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Helminth egg excretion in horses kept under tropical conditions—Prevalence, distribution and risk factors
Accepted Manuscript Title: Helminth egg excretion in horses kept under tropical conditions—Prevalence, distribution and risk factors Authors: J. Salas-Romero, K.A. G´omez-Cabrera, L.A. Aguilera-Valle, J.A. Bertot, J.E. Salas, A. Arenal, M.K. Nielsen PII: DOI: Reference:
S0304-4017(17)30282-0 http://dx.doi.org/doi:10.1016/j.vetpar.2017.06.014 VETPAR 8379
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
28-3-2017 10-6-2017 17-6-2017
Please cite this article as: Salas-Romero, J., G´omez-Cabrera, K.A., Aguilera-Valle, L.A., Bertot, J.A., Salas, J.E., Arenal, A., Nielsen, M.K., Helminth egg excretion in horses kept under tropical conditions—Prevalence, distribution and risk factors.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2017.06.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Helminth egg excretion in horses kept under tropical conditions – prevalence, distribution and risk factors
J. Salas-Romeroa*, K. A. Gómez-Cabreraa, L. A. Aguilera-Vallea, J. A. Bertota, J. E. Salasa, A. Arenala, M. K. Nielsenb
a
Facultad de Ciencias Agropecuarias, Universidad de Camagüey Ignacio Agramonte Loynáz,
Cuba. b
Department of Veterinary Science, University of Kentucky, M.H. Gluck Equine Research
Center, Lexington, Kentucky, USA.
* Corresponding author: Tel.: (53)32 26 1593 E-mail address: [email protected] (J. Salas-Romero)
Highlights
Sixty-three percent of equids were shedding over 1000 strongyle eggs per gram
All studied equid populations harbored Strongylus vulgaris
Strongylid egg shedding was associated with sex, collection month, operation type and days since last treatment
Ten percent were Parascaris spp. positive, associated with young age and absence of anthelmintic treatments
Abstract Increasing levels of anthelmintic resistance observed in equine cyathostomin parasites have led to recommendations of selective anthelmintic treatment strategies to lower the selection pressure favoring resistant populations. This principle is based on determining strongyle fecal egg counts from all herd members, and treating those exceeding a predetermined treatment cutoff. However, epidemiological information is lacking from horses kept under tropical conditions, where parasite burdens may be of a different composition and magnitude compared to those of horses kept under temperate climate conditions. The aim of the present work was to characterize the strongylid fauna in horses kept in tropical Camagüey, Cuba and identify risk factors associated with strongylid and ascarid egg counts. A total of 396 horses from eight different establishments were included in the study. Coprocultures revealed that Strongylus vulgaris and cyathostomins, sensu lato, were detected in all of those establishments. Prevalence and mean value of strongylid eggs per gram of feces were 97% and 1436, respectively. Eggs of Parascaris spp. were observed in 10% of horses. A multivariate mixed linear model identified sex (p=0.022), month (p=0.044), operation type (p=0.037) and time since last deworming (p<0.001) to be significantly associated in with the magnitude of strongylid fecal egg counts. A multivariate logistic regression identified horses less than two years of age (p=0.010) and horses not receiving anthelmintic treatment (p<0.001) to be significantly more likely to harbor Parascaris spp. parasites. The high magnitude and prevalence of strongylid fecal egg counts observed and the common occurrence of S. vulgaris suggest that
strongylid parasite burdens are substantially different from those typically observed in managed equines kept under more temperate conditions.
Keywords: Strongyles, Parascaris spp., prevalence, risk factors, tropical.
Introduction Worldwide, horses are exposed to a large number of gastrointestinal nematodes that can affect their health status. Animals that graze in contaminated areas, where parasite control is absent or suboptimal, can accumulate large numbers of parasites (Gawor, 1995; Kuzmina et al., 2012). Cyathostomin parasites are the cause of a severe disease complex known as larval cyathostominosis, which is a condition characterized by acute necrotizing typhlo-colitis and a profuse watery diarrhea (Love et al., 1999). The case-fatality rate of such cases has been reported to be around 50% (Reid et al., 1995). The occurrence of large strongyles has generally been observed to decline in managed horses over recent decades (Herd, 1990; Love and Duncan, 1991), although Strongylus vulgaris has been reported to be endemic in countries with strict regulations of anthelmintic usage, such as Denmark (Nielsen et al., 2012). This parasite is regarded as undesired due to its pathogenic potential as a cause of non-strangulating intestinal infarctions (Nielsen et al., 2016). Equine strongyle and ascarid parasites have developed resistance to all anthelmintics available on the market, and this constitutes an additional threat to the welfare of horses in many parts of the world (Peregrine et al., 2014). As a result, surveillance-based parasite control programs are now widely recommended to decrease the reliance on anthelmintic usage and slow down further development of anthelmintic resistance (Kaplan and Nielsen, 2010). Strongyle fecal egg counts are regarded as a cornerstone of equine parasite control, and studies have shown that adult horses are likely to maintain relatively consistent levels of egg shedding across time (Becher et al., 2010; Nielsen et al., 2006). The principle of targeted selective deworming has been widely recommended, in which only horses exceeding a pre-
determined strongyle egg count threshold will receive treatment. While this approach has been adopted in some parts of the world, such as Northern Europe (Nielsen et al., 2014a; Relf et al., 2012), many questions remain (Nielsen et al., 2014b). For example, little is known about application of a selective therapy-based system in underdeveloped regions outside Northern Europe and North America, where anthelmintic formulations are scarce and not always readily available and climatic conditions may be substantially different. Regardless of the region, it remains a challenge to ensure that control programs do not alter the strongyle species composition in favor of undesired large strongyle species (Nielsen et al., 2012). Equine parasite control in Cuba represents a unique challenge because none of the anthelmintic products available are formulated for equine usage (Salas-Romero et al., 2017b) and climatic conditions may favor heavy parasite infection pressures. Previous studies have documented that anthelmintic products widely used for equids include pelleted albendazole and injectable ivermectin products formulated for swine, carnivores, and ruminants (SalasRomero et al., 2017b). The aim of this study was to investigate strongyle and ascarid fecal egg count profiles in horses in Cuba and to identify risk factors associated with these variables.
