Does examination of fecal samples 24 hours after cestocide treatment increase the sensitivity of Anoplocephala spp. detection in naturally infected horses?

Abstract

Fecal samples were examined immediately before and 24 to 48 h after cestocide treatment for a comparative detection of tapeworm-positive horses. In early winter, 17 weanlings, 20 yearlings, 15 2-year-old horses, 24 breeding mares, and 2 stallions were treated with praziquantel in combination with a macrocyclic lactone. The horses were presumed to be naturally infected with tapeworms after pasture grazing. Fecal samples were collected before treatment (Day 0), at 24 or 48 h after treatment (Day 1–2), and 16 to 21 d after treatment (Day 16–21). A Wisconsin test was done on all fecal samples. Odds of detection of infection for all age groups increased by a factor of 2.04 [95% confidence interval (CI): 1.30 to 3.20] from Day 0 to Day 1–2 (P = 0.002).

Introduction

Tapeworm infection is widespread in North American horses. Prevalence studies have shown that tapeworms can be recovered in up to 64% of horses at necropsy (1–4). A recent Canadian study reported a seroprevalence of 58% in a selected subpopulation of horses (5). On some farms, up to 100% of the horses may be infected with tapeworms. Anoplocephala perfoliata is by far the most frequent species recovered from these horses (2).

Recent studies suggesting a correlation between tapeworm infections and colic in horses increased the concern of veterinarians and horse owners over tapeworms (6–10). The detection and treatment of tapeworms is the key to any control program. The sensitivity of commonly used fecal techniques for the diagnosis of Anoplocehala perfoliata infection, however, can be very low, especially in horses that have less than 100 tapeworms (11). IgG(T) subtype antibody response to the 12/13-kDa parasite excretory/secretory antigen is positively correlated with parasite intensity (12). Unfortunately, this serological test is not commercially available in Canada; therefore, Canadian equine veterinarians must rely on low sensitivity fecal techniques to detect tapeworm infection in horses.

In recent studies, Slocombe et al (13–15) reported an increased sensitivity of the Cornell-Wisconsin centrifugal flotation technique with fecal samples collected 24 h after treatment with cestocidal drugs compared with results obtained from pre-treatment fecal samples. The objective of this study was to compare the effectiveness of 2 different protocols based on fecal samples collected before or after cestocidal treatment for detection of tapeworm infection in horses.

Materials and methods

Animals and treatments

Breeding mares (n = 24), stallions (n = 2), 2-year-olds (n = 15), yearlings (n = 20), and weanling warmbloods of both sexes (n = 17), presumed to be naturally infected with tapeworms after pasture grazing, were included in the study. These horses were presumed to be infected because tapeworm-positive horses had been identified on that farm in the past. The farm was selected for the trial because the farm management was excellent and the owner agreed to collaborate. All horses were located on the same farm in the Ottawa valley, Ontario. All the mares and stallions and approximately 50% of the younger horses were treated PO with 2.5 mg/kg body weight (BW) of praziquantel and 0.4 mg/kg BW of moxidectin (MP) (Quest Plus Gel; Wyeth Animal Health, Guelph, Ontario). Because the younger horses were included in a comparative macrocyclic lactone trial, the 2-year-olds, yearlings, and weanling warmbloods were blocked by age and randomly allocated to one of the following groups: Group 1 — treated orally with MP, and Group 2 —treated orally with 1.5 mg/kg of praziquantel and 0.2 mg/kg of ivermectin (IP) (Equimax; Pfizer Animal Health, Montréal, Québec).

The horses were treated on December 13, 2006 (weanlings, stallions, and 17 mares), on December 14, 2006 (7 mares), or on December 19, 2006 (2-year-olds and yearlings) after removal from pasture by the farm manager. On the day of treatment, all the horses were put into stalls without feed to ensure that no feed was present in their mouths at the time of treatment. Each horse was individually weighed on a digital scale (Instaweight digital animal scale–model LCD-U2100U, indicator model 83-10; Norac System International, Saskatoon, Saskatchewan). The farm manager was instructed by his veterinarian on how to administer oral dewormers. The products were administered in the back of the mouth at the base of the tongue with prefilled syringes set at the upper nearest 25 kg mark (MP) or 100 kg mark (IP). The products were kept at room temperature until they were used for treatment. The farm manager collected the fecal samples, and the horses were then fed dry hay and commercial grain and housed for the winter.

