewormed at intervals longer than 6 months, and were treated for the last time more than 3 months before the start of the study. Horses with consistently low EPGs were more often male horses with no or limited access to pasture, that were dewormed at maximally 6-month intervals, and were aged between 6 and 23 years. The results are an indication that some horses have consistently low EPGs and perhaps could be used as non-treated animals in a selective anthelmintic treatment scheme aimed at the prevention of the development of anthelmintic resistance. # 2004 Elsevier B.V. All rights reserved. Keywords: Horse; Strongylosis; Faecal egg count; Consistency
* Corresponding author: Present address. Animal Sciences Group of Wageningen UR, P.O. Box 65, 8200 AB Lelystad, The Netherlands. Tel.: +31 320 238464; fax: +31 320 238961. E-mail address: [email protected] (D. Do¨pfer). 0304-4017/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2004.06.028
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1. Introduction Strongylosis is a common problem in horses where clinical symptoms vary between acute colic, diarrhea and chronic weight loss (Ogbourne, 1978; Love et al., 1999). The wide spread infections with especially the cyathostomins have rendered the use of anthelmintics (AH) indispensable for control of patent infections and reduction of pasture contamination (Herd, 1990; Reinemeyer, 1998). Due to frequent usage of the three groups of AH (benzimidazoles, pyrantel and avermectins/milbemycins) the problem of nematode resistance has become a worldwide worry (Herd, 1990; Wescott, 1986; Eysker and Vercruysse, 1990; Boersema et al., 1991, 1995; Chapman et al., 1996; Waller, 1997; Boersema et al., 2002). In order to reduce the risk of AH resistance, alternatives for AH therapy are necessary (Wescott, 1986; Vercruysse and Eysker, 1989). Selective AH treatment of horses, for example, results in a decrease of selection pressure by generating refugia for worms (Duncan and Love, 1991). Recently, the need to use selective AH treatment was pointed out again (Coles et al., 2003). However, this approach requires costly animal monitoring using repeated faecal examination. If faecal egg counts were predictable and repeatable enough to define the ‘low egg shedders’ within a herd, the costs for faecal examination in a scheme for selective AH treatment could be reduced considerably. The aim of this study was to evaluate whether such predictability of faecal egg counts could be demonstrated. Furthermore, attempts were made to find risk factors associated with high and low EPGs consistent over time. For this end, eighteen cohorts of horses were followed during six weeks while AH treatment was suspended.
2. Materials, methods and animals 2.1. Sampling and laboratory analysis Eighteen boarding stables cooperated voluntarily as cohorts during a study period of 6 weeks, between September and November 2001. Two faecal samples were taken rectally with an interval of 6 weeks time. At the start, 504 horses were included in the study and 20 dropped out due to sale, death, and absence on the second day of sampling, resulting in 484 horses being included in the analysis. They were included if the last treatment with pyrantel was longer than 6 weeks, with ivermectin longer than 3 months and with moxidectin longer than 6 months ago. Individual and stall records of the following variables were collected: identitiy number, breed, age, gender, body weight, AH used, interval of deworming, time elapsed since last dewormed, change of AH and time interval of change of AH, access to pasture, pasture management and deworming strategy for newly arrived horses. Faecal samples were taken rectally or collected from non-contaminated freshly deposited faeces on the ground. Transport and storage of samples was done at 4 8C. Laboratory analysis of samples occurred within maximally five days after sampling. Faecal egg counts were accomplished using a modified McMaster method with a detection level of 50 EPG. Strongyle eggs were counted in two counting chambers of a
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haemocytometer and saturated NaCl solution was used for dilution and flotation (Kassai, 1999). A pooled larval culture (incubation period: 10 days at 27 8C) per cohort and date of sampling was made using 10 g faeces of each horse. The Baermann technique was used for recovery of the third stage larvae (L3) (Kassai, 1999). The L3 were differentiated according to Janssens et al. (1989). In order to evaluate the quality of the faecal egg counts, six replicates were re-counted for 10 samples with a faecal egg count of 100 EPG. Subsequently, the mean and standard error of the mean were calculated for the six replicates for each sample. Faecal samples with EPGs previously determined to be 0, 50, 100, 200, 400, 800, 1600, and 2000 were analyzed twice using the McMaster technique. This procedure was repeated for two different technicians separately. 2.2. Statistical analysis The statistical analysis was done using SPSS 9.0.0 for Windows (SPSS Inc.). A high and low EPG was set to be higher (>100) or smaller or equal to 100 EPG, respectively following the recommendation to administer AH treatment to animals with EPGs higher than 100 (Herd, 1990). The categories high and low EPG were then transformed into four groups to compare first and second faecal egg counts: low-remainslow (1), low-becomes-high (2), high-becomes-low (3) and high-remains-high (4). Given the average and standard deviation of the repeated six-fold counting of previously determined EPGs the standard error of the mean was calculated and used as the tolerance, being the allowable error, for EPGs. Statistical testing for agreement between technicians and repeated sampling of previously determined EPGs of samples was done using ANOVA and correlation coefficients. A descriptive analysis generated the average and standard deviation for the variables, recorded per horse and stable. Two-by-two tables were tabulated and relative risks (RR) with 95% confidence intervals were calculated for the relationship between consistently high/low EPGs and a variable considered to be a potential risk factor (Kleinbaum et al., 1988; Ahlbom and Norell, 1990). Statistically significant factors in the 2 2 analysis were selected as independent variables for a logistic regression analysis to estimate regression coefficients and predict consistently high or low EPGs as outcome variable (Kleinbaum et al., 1988). Farm number was forced into the models as a fixed effect and based on significant relations in the 2 2 analysis interaction terms created for gender pasture, age pasture and breed pasture. The significance level was a = 5%. The variable ‘farm’ was forced into the model as a fixed effect to correct for over dispersion. A backstep method of selection for statistically significant coefficients was used. The goodness of fit was calculated for every selection step to monitor its changes. The coefficients from the logistic regression were used for the calculation of odds ratios and their 95% confidence intervals (Hosmer and Lemeshow, 1989). Risk factor and risk are not interpreted as causal effects for an outcome variable of infection in this study. A statistically significant risk factor has a numeric link with the variable of outcome in the sense that infection has been found more often in the exposed group compared to the non-exposed group.