Material and methods 2.1. Horses The study covered a period of 12 months, from March 2014 to March 2015. Three hundred ninety six horses from eight locations situated in the province of Camagüey were included (Table 1). The climate in this region is characterized as tropical savannah according to Köppen climate classification criteria, with a rainy season extending from May to October and a dry
season from November to April. The studied establishments included five larger farms, a group of horses kept at the local agricultural fair, horses used by local coachmen offering transportation services in Camagüey City, and a contingency of horses kept in communal paddocks/pastures by small-scale farmers in the more rural parts of the region. Information about anthelmintic treatments and grazing management was obtained through the veterinary records of the farms Galvis, San Vicente, and Alegre and horses from the agriculture fair. Information for the remaining participating horses was obtained by interviewing the owner/manager at each operation.
2.2. Coprological analysis Twenty g of feces were collected directly from the rectum of each horse, and placed in individually labelled plastic bags. They were refrigerated and transported to the laboratory, where they were processed within 72 h after collection. Fecal egg count was performed using the McMaster technique with a detection limit of 25 eggs per gram (EPG). Four grams of feces were placed in a container with 26 ml of saturated sucrose/NaCl (specific density of 1.21). The suspension was thoroughly homogenized with a wooden spatula and strained through a wire mesh to remove large debris. The strained suspension was then collected in a beaker and thoroughly mixed by stirring with the spatula. Then, 0.5 ml aliquots of the suspension were added to each of two chambers of a McMaster slide. After 10 min, parasite ova present under the two grids located within the chambers were counted under a light microscope at 100x magnification.
Pooled coprocultures were set up for each farm. Briefly, five g of feces were used from each sample with a value greater than 1500 EPG and then incubated for 14 days at room temperature (27-30 °C). Each pooled coproculture represented between three and ten horses. Third stage larvae were identified to genus and/or species level according to morphological criteria (Russell, 1948).
2.3. Risk factors The following information was recorded for each horse: Month of sampling, sex, breed (Arabian breeds and mixed breeds), age group (>2 years, 3-4 years, 5-6 years, 7-10 years), number of anthelmintic treatments in the previous year (no treatment, 1, >2 treatments), time since last anthelmintic treatment (<60 days, 60-120 days, 121-180 days, 180-365 days, >356 days), equine operation type (animal production, agricultural production, small scale farmer, and carriage & coach service), horse category (foal <12 months, 13-36 months, working male horse, broodmare, working mare), and management system (grazing, housing and daytime/nocturnal grazing).
2.2. Statistical analyses Two sets of analyses were carried out using SAS, version 9.4 (SAS Institute, Cary, North Carolina, USA). A multivariate mixed linear analysis was carried out with log-transformed strongyle fecal egg counts as the response variable and the risk factors described above as covariates and equine establishment origin as random effect. The model was constructed using forward addition and backward elimination of covariates. All covariates with a p-value of 0.20 or below
were kept in the model. In a second set of analyses, a multivariate logistic regression was carried out with Parascaris spp. presence (0/1) as the response variable. The same covariates were evaluated in this model, which was constructed in a similar fashion. Odds ratios were calculated for each significant covariate. Covariates were interpreted as significant at the 0.05 level.
3. Results A total of 396 horses present in eight establishments were included in the final data set. The mean age of participating horses was 5.1 years, ranging from seven months to 26 years.
3.1. Egg count prevalences A total of 385 horses (97%) had positive strongyle egg counts and 39 (10%) were ascarid egg positive. Mean and median strongyle egg counts were 1436 and 1237 EPG, respectively, with 63% of the horses having more than 1000 EPG. Of the total strongyle egg output in the data set, 80% was contributed by 217 (55%) of the horses. Eggs of Oxyuris equi were observed in samples of five horses and two samples had Anoplocephala spp. eggs.
3.2. Coprocultures Coproculture results are presented in Table 1. Cyathostomins and S. vulgaris were observed in all of the studied groups and others with widespread occurrence were Poteriostomum spp. and Trichostrongylus axei. Strongyloides westeri and S. edentatus were only identified in three farms. Strongylus equinus was not identified in any of the coprocultures.
3.2. Risk factor analysis Table 2 presents the FEC data broken down by selected categorical covariates evaluated in this study. Equine operation type (p=0.037), horse category (p=0.002), sex (p=0.022), collection month (p=0.044), and time since last deworming (p<0.001) were all significantly associated with the output variable. The pairwise comparisons revealed that female horses had significantly higher strongyle egg counts than males, samples collected during the month of October had significantly higher egg counts than those collected in March (p=0.030), and carriage horses owned by coachmen had significantly lower counts than horses belonging to rural farmers (p=0.032). Finally, horses dewormed within 60 days prior to the study had significantly lower egg counts than all other horses (p<0.01). In the logistic regression analysis of Parascaris spp. presence, age group (p=0.010) and treatment intensity (p<0.001) were both significantly associated with the outcome variable. Odds ratios for these covariates are presented in Table 3. Horses less than two years of age and horses not receiving anthelmintic treatment in the previous year were significantly more likely to harbor Parascaris spp. parasites.