Fecal examination

Fecal samples were collected from all horses on the day of treatment (Day 0), 24 h later (except for 19 mares for which the fecal samples were collected 48 h after treatment) (Day 1–2), 16 to 21 d after treatment (Day 16–21) and 144 to 155 d after treatment (Day 144–155). The fecal samples were sent by the farm manager to the Parasitology Laboratory of the Université de Montréal by overnight courier where they were stored in the refrigerator until examination the day after reception. The technician, who was blinded to treatment protocols, counted the tapeworm fecal eggs per 5 g of feces by performing a modified Wisconsin sugar centrifugation technique (17). Since the younger horses had been included in a macrocyclic lactone trial, strongyle fecal eggs were also counted to compare the efficacy of the 2 products. The results are reported elsewhere (16).

Statistical analysis

To analyze changes in prevalence through time for all horses, the results for weanlings and yearlings and then for mares and stallions were pooled given the very low number of animals or the very low prevalence of positive horses in some of these age groups. A repeated-measures logistic regression model, with type of horses as a between-subject factor and time (2 measures: t0 and t2) as a within-subject factor was used. The exact McNemar test was used to compare the prevalence of positive horses on Day 0 and Day 1–2 for each age category group. Logarithmic transformations [log 10(x + 1)] were done on tapeworm fecal egg counts to obtain a normal distribution of values. A linear repeated-measures model, with categories (5 levels) as between-subject factors and time (3 levels) as a within-subject factor, was used to analyze the data (SAS version 9.1, SAS Institute, Cary, North Carolina, USA). A priori contrasts, with Bonferroni sequential correction, were used to examine differences between means for each categorical variable. Differences were regarded as significant at a level of P < 0.05.

Results

When controlling for type of horses, odds of infection for all age groups increased by a factor of 2.04 [95% confidence interval (CI): 1.30 to 3.20] from time 0 to time 2 (P = 0.002). Within the different age categories, the prevalence of positive adults (mares + stallions) was statistically higher on Day 1–2 compared with the prevalence of positive adults on Day 0 (P = 0.03) (Table 1). The prevalence of positive horses in the 2-year-old and in the foal (weanlings + yearlings) groups was numerically higher on Day 1–2 than on Day 0; however, no statistically significant difference was reached for these age groups (P > 0.05). All the horses that were positive for tapeworm eggs on Day 0 were also positive on Day 1–2 (data not shown).

Table 1.

Prevalence of tapeworm positive horses within age categorie

	Number of horses	Prevalence (numbers and percentages of horses positive for tapeworm positive horses)
		Day 0 (Treatment day)	Day 1–2
		Wisconsin	Wisconsin
Mares + stallions	26	7.7%a (2)	30.8%b (7)
2-year-olds	15	66.7%a (10)	73.3%a (11)
Weanlings + yearlings	37	16.2%a (6)	21.6%a (8)

^a,bValues with different alphabetical superscripts within rows differ significantly (P < 0.05).

The mean tapeworm fecal egg count for all the horses was statistically higher at Day 1–2 than at Day 0 or Day 21 (P = 0.001) (Table 2). At Day 1–2, the mean fecal egg count was statistically higher in 2-year-olds and yearlings than in mares or weanlings (P = 0.001). The tapeworm fecal egg counts per 5 g of feces at Day 1–2 ranged from 0 to 110 for mares, 0 to 6 for stallions, 0 to 252 for 2-year-olds, 0 to 1105 for yearlings, and from 0 to 0 for weanlings. No tapeworm eggs were found in weanlings over the entire study. All the horses had negative tapeworm fecal egg counts at Day 16–21 and Day 144–155.

Table 2.