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3. Results 3.1. Samples Two times 484 faecal samples entered the analysis and EPGs had a right skewed distribution (Fig. 1). The EPGs one and two ranged from a minimum of zero to a maximum of 6650 EPG. The category with a first EPG 100 was n = 318 (65.7%) and first EPG > 100 was n = 166 (34.3%). The category with a second EPG 100 was n = 278 (57.4%)
Fig. 1. Frequency distribution of the EPG at the first (EPG1) and second (EPG2) visit of 484 horses sampled with 6 weeks interval after suspending anthelmintic treatment. The mean is the arithmetic mean.
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while the second EPG > 100 was n = 206 (42.6%). Most EPGs were consistently low n = 267 (55.2%) or high n = 155 (32.0%). Very few EPGs switched from high to low n = 11 (2.3%) or low to high n = 51 (10.5%). Animals with EPG1 100 had 12.7 times (95% C.I.: 7.1–22.5) more often an EPG2 100. 3.2. Quality of egg counts The 10 samples with an initial faecal egg count of 100 EPG that were re-examined in six-fold had an average EPG of 100 (S.D.: 21 EPG). Considering the two counting chambers used during the study, this resulted in a S.E.M. of 21/2 = 15 EPG. This meant that the category ‘‘low EPG’’ could be distinguished from ‘‘high EPG’’ with an acceptable error that is a tolerance of 15 EPG. In counting two chambers, the committed error to distinguish low and high EPG was acceptable, because in the worst case the EPG remained far below 200 EPG. The agreement between the two technicians while repeating the McMaster counting was good. The correlation coefficient of EPGs between two samplings was 0.83 for one technician and 0.82 for the second technician. The agreement between the two technicians when counting the same faecal sample was excellent (technician1 = 1.006 technician2 + 0.16, R2 = 0.99). The data is not shown. 3.3. Descriptive results and cross tabulation Geldings n = 217 (44.8%) and stallions four (0.8%) were joined into one category for gender as opposed to the mares n = 263 (54.3%). Body weight varied between a minimum of 150 kg and a maximum of 760 kg, (mean: 473 kg, S.D.: 105 kg). The body weight was evenly distributed over EPG categories and was not included during further testing. Nineteen breeds were categorized into two: the Dutch Warmblooded horses, Selle Francaise, Trakehner, Oldenburger, Westfalean, Belgian Warmblooded horse and Swiss Warmblooded horse, or the so-called ‘‘Western Sport breeds’’ comprising 227 horses (50%) as opposed to all other breeds, such as Arabians, New Forest, Welsh Pony, Frieseans, Shetlanders and the others. The age of the horses ranged between a minimum of 3 years and a maximum of 35 years, (mean: 12.1 years, S.D.: 6.1 years). They were categorized into horses younger than 5 years, n = 50 (10.3%), horses aged 6–23 years, n = 400 (82.7%), and horses older than 23 years, n = 34 (7.0%). There were neither foals nor yearlings in the study group. A group of 371 (76.7%) horses had access to pasture. Anthelmintics previously used were pyrantel 118 (24.4%), ivermectin 121 (25.0%), moxidectin 73 (15.1%), fenbendazole 135 (27.9), and no AH was used in 37 (7.6%) horses. The AH were categorized into effective (pyrantel, ivermectin, and moxidectin) 312 (64.5%) as opposed to not-effective (fenbendazole in a single dose) or no anthelmintics 172 (35.5%) horses. The interval of AH therapy was every 3 or 6 months, 402 (83.1%) in contrast to 12 months, sporadic or never 82 (16.2%) horses. A change of AH between effective AH (changed pyrantel to ivermectin, ivermectin to pyrantel or ivermectin to moxidectin) 254
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Table 1 Odds ratio and 95% confidence interval for the logistic regression for the independent variable: y = consistently high EPG (N = 484, d.f. 461) Variable
Farm Gender Last therapy Interval of therapy Age 6–23 years Gender pasture
Odds ratio
95% CI for Odds ratio
1.0110 0.2922 0.5095 0.3280 0.6889 3.7759
Lower
Upper
0.9698 0.1500 0.3212 0.1766 0.4886 2.3660
1.0540 0.5693 0.8084 0.6090 0.9713 6.0259
(50.6%) was compared to a change to fenbendazole (ivermectin to fenbendazole or pyrantel to fenbendazole) 239 (49.4%) horses. Last AH treatment shorter than 3 months ago 193 (39.9%) was opposed to AH therapy more than 3 months ago 291 (60.1%) horses. New arrivals received AH treatment immediately upon arrival 279 (57.6%) or together with the resident horses, sporadically or never 205 (42.4%) horses. The results of the 2 2 tables are not shown, but resulted in the selection of independent variables for the logistic regression. Furthermore, interaction terms were created, based on the 2 2 analysis: mares had more access to pasture compared to geldings and stallions (RR: 1.25 (95% C.I.: 1.13–1.39)). Animals between 3 and 5 years of age or older than 24 years, had more access to pasture (RR: 1.19 (95% C.I.: 1.06–1.34) and RR: 1.28 (95% C.I.: 1.15–1.41)). Western Sport breeds had less access to pasture (RR: 0.70 (95% C.I.: 0.63– 0.78)). 3.4. Results of the logistic regression Independent variables entering the logistic regression to model consistently high EPG were: farm, breed, age (6–23) years, gender, anthelmintic, last dewormed, change of anthelmintics, deworming upon arrival, deworming interval, access to pasture, gender pasture, age pasture, and breed pasture. The same regression was calculated using ‘‘consistently low EPG’’ as the dependent variable. The results are shown in Tables 1 and 2. Horses with access to pasture and
Table 2 Odds ratio and 95% confidence interval for the logistic regression for the independent variable: y = consistently low EPG (N = 484, d.f. 462) Variable
Odds ratio
95% CI for Odds ratio Lower
Upper