Discussion This study provided useful information about equine gastrointestinal helminth parasites present in managed horses kept under tropical conditions in Cuba. Notably, the large strongyle, S. vulgaris, was identified in all the studied establishments. This confirms previous observations made in the same region, where this parasite was identified in up to 50% of horses and in all
establishments investigated (Salas-Romero et al., 2014). This contrasts findings made elsewhere in the world, where Strongylus spp. parasites have become increasingly rare and often go completely undetected or prevalences remain below 5% (Boxell et al., 2004; Craven et al., 1998; Hoglund et al., 1997; Osterman Lind et al., 1999). A recent study has suggested that the presence of S. vulgaris is largely dependent on anthelmintic treatment intensity as it has yet to be reported resistant to any of the available anthelmintics (Nielsen et al., 2012). Thus, the findings presented here suggest that anthelmintic treatments in the studied establishments are too scarce to effectively prevent transmission of this parasite. Indeed, in this study, only 31% of the horses (Table 2) were dewormed more than once a year, which is in stark contrast to typical deworming regimens employed elsewhere (Relf et al., 2012; Robert et al., 2015). While the overall prevalence of strongyle eggs (97%) was expected, the magnitude of egg counts was considerably higher than often reported in managed horses (Relf et al., 2013). Strongyle egg shedding in horses is often reported to follow an 80/20 distribution, with about 20% of horses shedding 80% of the total egg output (Kaplan and Nielsen, 2010; Lester et al., 2013; Relf et al., 2013; Wood et al., 2012). The distribution observed in this study was very different with over half (55%) of equines excreting 80% of the output, and 84% of the horses exhibiting egg counts above 500 EPG. In fact, our results are more similar to those reported in working equids (Upjohn et al., 2010), where it is not unusual to observe more than 50% of animals with fecal egg counts higher than 1000 EPG. Thus, it is possible that climatic conditions in Cuba particularly favor strongyle parasite transmission. The general scarcity of affordable anthelmintic products in Cuba and the fact that the majority of horses enrolled in this study can
be classified as working equids are both likely to have contributed to the high strongyle egg count levels as well. Targeted selective treatments are recommended as a sustainable deworming strategy in which a strongyle EPG cutoff value for treatment is identified and only a proportion of horses are treated (Duncan and Love, 1991; Gomez and Georgi, 1991). Treatment thresholds are often chosen in the 200-500 EPG range (Uhlinger, 1993). However, it is clear that a cutoff value in this range would have resulted in the large majority of horses in this study receiving treatment. This may not constitute a problem for the development of anthelmintic resistance as long as treatment intensities are as low as they are reported herein. It remains possible that a higher cutoff value could be appropriate for the Camagüey region, but more research is required to evaluate this. However, the presence of potentially pathogenic parasites like S. vulgaris in all the studied establishments, strongly suggests that selective therapy should not be recommended as the targeted selective treatment strategy was developed to primarily control cyathostomin parasites (Nielsen et al., 2014b). The presence of T. axei in equine herds that did not graze with ruminants was an interesting observation. However, similar findings have been made in other recent studies in Cuban horses (Salas-Romero et al., 2014; 2017a; 2017b), so this appears to be an endemic parasite in this region. While strongyle results were different from those of studies conducted in managed horse establishments elsewhere in the world, Parascaris spp. results were in general agreement with other studies (Relf et al., 2013), being most prevalent in the younger age group (<2 years).
Despite the low anthelmintic treatment intensity observed across the study population, this study did offer some insight into the effects of anthelmintic treatment. It was observed that two thirds of horses with strongyle egg counts below 500 EPG were dewormed within the previous six months. Similarly, horses that were dewormed twice or more per year, had significantly lower strongyle fecal egg counts compared to those horses that received no anthelmintic treatment. A similar trend was observed with Parascaris spp. prevalence. In summary, the present study generated useful information that can be used by horse owners, veterinarians and parasitologists in order to identify appropriate parasite control programs in the studied establishments. Further studies should evaluate the effect of tropical conditions on equine nematodes, pasture infectivity, parasite prevalence and abundance to better identify optimum strategies for parasite control.
Conflict of interest statement The authors declare no conflict of interest.
References Becher, A.M., Mahling, M., Nielsen, M.K., Pfister, K., 2010. Selective anthelmintic therapy of horses in the Federal states of Bavaria (Germany) and Salzburg (Austria): An investigation into strongyle egg shedding consistency. Vet. Parasitol. 171, 116-122. Boxell, A.C., Gibson, K.T., Hobbs, R.P., Thompson, R.C., 2004. Occurrence of gastrointestinal parasites in horses in metropolitan Perth, Western Australia. Aust. Vet. J. 82, 91-95. Craven, J., Bjorn, H., Henriksen, S.A., Nansen, P., Larsen, M., Lendal, S., 1998. Survey of anthelmintic resistance on Danish horse farms, using 5 different methods of calculating faecal egg count reduction. Equine. Vet. J. 30, 289-293. Duncan, J.L., Love, S., 1991. Preliminary observations on an alternative strategy for the control of horse strongyles. Equine Vet. J. 23, 226-228. Gawor, J.J., 1995. The prevalence and abundance of internal parasites in working horses autopsied in Poland. Vet. Parasitol. 58, 99-108.
