Albanian j. agric. sci. ISSN: 2218-2020, (2012), (Special Edition) Copyright © Agricultural University of Tirana

EPIDEMIOLOGICAL STUDIES ON EQUINE CESTODES IN SOUTHERN ALBANIA: A PRELIMINARY STUDY PËLLUMB VELAJ1*, REZART POSTOLI 2, GANI MOKA3, DORINA RAPTI4 1

Regional Agricultural Directory of Gjirokastra

2

Faculty of Veterinary Medicine, Agricultural University of Tirana, Albania

3

Ministry of Agriculture, Food, and Consumer Protection

4

Ministry of Education and Sciences

* Author of correspondence: E-mail: [email protected]

Abstract: An epidemiological study on equine cestodosis was carried out in southern Albania. A total of 90 fecal samples from equids located in south Albania were studied from March 2012 to September 2012. Faecal samples were examined for cestode eggs using a double centrifugation/combined sedimentation–floatation technique. Five equids subjected to coprological examination, resulted positive, respectively for the presence of Anoplocephala spp. eggs at the coprological examination. We were interested in tapeworm prevalence as well as any possible variations observable in parasite population or dynamics. Seasonal prevalence of infection by Anoplocephala spp. was lower in spring (4.2%) than in summer (7%). Since prevalence of infection was significantly low, spring and summer seems to be the seasons when oncospheres would not be available to be eaten by mites. This epidemiological pattern seems to describe the dependence of Anoplocephala spp. to humidity in warm dry climate. However, data from spring and summer were not enough to determine the pattern of infection on the equine tapeworms.

Key words: equine cestodes, epidemiology, season, coprological examination 1. Introduction There are three types of equine tapeworms, the most common being the Anoplocephala perfoliata. Anoplocephala perfoliata is a cestode of the family Anoplocephalidae. Three species of tapeworms (Anoplocephala perfoliata, Anoplocephala magna, and Paranoplocephala mamillana) can be found in horses. These tapeworms live around the illeo-cecal junction. Less common are the largest species, A. magna, located in the posterior small intestine and the smallest species, P. mamillana, which lives in the anterior small intestine and occasionally the stomach. Most importantly, A. perfoliata has recently been implicated as a cause of colic [20, 24, 25, 26]. Numerous types of colic can be caused by this disturbance which can lead to horse injuries and large veterinary bills for horse owners. Proudman et al. (1998) stated that with increasing infection intensities of A. perfoliata there was an increasing risk of spasmodic colic. As for many parasites of veterinary importance, a poor or incomplete understanding of epidemiological characteristics can lead to poor, inefficient, and expensive treatment [18]. The International Conference 31 October 2012, Tirana

enormous variation in infection characteristics of A. perfoliata in horses sampled throughout the world is almost certainly linked to regional and local variation in environmental characteristics [11, 19]. Very generally, regions characterized by moist humid conditions year-round (e.g. New Zealand) tend to have high prevalence [2], whereas areas with more pronounced seasonality (e.g. Ontario, Canada) tend to have lower prevalence [29]. Inaccurate diagnosis likely also contributes to the magnitude of variation seen in the studies. Neither coprological nor serological methods are reliable enough at the moment to totally discount the presence of these parasites. Therefore, reliable A. perfoliata prevalence studies are based on postmortem data and their usefulness is usually limited by low numbers of animals or short time studies. Inaccuracies associated with detection of eggs in faeces are very well known for many parasites of veterinary importance [7, 33], and results of studies involving A. perfoliata are no exception [1, 3, 18, 32]. Validation studies of centrifugation/flotation techniques report sensitivities of 8% to 61%, although it has been reported to be 92% in horses with >20 worms [23]. Poor sensitivity

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is likely to be related to the small number of eggs present in the feces of infected horses [29], and the high specific gravity of the eggs that makes flotation difficult. Although there has been considerable work on the diagnosis of A. perfoliata infection using serodiagnostic assays [5, 13, 17, 28, 31], detection of A. perfoliata DNA by polymerase chain reaction (PCR) in faeces [8, 9, 31] or coproantigen detection using an antigen-capture enzyme-linked immunosorbent assay (ELISA) [12, 28], diagnosis under practical conditions is usually based on examination of faeces for tapeworm ova. The distribution of this tapeworm in Albania is unknown. Anecdotal reports confirm its presence in the prairie regions of Albania, but no information is available regarding seasonal and spatial variation in prevalence or intensity. The purpose of this study was to determine A. perfoliata prevalence in horses from selected regions in southern Albania and to evaluate whether factors such as season, management regime and habitat could explain variation in prevalence within selected regions. 2. Material and Methods 2.1 Faecal samples Faecal samples were collected from privatelyowned horses in southern Albania regions. Fresh faecal samples were collected during the months of March to September 2012, and were examined for cestode eggs using a double centrifugation/combined sedimentation–floatation technique. For those horses that were kept in individual corrals or stalls, the freshest faecal sample was collected. Samples were collected into re-sealable plastic bags, labelled and frozen at -20oC until analyzed. 2.2 Assay procedure Following thorough mixing of each faecal sample, 15 g subsamples were weighed out. Each subsample was mixed with about 40 ml of tap water. The faecal slurry then was sieved through a tea strainer and pressed with a teaspoon to recover as much of the water as possible. The resulting suspension was poured immediately into two conicalbottom 30-ml centrifuge tubes and centrifuged at 400 g for 10 min in a swinging-bucket centrifuge. The supernatant was removed with a water-jet aspirator pump and the sediment re-suspended in a small International Conference 31 October 2012, Tirana