Farm Pasture Interval of therapy Gender pasture Age 6–23 years
0.9730 0.2840 2.8397 0.6310 1.7599
0.9356 0.1281 1.5816 0.4500 1.2278
1.0120 0.6294 5.0985 0.8849 2.5227
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especially mares with access to pasture had a lower risk of having a consistently low EPG and a higher risk of having a consistently high EPG. Horses dewormed at intervals of 3 or 6 months had more frequently a consistently low EPG and less frequently a consistently high EPG compared to horses dewormed at longer intervals of time or not at all. Horses aged 6–23 years had more frequently a consistently low EPG than horses between 3 and 5 years or 24 and more years of age. Horses that were dewormed less than three months before the start of the study had a lower chance for a consistently high EPG compared to horses dewormed earlier. 3.5. Faecal iarval cultures On 12 of the 18 farms, cyathostomins were the only larval type cultured, whereas this was the main larval type (>79%) on five other farms. No larvae were cultured on farm 11 where animals were housed permanently and treated with an AH every 3 or 6 months. On two of the other farms not applying anthelmintics, 11% (farm 3) and 4% (farm 6) of the larvae were Strongylus vulgaris. Small proportions (up to 8%) of Poteriostomum and/or Triodontophorus were found on farms 3, 4, 6, 10 and 14. The cyathostomins were found most frequently. The results of the differentiation of larvae per farm are shown in Table 3. 3.6. Discussion A cohort of 484 horses was sampled twice at an interval of 6 weeks and faecal egg counts were determined in order to study the consistency of strongyle egg shedding. A consistently low EPG was found in 267 (55.2%) of the 484 horses, which was a surprisingly large group considering the sampling season (September–October) when infections Table 3 Differentiation of 100 larvae per faecal culture in percentages according to (Janssens et al., 1989) Farm
Cyathostominae (%)
Strongylus vulgaris (%)
Poteriostomum spp. (%)
Triodontoforus spp. (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
100 100 79 99 100 92 100 100 100 98 0 100 100 97 100 100 100 100
0 0 11 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0
0 0 2 0 0 4 0 0 0 2 0 0 0 3 0 0 0 0
0 0 8 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0
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acquired during the summer could result in high egg counts. The committed error while executing the EPG counts and distinguishing between low and high EPG was acceptable, because in the worst case EPGs remained below 200 EPG, an EPG level that is often chosen as the limit for AH therapy in horses (Uhlinger, 1993). That low EPGs were found in more than 50% of the horses at both visits indicates that indeed it is possible to select animals with a consistent low EPG. These ‘low egg shedders’ are candidates for exclusion from AH treatment in a strategy aiming at selective AH treatment in order to delay development of AH resistance. The finding of these ‘low egg shedders’ is not really surprising and the criterion of low egg shedding has been used before to apply selective AH treatment (Gomez and Georgi, 1991; Krecek et al., 1994). The study of Duncan and Love (1991) already demonstrated that a proportion of the horses hardly ever had to be treated even when the criteria for treatment was the finding of eggs using a McMaster method with a detection level of 50 EPG. Moreover, it is not surprising because selective breeding against susceptibility of small ruminants for gastrointestinal nematode infections appears to be a feasible alternative approach for worm control (Bishop and Stear, 2003). Also in cattle the occurrence of ‘low responders’ shedding low numbers of eggs in their faeces is well known, that is being a genetic trait (Albers, 1981; Gasbarre et al., 2001; Kanobana et al., 2001). This genetic component for resistance implies that low faecal egg counts are heritable and repeatable (Gruner et al., 2002). However, there are several factors that imply that part of the horses in the present study with consistently low faecal egg counts may not be true ‘low egg shedders’. Not having access to pasture was a risk factor for consistently low egg counts. On one farm this coincided with frequent AH treatment and the absence of infective larvae in faecal cultures. This indicates for these horses that are part of the reason why low egg counts were observed, was that acquisition of strongyle infections was low, or on the farm with negative larval cultures, very low. Vercruysse and Eysker (1989) already mentioned that the risk for high infections in permanently housed animals is not high when suitable hygienic measures are implemented. This probably implies that nematode control for permanently housed horses can be achieved by hygienic measures alone. Horses treated at intervals of three or six months had a higher risk of low faecal egg counts at both visits than horses treated at longer intervals or not at all. Again this higher risk for low EPGs may partially reflect low exposure to worms on the farm as opposed to horses were ‘low egg shedders’. The same is true for the correlation of low faecal egg counts with the last AH treatment within the last three months before the study. On the other hand, the consequence of no, or very limited AH treatment such as on farms three and six results in the presence of the highly pathogenic species Strongylus vulgaris. It is well established that this species is often eliminated when AH treatment, in particular with ivermectin or moxidectin, is performed on a regular basis (Herd, 1990). Horses aged 6–23 years of age more often had repeated low EPGs than younger or older horses. This is not surprising because the studies about the build-up of immunity confirms that acquisition of immunity correlates with age (Klei and Chapman, 1999) and that very aged animals may loose their immunity again. Obviously, animals for ‘no treatment’ in a selective AH treatment strategy should be selected from the group of adult, but not yet geriatric horses. If foals and yearlings had been part of the present study the most likely