Gomez, H.H., Georgi, J., 1991. Equine helminth infections: control by selective chemotherapy. Equine Vet. J. 23, 198-200. Herd, R., 1990. The changing world of worms: The rise of the cyathostomes and the decline of Strongylus vulgaris. Comp. Cont. Educ. Pract. 12, 732-736. Hoglund, J., Ljungstrom, B.L., Nilsson, O., Lundquist, H., Osterman, E., Uggla, A., 1997. Occurrence of Gasterophilus intestinalis and some parasitic nematodes of horses in Sweden. Acta Vet. Scand. 38, 157-165. Kaplan, R.M., Nielsen, M.K., 2010. An evidence‐based approach to equine parasite control: It ain't the 60s anymore. Equine Vet. Educ. 22, 306-316. Kornas, S., Cabaret, J., Skalska, M., Nowosad, B., 2010. Horse infection with intestinal helminths in relation to age, sex, access to grass and farm system. Vet. Parasitol. 174, 285-291. Kuzmina, T., Lyons, E., Tolliver, S., Dzeverin, I., Kharchenko, V., 2012. Fecundity of various species of strongylids (Nematoda: Strongylidae)—parasites of domestic horses. Parasitol. Res. 111, 2265-2271. Love, S., Duncan, J.L., 1991. Could the worms have turned? Equine Vet. J. 23, 152-154. Love, S., Murphy, D., Mellor, D., 1999. Pathogenicity of cyathostome infection. Vet. Parasitol. 85, 113-121. Nielsen, M., Reist, M., Kaplan, R., Pfister, K., van Doorn, D., Becher, A., 2014a. Equine parasite control under prescription-only conditions in Denmark–Awareness, knowledge, perception, and strategies applied. Vet. Parasitol. 204, 64-72. Nielsen, M., Pfister, K., von Samson-Himmelstjerna, G., 2014b. Selective therapy in equine parasite control—Application and limitations. Vet. Parasitol. 202, 95-103. Nielsen, M.K., Haaning, N., Olsen, S.N., 2006. Strongyle egg shedding consistency in horses on farms using selective therapy in Denmark. Vet. Parasitol. 135, 333-335. Nielsen, M.K., Vidyashankar, A., Olsen, S.N., Monrad, J., Thamsborg, S.M., 2012. Strongylus vulgaris associated with usage of selective therapy on Danish horse farms—Is it reemerging? Vet. Parasitol. 189, 260-266. Osterman Lind, E., Hoglund, J., Ljungstrom, B.L., Nilsson, O., Uggla, A., 1999. A field survey on the distribution of strongyle infections of horses in Sweden and factors affecting faecal egg counts. Equine Vet. J. 31, 68-72. Reid, S.W., Mair, T.S., Hillyer, M.H., Love, S., 1995. Epidemiological risk factors associated with a diagnosis of clinical cyathostomiasis in the horse. Equine Vet. J. 27, 127-130. Relf, V., Morgan, E., Hodgkinson, J., Matthews, J., 2012. A questionnaire study on parasite control practices on UK breeding Thoroughbred studs. Equine Vet. J. 44, 466-471. Relf, V., Morgan, E., Hodgkinson, J., Matthews, J., 2013. Helminth egg excretion with regard to age, gender and management practices on UK Thoroughbred studs. Parasitology 140, 641-652. Robert, M., Hu, W., Nielsen, M., Stowe, C., 2015. Attitudes towards implementation of surveillance‐based parasite control on Kentucky Thoroughbred farms–Current strategies, awareness and willingness‐to‐pay. Equine Vet. J. 47, 694-700. Russell, A.F., 1948. The development of helminthiasis in Thoroughbred foals. J. Comp. Path. Therap. 58, 107-127.
Salas-Romero, J., Batista, B., Villavicencio, L., Mencho, J.D., Llorens, Y.G., 2014. Prevalencia de nematodos intestinales y eficacia de Labiomec® en caballos de Camagüey, Cuba. Rev. Salud Anim. Vol. 36, 152-158. Salas-Romero, J., Gomez-Cabrera, K., Chicoy, Y., Yero, J.C., Valle, E., Salas, J.E., Arenal, A., 2017a. Especies de Ciatostomas Resistentes al Albendazol en Equinos, Cuba. Rev. Inv. Vet. Perú 28. Salas-Romero, J., Gomez-Cabrera, K., Molento, M.B., Lyons, E.T., Delgado, A., González, L., Arenal, A., Nielsen, M.K., 2017b. Efficacy of two extra-label anthelmintic formulations against equine strongyles in Cuba. Vet. Parasitol.: Reg. Stud. Rep. 8, 39-42. Uhlinger, C., 1993. Uses of fecal egg count data in equine practice. Compend. Contin. Educ. Pract. Vet. 15, 742–749. Upjohn, M.M., Shipton, K., Lerotholi, T., Attwood, G., Verheyen, K.L., 2010. Coprological prevalence and intensity of helminth infection in working horses in Lesotho. Trop. Anim. Health Prod. 42, 1655-1661.
Table 1. Identification of nematode infective larvae from pooled coprocultures performed for each equine establishment. Location Cyat S. S. O. P. T. G. T. S. vul ede rob spp. spp. capt axei wes Galvis X X X X X X X Triángulo tres X X X X X X San Vicente X X X X X X Feria Camagüey X X X X X X Jimaguayú X X X X X X Coachmen X X X X X Ranch Alegre X X X X X X Rural X X X X X X X Cyat: Cyathostomins; S. vul: Strongylus vulgaris; S. ede: Strongylus edentatus; O. rob: Oesophagodontus robustus; P. sp.: Poteriostomun sp.; T. spp.: Triodontophorus spp.; G. capt.: Gyalocephalus capitatus; T. axei: Trichostrongylus axei; S. wes.: Strongyloides westeri
Table 2. Descriptive data for strongyle and Parascaris spp. fecal egg count results among the 396 horses studied. Prevalence and mean eggs per gram (EPG) presented for different groups. Factors Age (years)
Sex Breed Pasture access Treatment intensity (per year) Time since last treatment (months) Summary (range) a Positive b
<2 3-4 5-6 7-10 ≥11 Male Female Mixed Arabian Grazing Housed Both 0 1 ≥2 <2 2-4 4-6 6-12 >12
Horses (n) 90 88 76 77 27 174 211 276 115 180 81 130 91 136 101 26 28 78 120 86 396
egg counts only These refer to a single case
Strongyles Prevalence % EPG 95.6 1540 98.9 1435 97.4 1484 100 1400 85.2 985 96.6 1267 97.6 1559 97.2 1119 100.0 1891 98.3 1549 100.0 1155 94.5 1263 100.0 1636 97.0 1395 94.0 1264 76.9 824 88.9 818 100.0 1125 99.1 1648 100.0 1676 1436 97.2 (0-4150)
Parascaris spp. Prevalence % EPGa 26.7 249 3.4 183 2.6 100 1.3 100b 3.7 150b 5.2 280 14.2 209 8.6 244 17.4 220 13.1 148 3.7 325 11.0 70 22.5 117 3.0 900 5.4 181 3.7 150b 10.4 275 1.7 1200 21.4 109 229 10.0 (0-2325)
Table 3. Odds ratio estimates for two significant covariates in the logistic regression with Parascaris spp. presence (0/1) as outcome variable. Covariate Comparison Odds ratio 95% Wald Confidence Interval <2 years vs. 7-10 years 18.04 1.92-168.98 Age group 3-4 years vs. 7-10 years 2.33 0.18-30.31 5-6 years vs. 7-10 years 1.47 0.08-26.79 1/year vs. 0/year 0.04 0.01-0.18 Treatment intensity >2/year vs. 0/year 0.10 0.02-0.54
S0304-4017(17)30282-0 http://dx.doi.org/doi:10.1016/j.vetpar.2017.06.014 VETPAR 8379
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
28-3-2017 10-6-2017 17-6-2017
Please cite this article as: Salas-Romero, J., G´omez-Cabrera, K.A., Aguilera-Valle, L.A., Bertot, J.A., Salas, J.E., Arenal, A., Nielsen, M.K., Helminth egg excretion in horses kept under tropical conditions—Prevalence, distribution and risk factors.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2017.06.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Helminth egg excretion in horses kept under tropical conditions – prevalence, distribution and risk factors
J. Salas-Romeroa*, K. A. Gómez-Cabreraa, L. A. Aguilera-Vallea, J. A. Bertota, J. E. Salasa, A. Arenala, M. K. Nielsenb
a
Facultad de Ciencias Agropecuarias, Universidad de Camagüey Ignacio Agramonte Loynáz,
Cuba. b
Department of Veterinary Science, University of Kentucky, M.H. Gluck Equine Research
Center, Lexington, Kentucky, USA.