amount of floatation solution using a vortexer. Resuspended samples were transferred to 15-ml conical-bottom centrifuge tubes. Additional floatation solution was added to the tubes until a slight convex meniscus formed, and a 22 mm X 2 2mm coverslip was placed on the top of each tube, in contact with the floatation solution. Tubes with coverslips were placed in a swinging-bucket centrifuge and spun for 10 min at 200 g. After the centrifuge came to a complete stop, the coverslips, with the attached suspension, were removed and placed on a microscope slide. The preparation (two coverslips per faecal subsample placed together on one microscope slide) was examined for cestode eggs using a compound light microscope at a magnification of X40 or X100. Based on morphological features all recovered cestode eggs were identified as Anoplocephala eggs. No efforts were made to distinguish the eggs of A. magna and A. perfoliata, which are similar in shape and size. However, differentiation is possible based on the structure of the so-called piriform apparatus. All Anoplocephala eggs were counted per preparation to derive an egg count per sample. For floatation, the following solutions were used: concentrated sugar solution, sp. g. 1.26 (prepared by dissolving 450 g sugar in 350 ml water). 3. Results and Discussion Total prevalence of infection by Anoplocephala spp. was around 5.6% (5 out of 90 horses, Table 1) while seasonal prevalence was 4.2% (2 out of 48 horses) in the spring and 7% in the summer (3 out of 42 horses). Infected horses will be continuously detected for monthly prevalences of infection in some periods throughout the study period (March 2012– May 2012, spring): first, from (June 2012 to September 2012, summer) second, from (October 2012 to December 2012, autumn) third, from (January 2013 to March 2013, winter) four, and from spring of 2013 onwards till winter 2013. Table 1: Total prevalence of infection by Anoplocephala spp. eggs (N=90) in equids in southern Albania from March 2012 to September 2012 Infection Anoplocephala spp.

Seasons Spring Summer Total

n/N (%) 2/48 (4.2) 3/42 (7.14) 5/90 (5.6)

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4. Conclusions The cestode Anoplocephala perfoliata is known to cause fatal colic in horses. The concern over potential A. perfoliata induced pathology in horses has led to increased efforts in characterizing basic epidemiological characteristics. A number of studies have documented general epidemiological patterns of A. perfoliata infection in horses. Previous surveys indicate enormous variation in infection characteristics of A. perfoliata in horses. Characterizing this variation and understanding its underlying causes is an important feature in the control, diagnosis and management of this tapeworm. The purpose of this study is to characterize the prevalence of A. perfoliata in privately owned horses sampled across southern Albania. Although the biological cycle is well known, no data are available on the epidemiological factors of this parasitism in Albania. This study has provided insight into the occurrence of Anoplocephala spp infection in horses in Southern Albania. Tapeworm eggs were detected in the feces of 5.6% of control horses. This was the first study to monitor horses kept for work purposes in realistic management settings in Albania. Despite the relatively low prevalence of cestodes in horses from southern Albania, the results of this preliminary study indicate significant variation between seasons. However, data from spring and summer were not enough to determine the pattern of infection on the equine tapeworms. The prevalence values observed in this study likely underestimate the true prevalence of Anoplocephala spp in southern Albania horses. This result is not surprising, as fecal diagnostic methods are considered to have very low sensitivity for detection of Anoplocephala spp infection, especially where only a few tapeworms are present [23, 29]. Neither coprological nor serological methods are reliable enough at the moment to totally discount the presence of these parasites. Therefore, reliable A. perfoliata prevalence studies are based on post mortem data and their usefulness is usually limited by low numbers of animals or short time studies. For example, few equine tapeworm prevalence studies include a whole year [21]. We are interested in tapeworm prevalence as well as any possible variations observable in parasite population or dynamics. It is likely that some proportion of infected horse may have had low worm burdens that could have led to misdiagnosis. Faecal International Conference 31 October 2012, Tirana