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outcome would have been that these categories, and in particular the yearlings, would have had the highest risk for high egg counts. Finally, the interval between both samplings was only six weeks. Obviously this implies that characterization of horses, as ‘low egg shedders’ are not definitive yet. Further confirmation would be necessary. Nevertheless, the study indicates that such ‘low egg shedders’ can be defined and are candidates for exclusion from AH treatment in a selective anthelmintic treatment system. In conclusion, the results indicate that candidate horses, for becoming ‘non-treated ’ animals in a selective AH treatment strategy with the aim to generate worm refugia can be selected by performing faecal examination of mature grazing horses. Costs of faecal examination can then be reduced considerably because only the horses that are not treated need to be examined to confirm that they are still excreting low numbers of eggs in their faeces. A cost-benefit analysis could help in optimizing the diagnostic costs and costs made during the preventive treatment as opposed to extra labor and costs made for treating and loosing horses due to (sub-)clinical disease caused by GI nematodes. It may be advisable to perform faecal examination for worm eggs more frequently on farms with a previous history of frequent AH treatment because of the possibility that horses may be misidentified as being ‘low egg shedders’. A major question obviously is which proportion of a herd should be left untreated. This needs further study, preferably by using a model for the population transmission dynamics of cyathotomines. The design of such a model would be worthwhile in the light of optimizing selective AH treatment in horses and reducing the risk of resistance development of GI nematodes against anthelmintics.
Acknowledgement The members of the boarding stables that collaborated during the study are thanked for their time and effort while sampling the horses.
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Coles, G.C., Eysker, M., Hodginson, J., Matthews, J.B., Kaplan, R.M., Klei, T.R., Sangster, N., 2003. Anthelmintic resistance and use of anthelmintics in horses. Vet. Rec. 153, 636. Duncan, J.L., Love, S., 1991. Preliminary observations on an alternative strategy for the control of horse strongyles. Equine Vet. J. 23, 226–228. Eysker, M., Vercruysse, J., 1990. Epidemiologie, chemotherapie, anthelmintica-resistentie en Preventie van Strongylidae-infecties bij het paard. Tijdschr. Diergeneeskd. 115, 891–907. Gasbarre, L.C., Leighton, E.A., Sonstegard, T., 2001. Role of the bovine immune system and genome in resistance to gastrointestinal nematodes. Vet. Parasitol. 98, 51–64. Gomez, H.H., Georgi, J.R., 1991. Equine helminth infections: control by selective chemotherapy. Equine Vet. J. 23 (3), 198–200. Gruner, L., Cortet, J., Sauve, C., Limouzin, C., Brunel, J.C., 2002. Evoluotion of nematode community in grazing sheep selected for resistance and susceptibility to Teladorsagia circumcincta and Trichostrongylus colubriformis: a four-year experiment. Vet. Parasitol. 109, 277–291. Herd, R.P., 1990. Equine parasite control–solutions to anthelmintic associated problems. Equine Vet. Educ. 2, 86–91. Hosmer, D.W., Lemeshow, S., 1989. Applied Logistic Regression,Wiley, New York, USA. Janssens, P.G., Vercruysse, J., Jansen, J., 1989. Wormen en wormziekten bij mens en huisdier. Samson Stafleu, Alphen ad Rijn/ Brussel/Belgium. Lab. Diagnostiek, Chapter 29. Kanobana, K., Vervelde, L., Veer, M. van der, Eysker, M., Ploeger, H.W., 2001. Characterisation of host responder types after a single Cooperia oncophora infection: kinetics of the systemic immune response. Parasite Immunol. 23, 641–653. Kassai, T., 1999. Veterinary Helminthology. Butterworth–Heinemann, 188–189. Klei, T.R., Chapman, M.R., 1999. Immunity in equine cyathostome infections. Vet. Parasitol. 85, 123–136. Kleinbaum, D.G., Kupper, L.L., Muller, K.E., 1988. Applied Regression Analysis and Other Multivariate Methods, second ed.Duxbure Press, Belmont, USA. Krecek, R.C., Guthrie, A.J., Van Nieuwenhuizen, L.C., Booth, L.M., 1994. A comparison between the effects of conventional and selective antiparasitic treatments on nematode parasites of horses from two management schemes. J. S. Afr. Vet. Assoc. 65 (3), 97–100. Love, S., Murphy, D., Mellor, D., 1999. Pathogenicity of cyathostome infections. Vet. Parasitol. 85, 115–122. Ogbourne, C.P., 1978. Pathogenesis of Cyathostome (Trichonema) Infections of the Horse. A Review Commonwealth Institute of Helminthology, Miscellaneous publication no. 5, p. 25. Reinemeyer, C., 1998. Practical and theoretical consequences of larvicidal therapy. Equine Prac. 20, 10–13. SPSS version 9.00 for Windows. SPSS Inc. Headquarters, 233 S. Wacker Drive, 11th floor, Chicago, Illinois, USA 60606. Uhlinger, C.A., 1993. Uses of faecal egg count data in equine practice. Comp. Cont. Educ. Pract. Vet. 15 (7), 42– 747. Vercruysse, J., Eysker, M., 1989. De diagnose van gastro-intestinale nematoden infecties bij het Paard. Vlaams Diergeneeskd. Tijdschr. 58, 36–43. Waller, P.J., 1997. Anthelmintic resistance. Vet. Parasitol. 72, 391–412. Wescott, R.B., 1986. Anthelmintics and drug resistance. Vet. Clin. North Am.: Equine Pract. 2, 367–380.