Geometric mean tapeworm fecal egg counts per 5 grams of feces

		Geometric mean tapeworm fecal eggs per 5 grams of feces
	Number of horses	Day 0 (CI)	Day 1–2 (CI)	Day 16–21 (CI)
Mares	24	0.14 (0–0.34)	0.821 (0.20–1.74)	0
Stallions	2	0	1.651,2 (0–1106)	0
2-year-olds	15	2.53 (1.06–5.06)	10.252 (3.70–25.96)	0
Yearlings	20	1.20 (0.31–2.69)	4.682 (1.05–14.74)	0
Weanlings	17	0	01	0
All horses	78	0.63a (0.35–0.96)	2.06b (1.14–3.38)	0a

CI–confidence interval.

^a,bValues with different alphabetical superscripts within rows differ significantly (P < 0.05).

^1,2Values with different numerical superscripts within columns differ significantly (P < 0.05).

Discussion

The results of this study are in agreement with other studies reporting an increased sensitivity of coprological techniques for diagnosing tapeworm infection in horses when fecal samples are taken at 24 h after treatment with a cestocidal drug (13–15,18). Slocombe (13) reported an increase in the sensitivity of the Cornell-Wisconsin centrifugation flotation technique from 62% in samples collected right before treatment to 100% for samples collected 24 h after cestocidal treatment. It could be speculated that the tapeworm prevalence found before and after treatment in that study were higher than those found herein because all the horses included in Slocombe’s study (13) were confirmed to be infected with tapeworms before the start of the trial, while the horses in our study were not. When looking at the different age categories of horses included in the study, it appears that the adult group is mainly responsible for the increased sensitivity of the test at 24 h post-treatment.

The increase in the mean fecal egg counts found at 24–48 h after treatment is in agreement with other studies (13–15,18). Slocombe (13) observed a 10× increase in the mean number of fecal egg counts excreted in the feces at 24 h after cestocidal treatment while we observed a 3× increase at 24–48 h. The lower increase found in our study could be partly explained by the fact that the fecal samples of 19 mares were collected at 48 h rather than at 24 h after treatment. In the Slocombe (13) study, the mean fecal egg count found in the feces was 22× lower for feces collected at 48 h post-treatment compared with feces collected at 24 h post-treatment.

Tapeworms are affected or killed shortly after praziquantel treatment and are then passed in the feces (14–15). The disintegration of the dead tapeworms allows for an increased egg excretion and dispersion. It can be speculated that the increase in mean fecal counts at 24 h is partially responsible for the increased sensitivity of the Wisconsin test as a higher egg output or more dispersed eggs make it more probable to recover at least some eggs.

Except for the weanling category, the younger horses (2-year-olds and yearlings) had the highest prevalence rate and the highest mean fecal egg counts. These findings are consistent with the results of an immunoepidemiologic study done by Proudman and Trees (12) in which peak mean worm burden was found in the 6 mo to 2-year-old age group. The development of acquired immunity in older horses might explain the lower tapeworm infection intensity found in the adult age group. For the youngest group, it could be speculated that the weanlings were mostly negative because their forage intake was lower during the pasture season.

For the 2-year-olds, a Wisconsin test done at 24 h after treatment did not significantly increase the prevalence rate observed right before treatment, probably because their category had a relatively high prevalence and high mean worm egg counts on Day 0. With a 4× increase in the percentage of positive adult horses detected at 24 to 48 h post-treatment compared to the pre-treatment, the fecal sampling of adult horses at 24 h after cestocidal treatment could be a valuable protocol for practitioners to help increase the sensitivity of coprological tests. This conclusion is in agreement with other studies (13–15,18). Based on Slocombe’s (13) results, it can be speculated that the increase in the percentage of positive adult horses detected after treatment would have been higher if all the mares had been sampled at 24 h post-treatment.

Fecal sampling at 24 h post-cestocidal treatment would assist practitioners in detecting positive adult horses and in obtaining a better estimate of the true prevalence of tapeworm infection in an adult population. Such a protocol would also lower the number of fecal samples required to assess the infection status of a herd and to determine the necessity of treatments. It might also help in identifying individual horses that are more susceptible to tapeworm infection and those requiring more regular treatments. Collection and examination of feces within 24 to 48 h after treatment with pyrantel or praziquantel have been used in modified critical tests to assess drug efficacy (13–14).