RC590 - corregido.pdf

Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido: 16/12/2024 Aprobado:19/02/2025 Publicado: 15/04/2025 hps://doi.org/10.52973/rcfcv-e35590 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 8 Comparison of Free and Liposomal Levamisole Anparasic Acvity in Sheep Comparación de la acvidad anparasitaria del levamisol libre y liposomal en ovejas 1 Department of Parasitology, Faculty of Veterinary Medicine, Balikesir University, Balıkesir, Türkiye. ²Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Balikesir University, Balıkesir, Türkiye. ³Department of Laboratory and Veterinary Health, Savastepe Vocaonal School, Balikesir University, Balıkesir, Türkiye *Corresponding Author: mehmet.ozuicli@balikesir.edu.tr, phone 0554 334 18 70 ABSTRACT Worldwide, parasic organisms residing in the digesve systems of sheep cause substanal economic losses. Various anparasic chemicals are employed to combat parasites, mostly levamisole. Nevertheless, parasites have developed resistance to the treatments employed in recent years. Consequently, sciensts are currently seeking more eﬀecve medicinal composions. The ulizaon of liposomes is one of the most extensively studied techniques to enhance pharmaceucal eﬃcacy. This study assessed the anparasic eﬃcacy of both free and liposomal levamisole. In this study four groups, each containing 12 animals, were formed: Group 1; free levamisole group (FLOG) received a single oral dose of free levamisole at 7.5 mg/kg; Group 2; liposomal levamisole group (LLOG) received a single oral dose of liposomal levamisole at 7.5 mg/kg; Group 3; (Posive Control: PCG) received a single oral dose of physiological serum at 7.5 mg/kg, and Group 4; served as the negave control (NCG). Fecal specimens were collected from the rectum into sterile containers on days 0, 7, 14, 21, and 28. The McMaster method was employed in the study to quanfy eggs per gram of feces (EPG) loading. The eﬃcacy of treatment groups was assessed using the Fecal Egg Count Reducon (FECR) formula. According to the FECR formula, Strongylids had a treatment eﬃcacy of 65.36% in the FLOG group and 70.67% in the LLOG group, Trichuris had 41.78% and 74.22%, and Nematodirus had 52.78% and 71.85%. The eﬃcacy of treatment was higher in the liposomal levamisole group compared to the free levamisole group. This study established the anparasic eﬃcacy of liposomal levamisole for the ﬁrst me. Further research are required to evaluate the anparasic eﬀects of liposomal levamisole through the administraon of varied and recurrent doses. Key words: Anparasic acvity; levamisole; liposome; sheep RESUMEN A nivel mundial los organismos parasitarios que residen en el aparato digesvo del ganado ovino causan cuanosas pérdidas económicas. Para combarlos se emplean diversos productos químicos anparasitarios, principalmente levamisol. Sin embargo, en los úlmos años los parásitos han desarrollado resistencia a los tratamientos empleados. En consecuencia, los cienﬁcos buscan actualmente composiciones medicinales más eﬁcaces. La ulización de liposomas es una de las técnicas más estudiadas para mejorar la eﬁcacia farmacéuca. En este estudio se formaron cuatro grupos, cada uno con 12 animales: Grupo 1; grupo de levamisol libre (FLOG) recibió una dosis oral única de levamisol libre a 7,5 mg/kg; Grupo 2; grupo de levamisol liposomal (LLOG) recibió una dosis oral única de levamisol liposomal a 7,5 mg/kg; Grupo 3; (control posivo: PCG) recibió una dosis oral única de suero ﬁsiológico a 7,5 mg/kg, y Grupo 4; sirvió como control negavo (NCG). Los días 0, 7, 14, 21 y 28 se recogieron muestras fecales del recto en recipientes estériles. En el estudio se empleó el método McMaster para cuanﬁcar la carga de huevos por gramo de heces (EPG). La eﬁcacia de los grupos de tratamiento se evaluó mediante la fórmula de reducción del recuento de huevos en heces (FECR). Según la fórmula FECR, Strongylids tuvo una eﬁcacia de tratamiento del 65,36% en el grupo FLOG y del 70,67% en el grupo LLOG, Trichuris del 41,78% y del 74,22%, y Nematodirus del 52,78% y del 71,85%. La eﬁcacia del tratamiento fue mayor en el grupo de levamisol liposomal en comparación con el grupo de levamisol libre. Este estudio estableció por primera vez la eﬁcacia anparasitaria del levamisol liposomal. Se requieren más invesgaciones para evaluar los efectos anparasitarios del levamisol liposomal mediante la administración de dosis variadas y recurrentes. Palabras clave: Acvidad anparasitaria; levamisol; liposoma; oveja Mehmet Özüiçli 1* , Hasan Susar 2 , Murat Çelebi 3 , Çağla Çelebi 2 , Hüseyin Şen 2 , İzzet Karahan 2