Shedding consistency of strongyle-type eggs in dutch boarding horses D. Do¨pfer*, C.M. Kerssens, Y.G.M. Meijer, J.H. Boersema, M. Eysker Division of Parasitology and Tropical Veterinary Diseases, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.165, 3508 TD, Utrecht, The Netherlands Received 6 February 2004; received in revised form 18 June 2004; accepted 26 June 2004
Abstract Faeces of 484 horses were sampled twice with an interval of 6 weeks while anthelmintic therapy was halted. Faecal eggs counts revealed that 267 (55.2%) horses had consistently low numbers of eggs per gram faeces (EPG) (EPG < 100 or = 100), 155 (32.0%) horses had consistently high EPGs (EPG > 100). Horses with consistently high EPGs were more often mares with access to pasture, aged less than 6 or more than 23 years, that were dewormed at intervals longer than 6 months, and were treated for the last time more than 3 months before the start of the study. Horses with consistently low EPGs were more often male horses with no or limited access to pasture, that were dewormed at maximally 6-month intervals, and were aged between 6 and 23 years. The results are an indication that some horses have consistently low EPGs and perhaps could be used as non-treated animals in a selective anthelmintic treatment scheme aimed at the prevention of the development of anthelmintic resistance. # 2004 Elsevier B.V. All rights reserved. Keywords: Horse; Strongylosis; Faecal egg count; Consistency
* Corresponding author: Present address. Animal Sciences Group of Wageningen UR, P.O. Box 65, 8200 AB Lelystad, The Netherlands. Tel.: +31 320 238464; fax: +31 320 238961. E-mail address: [email protected] (D. Do¨pfer). 0304-4017/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2004.06.028
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1. Introduction Strongylosis is a common problem in horses where clinical symptoms vary between acute colic, diarrhea and chronic weight loss (Ogbourne, 1978; Love et al., 1999). The wide spread infections with especially the cyathostomins have rendered the use of anthelmintics (AH) indispensable for control of patent infections and reduction of pasture contamination (Herd, 1990; Reinemeyer, 1998). Due to frequent usage of the three groups of AH (benzimidazoles, pyrantel and avermectins/milbemycins) the problem of nematode resistance has become a worldwide worry (Herd, 1990; Wescott, 1986; Eysker and Vercruysse, 1990; Boersema et al., 1991, 1995; Chapman et al., 1996; Waller, 1997; Boersema et al., 2002). In order to reduce the risk of AH resistance, alternatives for AH therapy are necessary (Wescott, 1986; Vercruysse and Eysker, 1989). Selective AH treatment of horses, for example, results in a decrease of selection pressure by generating refugia for worms (Duncan and Love, 1991). Recently, the need to use selective AH treatment was pointed out again (Coles et al., 2003). However, this approach requires costly animal monitoring using repeated faecal examination. If faecal egg counts were predictable and repeatable enough to define the ‘low egg shedders’ within a herd, the costs for faecal examination in a scheme for selective AH treatment could be reduced considerably. The aim of this study was to evaluate whether such predictability of faecal egg counts could be demonstrated. Furthermore, attempts were made to find risk factors associated with high and low EPGs consistent over time. For this end, eighteen cohorts of horses were followed during six weeks while AH treatment was suspended.