egg counts varied greatly according to the time of day the faeces were sampled and in the distribution of eggs in the faecal mass. Further, Nilsson et al. (1995) indicated that gravid tapeworm segments were sporadically discharged and were unequally distributed in the faecal mass, which could explain the lack of relationship between tapeworm intensities and egg detection [16, 23]. The lack of an accurate test for the diagnosis of A. perfoliata infection in individual horses has been a significant impediment to investigating various aspects of equine cestodiasis. To date, Proudman & Edwards (1992) have achieved the highest diagnostic sensitivity (61%) using a combined centrifugation/flotation technique. The sensitivity increased to 92% when horses harbouring 20 or fewer worms were eliminated from the sample. Hence, the test is a diagnostic tool for horses at the highest risk of clinical disease. This shortcoming emphasizes the need for more accurate diagnostic techniques. The result of this study is in line with previous survey of horses in Spain [19]. An epidemiological study on equine cestodosis from equids slaughtered in abattoirs was carried out in central Spain. Anoplocephala perfoliata was detected in 24% of the animals and Anoplocephala magna in 18%. Seasonal prevalence was significantly higher in autumn (37.5%) and winter (32.3%) than in spring (9.2%) and summer (10.8%). Results from this post mortem study showed great differences between sampling months. There were several months without any positive animals while in others the prevalence was very high. It seems that in our climatic conditions, tapeworm prevalence varies considerably throughout the year, showing a remarkable dependence on the environmental conditions. Seasonal differences could clearly be established, with the highest prevalence in autumn and winter. Results from this seasonal survey indicate that the transmission of Anoplocephala spp.in southern Albania will vary over the course of the year. We have interpreted the seasonal patterns of infection as follows, supporting the results of similar studies in Spain [19] and Sweden [11]. Survey studies in Spain [19] and Sweden [11] indicate an approximate annual cycle of infection. In general, encysted larval stages in mites are ingested during mid- to late-summer and require approximately 8-10 weeks to mature to the egg-producing stage. Transmission rates from egg to mite are unknown, but in temperate habitats, transmission likely occurs in spring/early summer when mites emerge from winter dormancy. Thus, the

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annual cycle repeats when fully-developed cysticercoids are available for ingestion in mid- to late summer. Following the period of A. perfoliata maturation during the fall months, the maximum number of eggs seeded onto pasture likely occurs over winter and spring. Hoglund et al. (1998) and Meana et al. (2005) showed that ingestion of infected mites occurs during summer grazing, followed by deposition of eggs onto pasture when worms mature in winter. It is likely that eggs would not be detected in the faeces until adult worms had developed over a period of 2-3 months [19, 34]. It could be deduced that eggs are available to be eaten by mites in winter and spring, and while in both seasons tapeworm burdens were equally low, egg quantities in the field seem to be higher in winter, when more horses are infected with tapeworms. It could be concluded that once the mites have ingested the eggs, they should be able to survive warm, dry summer conditions with cysticercoids developing inside, with the result of a high number of infected mites at the end of summer and, especially, in autumn. This lag period associated with the worm maturation explains the decrease in prevalence that we observed during spring and summer at all sites. A number of studies have documented the prevalence of A. perfoliata in different countries throughout the world. Reported prevalences vary from 14% to 81% as documented in several postmortem surveys [4, 10, 14, 15, 16, 22]. The highest prevalences reported in Europe came from wet, northern countries such as Sweden [21], the United Kingdom [22], France [6] or Germany [3]. In Mediterranean Basin countries, with a climate pattern similar to that of the Iberian Peninsula, lower prevalences are reported, Italy 14% [27] and Greece 0.4% [30]. The percentage of animals found with tapeworm eggs should be considered a minimum prevalence. The true infection rate should be considered to be much higher. This literature has been well reviewed, and indicates that high prevalences of infection occur in countries with temperate climates. Differences in the numbers of horses examined in each study and the methodology used mean that inter study comparisons should be made with caution. 6. References 1. Agneessens J, Debever P, Engelen S, Vercruysse J: The prevalence of Anoplocephala perfoliata in horses in Belgium, and evaluation of the International Conference 31 October 2012, Tirana