Anparasic Acvity of Levamisole with diﬀerent structures in Sheep / Özüiçli et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Türkiye’s economy and agricultural sector are heavily dependent on livestock farming [1]. Parasic infecons result in substanal losses in meat, milk, wool, and leather producon, hence impacng economic input [2]. Türkiye’s geographical posion and diverse climate create an ideal seng for parasic infecons. Epidemics produced by endoparasites in domesc animals are prevalent in Türkiye, resulng in considerable damage to the local sheep (Ovis aries) industry and the naonal economy [3]. The prevalence of helminths in sheep has been documented to range from 0.2% to 100% globally, according to studies ulizing necropsy and fecal analysis [4]. The prevalence of trematodes, cestodes, gastrointesnal nematodes, and lung nematodes in Türkiye was reported as 3.1-72.6% [5], 7.56-21% [6], 0.39-100% [7], and 7.8-34% [8], respecvely. Levamisole (Tetramisole) is a compound belonging to the Imidazothiazole derivave class of anthelminc agents, parcularly eﬀecve against nematodes that invade the gastrointesnal tract and lungs [9]. It has been used in the control of Haemonchus spp., Trichostrongylus axei, Ostertagia spp., Nematodirus spp., Cooperia spp., Bunostomum trigonocephalum, Strongyloides spp., Oesophagostomum spp., Chabera spp., and Dictyocaulus spp. Levamisole is a combinaon of D- and L- isomers of Tetramisole, exhibing a nematodicides acvity and resembling pyrantel in its mechanism of acon [10]. To ascertain that the anthelminc eﬃcacy of the drugs is aributable to the L-isomer, the toxic eﬀects associated with the D-isomer were migated, and the conﬁdence interval was expanded by employing solely the L-isomer. Levamisole funcons as an agonist at niconic-cholinergic neuromuscular juncons, targeng nematodes in people and animals at low doses, leading to muscle contracon and subsequent paralysis of the parasites. At elevated concentraons, it interferes with intermediate metabolism by inhibing the enzyme fumarate reductase [11 , 12]. Resistance to anparasic drugs is becoming a signiﬁcant public health issue. With prolonged use at advised dosages, resistance may gradually develop against anparasic medicaons capable of signiﬁcantly reducing the parasite populaon within the host. Prolonged medicaon administraon can result in the development of resistance in parasites over me. Novel pharmaceucals must be produced to address this issue [13 , 14]. The potenal ways by which liposomes may prevent parasite resistance might be brieﬂy stated as follows: Diverse phospholipids possess the capacity to act as intracellular messengers in regulang innate and adapve immune responses via various mechanisms, including the acvaon of anmicrobial and anparasic enzymac pathways, the regulaon of fusion- ﬁssion events between endosomes inﬂuencing phagosome maturaon and/or the angen presentaon pathway, and the modulaon of the inﬂammatory response [15 , 16]. This study aimed to examine the parasicidal eﬀects of free levamisole versus liposomal levamisole. As parasites acquire resistance to exisng medicaons, novel pharmacological agents are required. This invesgaon has resulted in the development of a liposomal formulaon of levamisole, which has potenal for applicaon in veterinary medicine, and its eﬃcacy has been established. MATERIALS AND METHODS Study area The study was conducted in Balıkesir province in Türkiye. Balıkesir is at latude 39.6484° N and longitude 27.8826° E coordinates. Preparaon of free levamisole soluon The mean weight of the animals (Merino sheep from Türkiye, aged 1 to 2 years) included in the study was 60 kg. Considering that levamisole was to be administered at a dosage of 7.5 mg/ kg, 450 mg of the substance was measured. The measured levamisole was dissolved in 10 mL of dislled water and delivered orally to the animals. Levamisole was used orally in this study. Levamisole has been used orally in previous studies. In sheep, Gokbulut et al. [17] 7.5 mg/kg and Fernandez et al. [18] 5-10 mg/kg doses were