2. Materials, methods and animals 2.1. Sampling and laboratory analysis Eighteen boarding stables cooperated voluntarily as cohorts during a study period of 6 weeks, between September and November 2001. Two faecal samples were taken rectally with an interval of 6 weeks time. At the start, 504 horses were included in the study and 20 dropped out due to sale, death, and absence on the second day of sampling, resulting in 484 horses being included in the analysis. They were included if the last treatment with pyrantel was longer than 6 weeks, with ivermectin longer than 3 months and with moxidectin longer than 6 months ago. Individual and stall records of the following variables were collected: identitiy number, breed, age, gender, body weight, AH used, interval of deworming, time elapsed since last dewormed, change of AH and time interval of change of AH, access to pasture, pasture management and deworming strategy for newly arrived horses. Faecal samples were taken rectally or collected from non-contaminated freshly deposited faeces on the ground. Transport and storage of samples was done at 4 8C. Laboratory analysis of samples occurred within maximally five days after sampling. Faecal egg counts were accomplished using a modified McMaster method with a detection level of 50 EPG. Strongyle eggs were counted in two counting chambers of a
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haemocytometer and saturated NaCl solution was used for dilution and flotation (Kassai, 1999). A pooled larval culture (incubation period: 10 days at 27 8C) per cohort and date of sampling was made using 10 g faeces of each horse. The Baermann technique was used for recovery of the third stage larvae (L3) (Kassai, 1999). The L3 were differentiated according to Janssens et al. (1989). In order to evaluate the quality of the faecal egg counts, six replicates were re-counted for 10 samples with a faecal egg count of 100 EPG. Subsequently, the mean and standard error of the mean were calculated for the six replicates for each sample. Faecal samples with EPGs previously determined to be 0, 50, 100, 200, 400, 800, 1600, and 2000 were analyzed twice using the McMaster technique. This procedure was repeated for two different technicians separately. 2.2. Statistical analysis The statistical analysis was done using SPSS 9.0.0 for Windows (SPSS Inc.). A high and low EPG was set to be higher (>100) or smaller or equal to 100 EPG, respectively following the recommendation to administer AH treatment to animals with EPGs higher than 100 (Herd, 1990). The categories high and low EPG were then transformed into four groups to compare first and second faecal egg counts: low-remainslow (1), low-becomes-high (2), high-becomes-low (3) and high-remains-high (4). Given the average and standard deviation of the repeated six-fold counting of previously determined EPGs the standard error of the mean was calculated and used as the tolerance, being the allowable error, for EPGs. Statistical testing for agreement between technicians and repeated sampling of previously determined EPGs of samples was done using ANOVA and correlation coefficients. A descriptive analysis generated the average and standard deviation for the variables, recorded per horse and stable. Two-by-two tables were tabulated and relative risks (RR) with 95% confidence intervals were calculated for the relationship between consistently high/low EPGs and a variable considered to be a potential risk factor (Kleinbaum et al., 1988; Ahlbom and Norell, 1990). Statistically significant factors in the 2 2 analysis were selected as independent variables for a logistic regression analysis to estimate regression coefficients and predict consistently high or low EPGs as outcome variable (Kleinbaum et al., 1988). Farm number was forced into the models as a fixed effect and based on significant relations in the 2 2 analysis interaction terms created for gender pasture, age pasture and breed pasture. The significance level was a = 5%. The variable ‘farm’ was forced into the model as a fixed effect to correct for over dispersion. A backstep method of selection for statistically significant coefficients was used. The goodness of fit was calculated for every selection step to monitor its changes. The coefficients from the logistic regression were used for the calculation of odds ratios and their 95% confidence intervals (Hosmer and Lemeshow, 1989). Risk factor and risk are not interpreted as causal effects for an outcome variable of infection in this study. A statistically significant risk factor has a numeric link with the variable of outcome in the sense that infection has been found more often in the exposed group compared to the non-exposed group.
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3. Results 3.1. Samples Two times 484 faecal samples entered the analysis and EPGs had a right skewed distribution (Fig. 1). The EPGs one and two ranged from a minimum of zero to a maximum of 6650 EPG. The category with a first EPG 100 was n = 318 (65.7%) and first EPG > 100 was n = 166 (34.3%). The category with a second EPG 100 was n = 278 (57.4%)
Fig. 1. Frequency distribution of the EPG at the first (EPG1) and second (EPG2) visit of 484 horses sampled with 6 weeks interval after suspending anthelmintic treatment. The mean is the arithmetic mean.