* Corresponding author: Tel.: (53)32 26 1593 E-mail address: [email protected] (J. Salas-Romero)
Highlights
Sixty-three percent of equids were shedding over 1000 strongyle eggs per gram
All studied equid populations harbored Strongylus vulgaris
Strongylid egg shedding was associated with sex, collection month, operation type and days since last treatment
Ten percent were Parascaris spp. positive, associated with young age and absence of anthelmintic treatments
Abstract Increasing levels of anthelmintic resistance observed in equine cyathostomin parasites have led to recommendations of selective anthelmintic treatment strategies to lower the selection pressure favoring resistant populations. This principle is based on determining strongyle fecal egg counts from all herd members, and treating those exceeding a predetermined treatment cutoff. However, epidemiological information is lacking from horses kept under tropical conditions, where parasite burdens may be of a different composition and magnitude compared to those of horses kept under temperate climate conditions. The aim of the present work was to characterize the strongylid fauna in horses kept in tropical Camagüey, Cuba and identify risk factors associated with strongylid and ascarid egg counts. A total of 396 horses from eight different establishments were included in the study. Coprocultures revealed that Strongylus vulgaris and cyathostomins, sensu lato, were detected in all of those establishments. Prevalence and mean value of strongylid eggs per gram of feces were 97% and 1436, respectively. Eggs of Parascaris spp. were observed in 10% of horses. A multivariate mixed linear model identified sex (p=0.022), month (p=0.044), operation type (p=0.037) and time since last deworming (p<0.001) to be significantly associated in with the magnitude of strongylid fecal egg counts. A multivariate logistic regression identified horses less than two years of age (p=0.010) and horses not receiving anthelmintic treatment (p<0.001) to be significantly more likely to harbor Parascaris spp. parasites. The high magnitude and prevalence of strongylid fecal egg counts observed and the common occurrence of S. vulgaris suggest that
strongylid parasite burdens are substantially different from those typically observed in managed equines kept under more temperate conditions.
Keywords: Strongyles, Parascaris spp., prevalence, risk factors, tropical.
Introduction Worldwide, horses are exposed to a large number of gastrointestinal nematodes that can affect their health status. Animals that graze in contaminated areas, where parasite control is absent or suboptimal, can accumulate large numbers of parasites (Gawor, 1995; Kuzmina et al., 2012). Cyathostomin parasites are the cause of a severe disease complex known as larval cyathostominosis, which is a condition characterized by acute necrotizing typhlo-colitis and a profuse watery diarrhea (Love et al., 1999). The case-fatality rate of such cases has been reported to be around 50% (Reid et al., 1995). The occurrence of large strongyles has generally been observed to decline in managed horses over recent decades (Herd, 1990; Love and Duncan, 1991), although Strongylus vulgaris has been reported to be endemic in countries with strict regulations of anthelmintic usage, such as Denmark (Nielsen et al., 2012). This parasite is regarded as undesired due to its pathogenic potential as a cause of non-strangulating intestinal infarctions (Nielsen et al., 2016). Equine strongyle and ascarid parasites have developed resistance to all anthelmintics available on the market, and this constitutes an additional threat to the welfare of horses in many parts of the world (Peregrine et al., 2014). As a result, surveillance-based parasite control programs are now widely recommended to decrease the reliance on anthelmintic usage and slow down further development of anthelmintic resistance (Kaplan and Nielsen, 2010). Strongyle fecal egg counts are regarded as a cornerstone of equine parasite control, and studies have shown that adult horses are likely to maintain relatively consistent levels of egg shedding across time (Becher et al., 2010; Nielsen et al., 2006). The principle of targeted selective deworming has been widely recommended, in which only horses exceeding a pre-
determined strongyle egg count threshold will receive treatment. While this approach has been adopted in some parts of the world, such as Northern Europe (Nielsen et al., 2014a; Relf et al., 2012), many questions remain (Nielsen et al., 2014b). For example, little is known about application of a selective therapy-based system in underdeveloped regions outside Northern Europe and North America, where anthelmintic formulations are scarce and not always readily available and climatic conditions may be substantially different. Regardless of the region, it remains a challenge to ensure that control programs do not alter the strongyle species composition in favor of undesired large strongyle species (Nielsen et al., 2012). Equine parasite control in Cuba represents a unique challenge because none of the anthelmintic products available are formulated for equine usage (Salas-Romero et al., 2017b) and climatic conditions may favor heavy parasite infection pressures. Previous studies have documented that anthelmintic products widely used for equids include pelleted albendazole and injectable ivermectin products formulated for swine, carnivores, and ruminants (SalasRomero et al., 2017b). The aim of this study was to investigate strongyle and ascarid fecal egg count profiles in horses in Cuba and to identify risk factors associated with these variables.