studied and no side eﬀects were reported. Preparaon of liposomal levamisole formulaon Liposomes were synthesized using the speciﬁc technique [19]. Soya lecithin (L-α-Lecithin, Sigma – Aldrich, USA) and Cholesterol ((CL), Acros Organics, Belgium) were measured in a 3:1 rao. 450 mg of levamisole ((LVM), Santa Cruz Biotechnology – sc – 205730, USA) was included into this mixture. Chloroform (Sigma-Aldrich, USA) and methanol (Sigma-Aldrich, USA) were combined in a 1:1 rao to solubilize the compounds. The mixture was placed in a volalisaon ﬂask and subjected to volalisaon in a rotary evaporator (Isolab Laborgeräte GmbH 605.01.001, Germany) at 37 °C for 60 min at a speed of 8.77 G to produce a lipid ﬁlm. To eliminate the lipid coang, 10 mL of phosphate-buﬀered water (Oxoid – BR0014G UK) was introduced, and the ﬂask was spun without applying vacuum. The entryway of the evaporaon ﬂask was securely sealed with paraﬁlm, vortexed (Vortex MS 3 basic ika 3617000, Germany) for 2 min, and subsequently placed in an ultrasonic bath (MEDISSON, Türkiye) for 5 min to diminish the parcle size. The liposomes were centrifuged (ALLEGRA–X64R, USA) at 3043.76 G for 1 h to remove free levamisole that was not incorporated into the liposomes. The liposomal fracon that precipitated at the boom was extracted and preserved in a refrigerator (Arçelik 270530EB, Türkiye) at 4°C. The steps of liposome preparaon are shown in FİG. 1. Analysis of liposomal levamisole formulaon Parcle size (PS), polydispersity index (PDI), zeta potenal (ZP) and encapsulaon eﬃciency (EE) analyses are based on a predeﬁned methodology [20]. Animal management The research procedure was performed with the permission of Balıkesir University Animal Experiments Local Ethics Commiee (Date: 01.08.2024, Decision Number: 2024/8-5). Forty-eight Merino sheep, aged 1 to 2 years, were ulized on a sheep farm situated in Türkiye. 2 of 8

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 1. Steps of Levamisole Lyposomal Preparaon. 1: Dissoluon of substances, 2: Ob- taining lipid ﬁlm in rotary evaporator, 3: Obtained lipid ﬁlm, 4: Removal of lipid ﬁlm, 5: Centrifugaon to remove free drug, 6: Liposomal levamisole, 7: Oral administraon animals Experimental Design Idenﬁcaon of posive and negave animals Fecal samples, approximately ﬁve g each, were collected from the rectum of 100 Merino sheep aged 1-2 years, all of which had not undergone anparasic treatment prior to sampling, using sterile gloves or carrier bags. Fecal samples were examined using the Fulleborn saturated saline technique [21]. Determinaon of eggs loads of fecal samples To evaluate the eﬀecveness of free and liposomal levamisole against gastrointesnal parasites in both treatment groups, fecal samples were collected weekly for up to four weeks aﬅer treatment. Egg counts, expressed as eggs per g of feces (EPG), were measured using the modiﬁed McMaster’s technique, which has a sensivity of 50 EPG [22]. With this method, egg loads were assessed for each animal on day 0 (baseline) and at 7, 14, 21, and 28 days post-treatment. Treatment and control groups The study involved 48 animals, categorized into four groups according to their egg counts, with 12 animals assigned to each group. The study groups were structured as outlined below: Group 1 (FLOG) was administered a single oral dose of 7.5 mg/ kg free levamisole; Group 2 (LLOG) received a single oral dose of 7.5 mg/kg liposomal levamisole; Group 3 (Posive Control: PCG) was given a single oral dose of 7.5 mg/kg physiological serum; and the negave control group (NCG) was included. Following the collecon of feces in the morning, samples were transported to the laboratory under cold chain condions, where the McMaster method was applied. The treatment groups and posive control group were designed to be homogeneous regarding total EPG count. Fecal samples were taken from 100 sheep and 48 animals were included in the study considering the amount of EPG. Treatments applicaon day All treatment protocols were administered to the treatment groups on day 0 (baseline) as a single oral dose. Calculaon of treatment eﬃcacy The eﬃcacy of treatment groups was assessed using the subsequent formula: Fecal Egg Count Reducon (FECR)% = 100 – ( × 100) [22]. Day (d) 28 was designated as the conclusive count of parasite eggs, whereas d 0 was recognized as the inial