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while the second EPG > 100 was n = 206 (42.6%). Most EPGs were consistently low n = 267 (55.2%) or high n = 155 (32.0%). Very few EPGs switched from high to low n = 11 (2.3%) or low to high n = 51 (10.5%). Animals with EPG1 100 had 12.7 times (95% C.I.: 7.1–22.5) more often an EPG2 100. 3.2. Quality of egg counts The 10 samples with an initial faecal egg count of 100 EPG that were re-examined in six-fold had an average EPG of 100 (S.D.: 21 EPG). Considering the two counting chambers used during the study, this resulted in a S.E.M. of 21/2 = 15 EPG. This meant that the category ‘‘low EPG’’ could be distinguished from ‘‘high EPG’’ with an acceptable error that is a tolerance of 15 EPG. In counting two chambers, the committed error to distinguish low and high EPG was acceptable, because in the worst case the EPG remained far below 200 EPG. The agreement between the two technicians while repeating the McMaster counting was good. The correlation coefficient of EPGs between two samplings was 0.83 for one technician and 0.82 for the second technician. The agreement between the two technicians when counting the same faecal sample was excellent (technician1 = 1.006 technician2 + 0.16, R2 = 0.99). The data is not shown. 3.3. Descriptive results and cross tabulation Geldings n = 217 (44.8%) and stallions four (0.8%) were joined into one category for gender as opposed to the mares n = 263 (54.3%). Body weight varied between a minimum of 150 kg and a maximum of 760 kg, (mean: 473 kg, S.D.: 105 kg). The body weight was evenly distributed over EPG categories and was not included during further testing. Nineteen breeds were categorized into two: the Dutch Warmblooded horses, Selle Francaise, Trakehner, Oldenburger, Westfalean, Belgian Warmblooded horse and Swiss Warmblooded horse, or the so-called ‘‘Western Sport breeds’’ comprising 227 horses (50%) as opposed to all other breeds, such as Arabians, New Forest, Welsh Pony, Frieseans, Shetlanders and the others. The age of the horses ranged between a minimum of 3 years and a maximum of 35 years, (mean: 12.1 years, S.D.: 6.1 years). They were categorized into horses younger than 5 years, n = 50 (10.3%), horses aged 6–23 years, n = 400 (82.7%), and horses older than 23 years, n = 34 (7.0%). There were neither foals nor yearlings in the study group. A group of 371 (76.7%) horses had access to pasture. Anthelmintics previously used were pyrantel 118 (24.4%), ivermectin 121 (25.0%), moxidectin 73 (15.1%), fenbendazole 135 (27.9), and no AH was used in 37 (7.6%) horses. The AH were categorized into effective (pyrantel, ivermectin, and moxidectin) 312 (64.5%) as opposed to not-effective (fenbendazole in a single dose) or no anthelmintics 172 (35.5%) horses. The interval of AH therapy was every 3 or 6 months, 402 (83.1%) in contrast to 12 months, sporadic or never 82 (16.2%) horses. A change of AH between effective AH (changed pyrantel to ivermectin, ivermectin to pyrantel or ivermectin to moxidectin) 254
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Table 1 Odds ratio and 95% confidence interval for the logistic regression for the independent variable: y = consistently high EPG (N = 484, d.f. 461) Variable
Farm Gender Last therapy Interval of therapy Age 6–23 years Gender pasture
Odds ratio
95% CI for Odds ratio
1.0110 0.2922 0.5095 0.3280 0.6889 3.7759
Lower
Upper
0.9698 0.1500 0.3212 0.1766 0.4886 2.3660
1.0540 0.5693 0.8084 0.6090 0.9713 6.0259
(50.6%) was compared to a change to fenbendazole (ivermectin to fenbendazole or pyrantel to fenbendazole) 239 (49.4%) horses. Last AH treatment shorter than 3 months ago 193 (39.9%) was opposed to AH therapy more than 3 months ago 291 (60.1%) horses. New arrivals received AH treatment immediately upon arrival 279 (57.6%) or together with the resident horses, sporadically or never 205 (42.4%) horses. The results of the 2 2 tables are not shown, but resulted in the selection of independent variables for the logistic regression. Furthermore, interaction terms were created, based on the 2 2 analysis: mares had more access to pasture compared to geldings and stallions (RR: 1.25 (95% C.I.: 1.13–1.39)). Animals between 3 and 5 years of age or older than 24 years, had more access to pasture (RR: 1.19 (95% C.I.: 1.06–1.34) and RR: 1.28 (95% C.I.: 1.15–1.41)). Western Sport breeds had less access to pasture (RR: 0.70 (95% C.I.: 0.63– 0.78)). 3.4. Results of the logistic regression Independent variables entering the logistic regression to model consistently high EPG were: farm, breed, age (6–23) years, gender, anthelmintic, last dewormed, change of anthelmintics, deworming upon arrival, deworming interval, access to pasture, gender pasture, age pasture, and breed pasture. The same regression was calculated using ‘‘consistently low EPG’’ as the dependent variable. The results are shown in Tables 1 and 2. Horses with access to pasture and
Table 2 Odds ratio and 95% confidence interval for the logistic regression for the independent variable: y = consistently low EPG (N = 484, d.f. 462) Variable
Odds ratio
95% CI for Odds ratio Lower
Upper
Farm Pasture Interval of therapy Gender pasture Age 6–23 years
0.9730 0.2840 2.8397 0.6310 1.7599
0.9356 0.1281 1.5816 0.4500 1.2278
1.0120 0.6294 5.0985 0.8849 2.5227
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especially mares with access to pasture had a lower risk of having a consistently low EPG and a higher risk of having a consistently high EPG. Horses dewormed at intervals of 3 or 6 months had more frequently a consistently low EPG and less frequently a consistently high EPG compared to horses dewormed at longer intervals of time or not at all. Horses aged 6–23 years had more frequently a consistently low EPG than horses between 3 and 5 years or 24 and more years of age. Horses that were dewormed less than three months before the start of the study had a lower chance for a consistently high EPG compared to horses dewormed earlier. 3.5. Faecal iarval cultures On 12 of the 18 farms, cyathostomins were the only larval type cultured, whereas this was the main larval type (>79%) on five other farms. No larvae were cultured on farm 11 where animals were housed permanently and treated with an AH every 3 or 6 months. On two of the other farms not applying anthelmintics, 11% (farm 3) and 4% (farm 6) of the larvae were Strongylus vulgaris. Small proportions (up to 8%) of Poteriostomum and/or Triodontophorus were found on farms 3, 4, 6, 10 and 14. The cyathostomins were found most frequently. The results of the differentiation of larvae per farm are shown in Table 3. 3.6. Discussion A cohort of 484 horses was sampled twice at an interval of 6 weeks and faecal egg counts were determined in order to study the consistency of strongyle egg shedding. A consistently low EPG was found in 267 (55.2%) of the 484 horses, which was a surprisingly large group considering the sampling season (September–October) when infections Table 3 Differentiation of 100 larvae per faecal culture in percentages according to (Janssens et al., 1989) Farm