Material and methods 2.1. Horses The study covered a period of 12 months, from March 2014 to March 2015. Three hundred ninety six horses from eight locations situated in the province of Camagüey were included (Table 1). The climate in this region is characterized as tropical savannah according to Köppen climate classification criteria, with a rainy season extending from May to October and a dry
season from November to April. The studied establishments included five larger farms, a group of horses kept at the local agricultural fair, horses used by local coachmen offering transportation services in Camagüey City, and a contingency of horses kept in communal paddocks/pastures by small-scale farmers in the more rural parts of the region. Information about anthelmintic treatments and grazing management was obtained through the veterinary records of the farms Galvis, San Vicente, and Alegre and horses from the agriculture fair. Information for the remaining participating horses was obtained by interviewing the owner/manager at each operation.
2.2. Coprological analysis Twenty g of feces were collected directly from the rectum of each horse, and placed in individually labelled plastic bags. They were refrigerated and transported to the laboratory, where they were processed within 72 h after collection. Fecal egg count was performed using the McMaster technique with a detection limit of 25 eggs per gram (EPG). Four grams of feces were placed in a container with 26 ml of saturated sucrose/NaCl (specific density of 1.21). The suspension was thoroughly homogenized with a wooden spatula and strained through a wire mesh to remove large debris. The strained suspension was then collected in a beaker and thoroughly mixed by stirring with the spatula. Then, 0.5 ml aliquots of the suspension were added to each of two chambers of a McMaster slide. After 10 min, parasite ova present under the two grids located within the chambers were counted under a light microscope at 100x magnification.
Pooled coprocultures were set up for each farm. Briefly, five g of feces were used from each sample with a value greater than 1500 EPG and then incubated for 14 days at room temperature (27-30 °C). Each pooled coproculture represented between three and ten horses. Third stage larvae were identified to genus and/or species level according to morphological criteria (Russell, 1948).
2.3. Risk factors The following information was recorded for each horse: Month of sampling, sex, breed (Arabian breeds and mixed breeds), age group (>2 years, 3-4 years, 5-6 years, 7-10 years), number of anthelmintic treatments in the previous year (no treatment, 1, >2 treatments), time since last anthelmintic treatment (<60 days, 60-120 days, 121-180 days, 180-365 days, >356 days), equine operation type (animal production, agricultural production, small scale farmer, and carriage & coach service), horse category (foal <12 months, 13-36 months, working male horse, broodmare, working mare), and management system (grazing, housing and daytime/nocturnal grazing).
2.2. Statistical analyses Two sets of analyses were carried out using SAS, version 9.4 (SAS Institute, Cary, North Carolina, USA). A multivariate mixed linear analysis was carried out with log-transformed strongyle fecal egg counts as the response variable and the risk factors described above as covariates and equine establishment origin as random effect. The model was constructed using forward addition and backward elimination of covariates. All covariates with a p-value of 0.20 or below
were kept in the model. In a second set of analyses, a multivariate logistic regression was carried out with Parascaris spp. presence (0/1) as the response variable. The same covariates were evaluated in this model, which was constructed in a similar fashion. Odds ratios were calculated for each significant covariate. Covariates were interpreted as significant at the 0.05 level.
3. Results A total of 396 horses present in eight establishments were included in the final data set. The mean age of participating horses was 5.1 years, ranging from seven months to 26 years.
3.1. Egg count prevalences A total of 385 horses (97%) had positive strongyle egg counts and 39 (10%) were ascarid egg positive. Mean and median strongyle egg counts were 1436 and 1237 EPG, respectively, with 63% of the horses having more than 1000 EPG. Of the total strongyle egg output in the data set, 80% was contributed by 217 (55%) of the horses. Eggs of Oxyuris equi were observed in samples of five horses and two samples had Anoplocephala spp. eggs.
3.2. Coprocultures Coproculture results are presented in Table 1. Cyathostomins and S. vulgaris were observed in all of the studied groups and others with widespread occurrence were Poteriostomum spp. and Trichostrongylus axei. Strongyloides westeri and S. edentatus were only identified in three farms. Strongylus equinus was not identified in any of the coprocultures.
3.2. Risk factor analysis Table 2 presents the FEC data broken down by selected categorical covariates evaluated in this study. Equine operation type (p=0.037), horse category (p=0.002), sex (p=0.022), collection month (p=0.044), and time since last deworming (p<0.001) were all significantly associated with the output variable. The pairwise comparisons revealed that female horses had significantly higher strongyle egg counts than males, samples collected during the month of October had significantly higher egg counts than those collected in March (p=0.030), and carriage horses owned by coachmen had significantly lower counts than horses belonging to rural farmers (p=0.032). Finally, horses dewormed within 60 days prior to the study had significantly lower egg counts than all other horses (p<0.01). In the logistic regression analysis of Parascaris spp. presence, age group (p=0.010) and treatment intensity (p<0.001) were both significantly associated with the outcome variable. Odds ratios for these covariates are presented in Table 3. Horses less than two years of age and horses not receiving anthelmintic treatment in the previous year were significantly more likely to harbor Parascaris spp. parasites.