quanty (baseline) of parasite eggs. Stascal analysis All data were evaluated by SPSS 25.0 (IBM Corp, Armonk, NY, USA). The Friedman test was applied for intra-group comparisons, while the Kruskal-Wallis test was ulized for inter-group comparisons. P-values below 0.05 signify that the diﬀerence is stascally signiﬁcant [23]. RESULTS AND DISCUSSIONS In this study, the mean parcle size of liposomal levamisole 156.4±2.8 nanometer (nm), with a zeta potenal of -47.6, encapsulaon eﬃciency of 92.47%, and a polydispersity index of 0.128 were found. The quality control assessments of the prepared liposomes indicated their suitability for animal use. They exhibited a diminuve parcle size, elevated electrokinec potenal, monodispersity, and colloidal stability. Due to drug resistance, liposome-encapsulated pharmaceucals studies on levamisole have gained momentum. Numerous studies have examined the anparasic eﬀects of levamisole, leading to the development of various formulaons for animal use [24 , 25 , 26 , 27]. Nevertheless, no research has compared the anparasic eﬃcacy of free and liposomal formulaons of levamisole.The advancement of novel drug delivery systems is crucial for establishing therapeuc alternaves that enhance pharmacological responses and reduce side eﬀects [28]. This study evaluated a liquid liposome formulaon as a delivery system for levamisole hydrochloride to address parasic infecons. Liposomal levamisole represents a promising alternave for the treatment and prevenon of parasites in sheep. The present study yielded superior results compared to previous studies. In this study, the parcle size of liposomal levamisole was determined to be 156.4±2.8 nm, with a zeta potenal of -47.6 mV, an encapsulaon eﬃciency of 92.47%, and a polydispersity index of 0.128. Researchers found that the PDI of alizarin liposomes was 0.445-0.609, ZP was between -51.8 and -38.6 milivolt (mV), EE was 4-45%, and PS was 451- 1031 nanometer (nm) [29]. Another researchers found the PDI value of astaxanthin liposomes to be 0.31±0.04, the ZP value to be -37.23 mV, the EE value to be 89.45%, and the PS value to be 101.21±16.67 nm [30]. Also, the ZP of ciproﬂoxacin liposomes to be 23.2±2 mV, the EE to be 76.17±1.8% and the PS to be 183±3 nm were found [31]. The substances and amounts used may have contributed to the diﬀerent results. In this study, Strongylids, Trichuris, and Nematodirus groups, the eﬃcacies in the FLOG and LLOG treatment groups were determined as (65.36%-70.67%, 41.78%-74.22%, 52.78%- 71.85%), respecvely. In the study, liposomal levamisole was found to have more treatment eﬃcacy than free levamisole. It was thought that the reason for this may be the increase in the eﬃcacy of levamisole due to the encapsulaon process with liposome. 3 of 8

Anparasic Acvity of Levamisole with diﬀerent structures in Sheep / Özüiçli et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico A study was conducted to assess the eﬃcacy of a triclabendazole- levamisole combinaon in treang endoparasic infecons in sheep. The number of helminth eggs in feces was evaluated before and aﬅer treatment, and the eﬃcacy of triclabendazole-levamisole was found to be 96.9% for Fasciola spp., 87.1% for Moniezia spp., 83.3% for Trichuris ovis, and 99.9% for gastrointesnal nematodes [32]. Addionally, the eﬀecveness of levamisole in treang gastrointesnal nematodosis in sheep was examined. A total of 30 sheep with fecal egg counts exceeding 150 eggs per gram (EPG) were randomly selected. The study reported a signiﬁcant reducon (P<0.01) in the mean fecal egg count (FEC) in the treated groups compared with the pre-treatment FEC. In contrast, the diﬀerence between the pre-treatment and post-treatment FEC in the control group was not stascally signiﬁcant. In this study, levamisole was 99.52% eﬀecve against gastrointesnal nematodes in Garole sheep [33]. In a diﬀerent study, the combined treatment of moxidecn and levamisole was invesgated for its eﬃcacy against mul-resistant gastrointesnal nematodes. The researchers observed eﬃcacy rates of 84.3% (Haemonchus contortus), 100% (Teladorsagia circumcincta and Trichostrongylus axei), and 97.4% (T. colubriformis) in the ﬁrst year of drug administraon. Following four years of repeated administraon of the combined treatment, eﬃcacy remained high (100%) for all species, except for