Cyathostominae (%)
Strongylus vulgaris (%)
Poteriostomum spp. (%)
Triodontoforus spp. (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
100 100 79 99 100 92 100 100 100 98 0 100 100 97 100 100 100 100
0 0 11 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0
0 0 2 0 0 4 0 0 0 2 0 0 0 3 0 0 0 0
0 0 8 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0
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acquired during the summer could result in high egg counts. The committed error while executing the EPG counts and distinguishing between low and high EPG was acceptable, because in the worst case EPGs remained below 200 EPG, an EPG level that is often chosen as the limit for AH therapy in horses (Uhlinger, 1993). That low EPGs were found in more than 50% of the horses at both visits indicates that indeed it is possible to select animals with a consistent low EPG. These ‘low egg shedders’ are candidates for exclusion from AH treatment in a strategy aiming at selective AH treatment in order to delay development of AH resistance. The finding of these ‘low egg shedders’ is not really surprising and the criterion of low egg shedding has been used before to apply selective AH treatment (Gomez and Georgi, 1991; Krecek et al., 1994). The study of Duncan and Love (1991) already demonstrated that a proportion of the horses hardly ever had to be treated even when the criteria for treatment was the finding of eggs using a McMaster method with a detection level of 50 EPG. Moreover, it is not surprising because selective breeding against susceptibility of small ruminants for gastrointestinal nematode infections appears to be a feasible alternative approach for worm control (Bishop and Stear, 2003). Also in cattle the occurrence of ‘low responders’ shedding low numbers of eggs in their faeces is well known, that is being a genetic trait (Albers, 1981; Gasbarre et al., 2001; Kanobana et al., 2001). This genetic component for resistance implies that low faecal egg counts are heritable and repeatable (Gruner et al., 2002). However, there are several factors that imply that part of the horses in the present study with consistently low faecal egg counts may not be true ‘low egg shedders’. Not having access to pasture was a risk factor for consistently low egg counts. On one farm this coincided with frequent AH treatment and the absence of infective larvae in faecal cultures. This indicates for these horses that are part of the reason why low egg counts were observed, was that acquisition of strongyle infections was low, or on the farm with negative larval cultures, very low. Vercruysse and Eysker (1989) already mentioned that the risk for high infections in permanently housed animals is not high when suitable hygienic measures are implemented. This probably implies that nematode control for permanently housed horses can be achieved by hygienic measures alone. Horses treated at intervals of three or six months had a higher risk of low faecal egg counts at both visits than horses treated at longer intervals or not at all. Again this higher risk for low EPGs may partially reflect low exposure to worms on the farm as opposed to horses were ‘low egg shedders’. The same is true for the correlation of low faecal egg counts with the last AH treatment within the last three months before the study. On the other hand, the consequence of no, or very limited AH treatment such as on farms three and six results in the presence of the highly pathogenic species Strongylus vulgaris. It is well established that this species is often eliminated when AH treatment, in particular with ivermectin or moxidectin, is performed on a regular basis (Herd, 1990). Horses aged 6–23 years of age more often had repeated low EPGs than younger or older horses. This is not surprising because the studies about the build-up of immunity confirms that acquisition of immunity correlates with age (Klei and Chapman, 1999) and that very aged animals may loose their immunity again. Obviously, animals for ‘no treatment’ in a selective AH treatment strategy should be selected from the group of adult, but not yet geriatric horses. If foals and yearlings had been part of the present study the most likely
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outcome would have been that these categories, and in particular the yearlings, would have had the highest risk for high egg counts. Finally, the interval between both samplings was only six weeks. Obviously this implies that characterization of horses, as ‘low egg shedders’ are not definitive yet. Further confirmation would be necessary. Nevertheless, the study indicates that such ‘low egg shedders’ can be defined and are candidates for exclusion from AH treatment in a selective anthelmintic treatment system. In conclusion, the results indicate that candidate horses, for becoming ‘non-treated ’ animals in a selective AH treatment strategy with the aim to generate worm refugia can be selected by performing faecal examination of mature grazing horses. Costs of faecal examination can then be reduced considerably because only the horses that are not treated need to be examined to confirm that they are still excreting low numbers of eggs in their faeces. A cost-benefit analysis could help in optimizing the diagnostic costs and costs made during the preventive treatment as opposed to extra labor and costs made for treating and loosing horses due to (sub-)clinical disease caused by GI nematodes. It may be advisable to perform faecal examination for worm eggs more frequently on farms with a previous history of frequent AH treatment because of the possibility that horses may be misidentified as being ‘low egg shedders’. A major question obviously is which proportion of a herd should be left untreated. This needs further study, preferably by using a model for the population transmission dynamics of cyathotomines. The design of such a model would be worthwhile in the light of optimizing selective AH treatment in horses and reducing the risk of resistance development of GI nematodes against anthelmintics.
Acknowledgement The members of the boarding stables that collaborated during the study are thanked for their time and effort while sampling the horses.
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