Discussion This study provided useful information about equine gastrointestinal helminth parasites present in managed horses kept under tropical conditions in Cuba. Notably, the large strongyle, S. vulgaris, was identified in all the studied establishments. This confirms previous observations made in the same region, where this parasite was identified in up to 50% of horses and in all
establishments investigated (Salas-Romero et al., 2014). This contrasts findings made elsewhere in the world, where Strongylus spp. parasites have become increasingly rare and often go completely undetected or prevalences remain below 5% (Boxell et al., 2004; Craven et al., 1998; Hoglund et al., 1997; Osterman Lind et al., 1999). A recent study has suggested that the presence of S. vulgaris is largely dependent on anthelmintic treatment intensity as it has yet to be reported resistant to any of the available anthelmintics (Nielsen et al., 2012). Thus, the findings presented here suggest that anthelmintic treatments in the studied establishments are too scarce to effectively prevent transmission of this parasite. Indeed, in this study, only 31% of the horses (Table 2) were dewormed more than once a year, which is in stark contrast to typical deworming regimens employed elsewhere (Relf et al., 2012; Robert et al., 2015). While the overall prevalence of strongyle eggs (97%) was expected, the magnitude of egg counts was considerably higher than often reported in managed horses (Relf et al., 2013). Strongyle egg shedding in horses is often reported to follow an 80/20 distribution, with about 20% of horses shedding 80% of the total egg output (Kaplan and Nielsen, 2010; Lester et al., 2013; Relf et al., 2013; Wood et al., 2012). The distribution observed in this study was very different with over half (55%) of equines excreting 80% of the output, and 84% of the horses exhibiting egg counts above 500 EPG. In fact, our results are more similar to those reported in working equids (Upjohn et al., 2010), where it is not unusual to observe more than 50% of animals with fecal egg counts higher than 1000 EPG. Thus, it is possible that climatic conditions in Cuba particularly favor strongyle parasite transmission. The general scarcity of affordable anthelmintic products in Cuba and the fact that the majority of horses enrolled in this study can
be classified as working equids are both likely to have contributed to the high strongyle egg count levels as well. Targeted selective treatments are recommended as a sustainable deworming strategy in which a strongyle EPG cutoff value for treatment is identified and only a proportion of horses are treated (Duncan and Love, 1991; Gomez and Georgi, 1991). Treatment thresholds are often chosen in the 200-500 EPG range (Uhlinger, 1993). However, it is clear that a cutoff value in this range would have resulted in the large majority of horses in this study receiving treatment. This may not constitute a problem for the development of anthelmintic resistance as long as treatment intensities are as low as they are reported herein. It remains possible that a higher cutoff value could be appropriate for the Camagüey region, but more research is required to evaluate this. However, the presence of potentially pathogenic parasites like S. vulgaris in all the studied establishments, strongly suggests that selective therapy should not be recommended as the targeted selective treatment strategy was developed to primarily control cyathostomin parasites (Nielsen et al., 2014b). The presence of T. axei in equine herds that did not graze with ruminants was an interesting observation. However, similar findings have been made in other recent studies in Cuban horses (Salas-Romero et al., 2014; 2017a; 2017b), so this appears to be an endemic parasite in this region. While strongyle results were different from those of studies conducted in managed horse establishments elsewhere in the world, Parascaris spp. results were in general agreement with other studies (Relf et al., 2013), being most prevalent in the younger age group (<2 years).
Despite the low anthelmintic treatment intensity observed across the study population, this study did offer some insight into the effects of anthelmintic treatment. It was observed that two thirds of horses with strongyle egg counts below 500 EPG were dewormed within the previous six months. Similarly, horses that were dewormed twice or more per year, had significantly lower strongyle fecal egg counts compared to those horses that received no anthelmintic treatment. A similar trend was observed with Parascaris spp. prevalence. In summary, the present study generated useful information that can be used by horse owners, veterinarians and parasitologists in order to identify appropriate parasite control programs in the studied establishments. Further studies should evaluate the effect of tropical conditions on equine nematodes, pasture infectivity, parasite prevalence and abundance to better identify optimum strategies for parasite control.
Conflict of interest statement The authors declare no conflict of interest.
References Becher, A.M., Mahling, M., Nielsen, M.K., Pfister, K., 2010. Selective anthelmintic therapy of horses in the Federal states of Bavaria (Germany) and Salzburg (Austria): An investigation into strongyle egg shedding consistency. Vet. Parasitol. 171, 116-122. Boxell, A.C., Gibson, K.T., Hobbs, R.P., Thompson, R.C., 2004. Occurrence of gastrointestinal parasites in horses in metropolitan Perth, Western Australia. Aust. Vet. J. 82, 91-95. Craven, J., Bjorn, H., Henriksen, S.A., Nansen, P., Larsen, M., Lendal, S., 1998. Survey of anthelmintic resistance on Danish horse farms, using 5 different methods of calculating faecal egg count reduction. Equine. Vet. J. 30, 289-293. Duncan, J.L., Love, S., 1991. Preliminary observations on an alternative strategy for the control of horse strongyles. Equine Vet. J. 23, 226-228. Gawor, J.J., 1995. The prevalence and abundance of internal parasites in working horses autopsied in Poland. Vet. Parasitol. 58, 99-108.