diagnostic sedimentation/flotation technique. Vlaams Diergeneeskundig Tijdschrift 1998, 67 (1): 27-31. 2. Bain SA, Kelly JD: Prevalence and pathogenicity of Anoplocephala perfoliata in a horse population in South Auckland. New Zealand Veterinary Journal 1977, 25: 27-28. 3. Beelitz P, Gothe R: Tapeworm infections in slaughter horses from Upper Bavaria: prevalence and worm burden as well as correlation between coprological diagnosis and infection with adult cestodes. Pferdeheilkunde 2001, 17(5): 423-428. 4. Benton RE, Lyons ET: Survey in central Kentucky for prevalence of Anoplocephala perfoliata in horses at necropsy in 1992. Veterinary Parasitology 1994, 55: 81-86. 5. Boswinkel M, Van Oldruitenborgh-Oosterbaan M.M.S: Correlation between colic and antibody levels against Anoplocephala perfoliata in horses in the Netherlands. Tijdschr Diergeneeskd 2007, 132: 508-512. 6. Collobert C, Fleury C,Valognes A, Pedaille F: Prevalence du teniasis chezles equides en France. Prat. Vet. Equine 1997, 29: 149–158. 7. Cringoli G, Rinaldi L, Veneziano V, Capelli G, Scala A: The influence of flotation solution, sample dilution and the choice of McMaster slide area (volume) on the reliability of the McMaster technique in estimating the faecal egg counts of gastrointestinal strongyles and Dicrocoelium dendriticum in sheep. Veterinary Parasitology 2004, 123: 121-131. 8. Chlastáková I, Vavrouchováč E, Kamler M, Bodeček Š, & Koudela B: Comparison of coprological and molecular techniques for the diagnosis of Anoplocephala perfoliata infection of the horse. World Association for the Advancement of Veterinary Parasitology Congress, Calgary, Canada 2009, 8–15 August, Abstracts: 161. 9. Drogemuller M, Beelitz P, Pfister K, Schnieder T, Von Samson-Himmelstjerna G: Amplification of ribosomal DNA of Anoplocephalidae: Anoplocephala perfoliata diagnosis by PCR as a possible alternative to coprological methods. Veterinary Parasitology 2004, 124: 205-215. 10. Fogarty U, Del Piero F, Purnell RE, Mosurski KR: Incidence of Anoplocephala perfoliata in horses examined at an Irish abattoir. Veterinary Record 1994, 134: 515-518.

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in Sweden: prevalence, infection levels and intestinal lesions. Acta Vet. Scand 1995, 3: 319328. 22. Owen R, Jagger D.W & Quan-Taylor R. Prevalence of Anoplocephala perfoliata in horses and ponies in Clwyd, Powys and adjacent English marshes. The Veterinary Record 1988, 123: 562–563. 23. Proudman CJ, Edwards GB: Validation of a centrifugation/flotation technique for the diagnosis of equine cestodiasis. Veterinary Record 1992, 131:71-72. 24. Proudman CJ, Edwards GB: Are tapeworms associated with equine colic? A case control study. Equine Veterinary Journal 1993, 25: 224226. 25. Proudman CJ, French NP, Trees AJ: Tapeworm infection is a significant risk factor for spasmodic colic and ileal impaction colic in the horse. Equine Veterinary Journal 1998, 30: 194199. 26. Proudman CJ, Trees AJ: Tapeworms as a Cause of Intestinal Disease in Horses. Parasitology Today 1999, 15: 156-159. 27. Scala A, Cancedda M, Spagnesi M, Guberti V, De Marco M.A: Factors affecting the survival of ‘‘wild’’ horses on the Giara tableland (Sardinia): endoparasitic diseases. Ricerche di Biologia della Selvaggina 1994, 24 (Suppl.): 111– 117 (abstract in VETCD: database from CAB International, Oxfordshire, UK. Accession number 970805553, updated 980216). 28. Skotarek SL.: Epidemiology and diagnosis of Anoplocephala perfoliata in horses from Southern Alberta, Canada. MSc thesis. University of Lethbridge, Canada. 2008. 29. Slocombe JO: Prevalence and treatment of tapeworms in horses. Canadian Veterinary Journal 1979, 20: 136-140. 30. Sotiraki ST, Badouvas AG and Himonas CA: A survey on the prevalence of internal parasites of equines in Macedonia and Thessalia-Greece. Journal of Equine Veterinary Science 1997, 10: 550-552. 31. Traversa D, Fichi G, Campigli M, Rondolotti A, Iorio R, Proudman C.J, Pellegrini D, Perrucci S: A comparison of coprological, serological and molecular methods for the diagnosis of horse infection with Anoplocephala perfoliata

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33. Ward M P, Lyndal-Murphy M, Baldock F C: Evaluation of a composite method for counting helminth eggs in cattle faeces. Veterinary Parasitology 1997, 73: 181-187. 34. Worley DE, Jacobson RH, Barrett RE: The chronology of tapeworm (Moniezia expansa) acquisition by sheep on summer ranges in Montana and Idaho. Proceedings of the Helminthological Society of Washington 1974, 41 (1): 19-22.

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