Gomez, H.H., Georgi, J., 1991. Equine helminth infections: control by selective chemotherapy. Equine Vet. J. 23, 198-200. Herd, R., 1990. The changing world of worms: The rise of the cyathostomes and the decline of Strongylus vulgaris. Comp. Cont. Educ. Pract. 12, 732-736. Hoglund, J., Ljungstrom, B.L., Nilsson, O., Lundquist, H., Osterman, E., Uggla, A., 1997. Occurrence of Gasterophilus intestinalis and some parasitic nematodes of horses in Sweden. Acta Vet. Scand. 38, 157-165. Kaplan, R.M., Nielsen, M.K., 2010. An evidence‐based approach to equine parasite control: It ain't the 60s anymore. Equine Vet. Educ. 22, 306-316. Kornas, S., Cabaret, J., Skalska, M., Nowosad, B., 2010. Horse infection with intestinal helminths in relation to age, sex, access to grass and farm system. Vet. Parasitol. 174, 285-291. Kuzmina, T., Lyons, E., Tolliver, S., Dzeverin, I., Kharchenko, V., 2012. Fecundity of various species of strongylids (Nematoda: Strongylidae)—parasites of domestic horses. Parasitol. Res. 111, 2265-2271. Love, S., Duncan, J.L., 1991. Could the worms have turned? Equine Vet. J. 23, 152-154. Love, S., Murphy, D., Mellor, D., 1999. Pathogenicity of cyathostome infection. Vet. Parasitol. 85, 113-121. Nielsen, M., Reist, M., Kaplan, R., Pfister, K., van Doorn, D., Becher, A., 2014a. Equine parasite control under prescription-only conditions in Denmark–Awareness, knowledge, perception, and strategies applied. Vet. Parasitol. 204, 64-72. Nielsen, M., Pfister, K., von Samson-Himmelstjerna, G., 2014b. Selective therapy in equine parasite control—Application and limitations. Vet. Parasitol. 202, 95-103. Nielsen, M.K., Haaning, N., Olsen, S.N., 2006. Strongyle egg shedding consistency in horses on farms using selective therapy in Denmark. Vet. Parasitol. 135, 333-335. Nielsen, M.K., Vidyashankar, A., Olsen, S.N., Monrad, J., Thamsborg, S.M., 2012. Strongylus vulgaris associated with usage of selective therapy on Danish horse farms—Is it reemerging? Vet. Parasitol. 189, 260-266. Osterman Lind, E., Hoglund, J., Ljungstrom, B.L., Nilsson, O., Uggla, A., 1999. A field survey on the distribution of strongyle infections of horses in Sweden and factors affecting faecal egg counts. Equine Vet. J. 31, 68-72. Reid, S.W., Mair, T.S., Hillyer, M.H., Love, S., 1995. Epidemiological risk factors associated with a diagnosis of clinical cyathostomiasis in the horse. Equine Vet. J. 27, 127-130. Relf, V., Morgan, E., Hodgkinson, J., Matthews, J., 2012. A questionnaire study on parasite control practices on UK breeding Thoroughbred studs. Equine Vet. J. 44, 466-471. Relf, V., Morgan, E., Hodgkinson, J., Matthews, J., 2013. Helminth egg excretion with regard to age, gender and management practices on UK Thoroughbred studs. Parasitology 140, 641-652. Robert, M., Hu, W., Nielsen, M., Stowe, C., 2015. Attitudes towards implementation of surveillance‐based parasite control on Kentucky Thoroughbred farms–Current strategies, awareness and willingness‐to‐pay. Equine Vet. J. 47, 694-700. Russell, A.F., 1948. The development of helminthiasis in Thoroughbred foals. J. Comp. Path. Therap. 58, 107-127.
Salas-Romero, J., Batista, B., Villavicencio, L., Mencho, J.D., Llorens, Y.G., 2014. Prevalencia de nematodos intestinales y eficacia de Labiomec® en caballos de Camagüey, Cuba. Rev. Salud Anim. Vol. 36, 152-158. Salas-Romero, J., Gomez-Cabrera, K., Chicoy, Y., Yero, J.C., Valle, E., Salas, J.E., Arenal, A., 2017a. Especies de Ciatostomas Resistentes al Albendazol en Equinos, Cuba. Rev. Inv. Vet. Perú 28. Salas-Romero, J., Gomez-Cabrera, K., Molento, M.B., Lyons, E.T., Delgado, A., González, L., Arenal, A., Nielsen, M.K., 2017b. Efficacy of two extra-label anthelmintic formulations against equine strongyles in Cuba. Vet. Parasitol.: Reg. Stud. Rep. 8, 39-42. Uhlinger, C., 1993. Uses of fecal egg count data in equine practice. Compend. Contin. Educ. Pract. Vet. 15, 742–749. Upjohn, M.M., Shipton, K., Lerotholi, T., Attwood, G., Verheyen, K.L., 2010. Coprological prevalence and intensity of helminth infection in working horses in Lesotho. Trop. Anim. Health Prod. 42, 1655-1661.
Table 1. Identification of nematode infective larvae from pooled coprocultures performed for each equine establishment. Location Cyat S. S. O. P. T. G. T. S. vul ede rob spp. spp. capt axei wes Galvis X X X X X X X Triángulo tres X X X X X X San Vicente X X X X X X Feria Camagüey X X X X X X Jimaguayú X X X X X X Coachmen X X X X X Ranch Alegre X X X X X X Rural X X X X X X X Cyat: Cyathostomins; S. vul: Strongylus vulgaris; S. ede: Strongylus edentatus; O. rob: Oesophagodontus robustus; P. sp.: Poteriostomun sp.; T. spp.: Triodontophorus spp.; G. capt.: Gyalocephalus capitatus; T. axei: Trichostrongylus axei; S. wes.: Strongyloides westeri
Table 2. Descriptive data for strongyle and Parascaris spp. fecal egg count results among the 396 horses studied. Prevalence and mean eggs per gram (EPG) presented for different groups. Factors Age (years)
Sex Breed Pasture access Treatment intensity (per year) Time since last treatment (months) Summary (range) a Positive b
<2 3-4 5-6 7-10 ≥11 Male Female Mixed Arabian Grazing Housed Both 0 1 ≥2 <2 2-4 4-6 6-12 >12
Horses (n) 90 88 76 77 27 174 211 276 115 180 81 130 91 136 101 26 28 78 120 86 396
egg counts only These refer to a single case
Strongyles Prevalence % EPG 95.6 1540 98.9 1435 97.4 1484 100 1400 85.2 985 96.6 1267 97.6 1559 97.2 1119 100.0 1891 98.3 1549 100.0 1155 94.5 1263 100.0 1636 97.0 1395 94.0 1264 76.9 824 88.9 818 100.0 1125 99.1 1648 100.0 1676 1436 97.2 (0-4150)
Parascaris spp. Prevalence % EPGa 26.7 249 3.4 183 2.6 100 1.3 100b 3.7 150b 5.2 280 14.2 209 8.6 244 17.4 220 13.1 148 3.7 325 11.0 70 22.5 117 3.0 900 5.4 181 3.7 150b 10.4 275 1.7 1200 21.4 109 229 10.0 (0-2325)
Table 3. Odds ratio estimates for two significant covariates in the logistic regression with Parascaris spp. presence (0/1) as outcome variable. Covariate Comparison Odds ratio 95% Wald Confidence Interval <2 years vs. 7-10 years 18.04 1.92-168.98 Age group 3-4 years vs. 7-10 years 2.33 0.18-30.31 5-6 years vs. 7-10 years 1.47 0.08-26.79 1/year vs. 0/year 0.04 0.01-0.18 Treatment intensity >2/year vs. 0/year 0.10 0.02-0.54