T. colubriformis, where eﬃcacy decreased to 58%. In the inial applicaon, eﬃcacy was 99% for MOX, 85% for LEV, and 100% for MOX+LEV. The co- administraon of MOX and LEV was found to result in a signiﬁcantly higher anthelminc eﬀect (87%) than either MOX (42%) or LEV (69%) alone over the four-year trial period [34]. According to the results of this study, Strongylids (FLOG FECR=65.36%, LLOG FECR=70.67%), Trichuris (FLOG FECR=41.78%, LLOG FECR=74.22%), and Nematodirus (FLOG FECR=52.78%, LLOG=71.85%) were found in TABLES I, II, and III, respecvely. In this study, the eﬃcacy of levamisole was found to be lower than the eﬃcacies found in the studies menoned above. This diﬀerence may have been caused by the diﬀerent sheep breeds studied, the season in which the study was carried out, humidity and rainfall rates depending on the season, the method of administraon of the drugs. The development of parasite resistance to levamisole may also have contributed to this issue. A study assessed the eﬃcacy of ivermecn, albendazole, and levamisole, revealing eﬀecveness rates of 58.5, 70.1, and 85.8%, respecvely. These ﬁndings demonstrate that nematodes have acquired resistance to all treatments [35]. In another study, anthelmenc resistance was detected in sheep ﬂocks [36]. To elucidate this situaon, drug resistance studies must be conducted on a greater number of animals and herds across various regions within the same country, or even in diﬀerent countries, by diverse researchers, with subsequent comparison of the results. In this study, administraon of free and liposomal levamisole orally at a dose of 7.5 mg/kg to animals did not result in any local or systemic adverse eﬀects. In a study, the horse (Equus caballus) displayed symptoms of levamisole toxicity, including depression, recumbency, frequent urinaon, and the presence of signs indicave of labor. Addionally, the horse exhibited hyperemia of the conjuncvae, a palpebral reﬂex, constricted pupils, and lacrimaon [37]. A ﬂock of sheep also showed signs of acute levamisole-fenbendazole intoxicaon. The animals displayed symptoms such as mucosal congeson, depression, anorexia, convulsions, ataxia, and drooling. Of the animals, 12 died prior to treatment, and ﬁve died aﬅer treatment [38]. Friesian calves (Bos taurus taurus) exhibited a range of severe niconic-type symptoms, including hypersalivaon, foaming at the mouth, muscle spasms, a tendency to lie down, and rapid respiraon [39]. In this study, no clinical signs was observed in the treatment and control groups and no animal loss was experienced. The EPG loads and treatment eﬃciencies of Strongylids, Trichuris, and Nematodirus infecon at days 0, 7, 14, 21, and 28 were displayed in TABLE I, TABLE II, and TABLE III, respecvely. Also, posive and negave control groups total (Strongylids, Trichuris, and Nematodirus) EPG loads were given in TABLE IV. In both the FLOG and LLOG treatment groups of Strongylids, the total number of EPGs decreased from d 0 to d 28. In the study, the 0-28 d FECR value was 65.36% in the FLOG group and 70.67% in the LLOG group (TABLE I). TABLE I. The EPG loads and treatment eﬃciencies of Strongylids infecon at days 0, 7, 14, 21, and 28 Days FLOG EPG Loads LLOG EPG Loads D 0 76.950 72.800 D 7 60.650 48.650 D 14 50.450 40.900 D 21 38.450 32.750 D 28 26.650 21.350 Days Treatment Groups’ Eﬃcacies Determined According to the Days 0–7 FLOG FECR % = 21.18 LLOG FECR % = 33.17 0–14 FLOG FECR % = 34.43 LLOG FECR % = 43.81 0–21 FLOG FECR % = 50.03 LLOG FECR % = 55.01 0–28 FLOG FECR % = 65.36 LLOG FECR % = 70.67 D: Day, EPG: Eggs per gram of feces, FLOG: Free levamizole group, LLOG: Liposomal levamizole group, FECR: Fecal egg count reducon In both the FLOG and LLOG treatment groups of Trichuris, the total number of EPGs decreased from d 0 to d 28. In the study, the 0-28 d FECR value was 41.78% in the FLOG group and 74.22% in the LLOG group (TABLE II). TABLE II. The EPG loads and treatment eﬃciencies of Trichuris infecon at days 0, 7, 14, 21, and 28 Days FLOG EPG Loads LLOG EPG Loads D 0 68.450 75.450 D 7 62.450 60.250 D 14 55.750 45.550 D 21 44.650 25.350 D 28 39.850 19.450 Days Treatment Groups’ Eﬃcacies Determined According to the Days 0–7 FLOG FECR % = 8.76 LLOG FECR % = 20.14 0–14 FLOG FECR % = 18.55 LLOG FECR % = 39.62 0–21 FLOG FECR % = 34.76 LLOG FECR % = 66.40 0–28 FLOG FECR % = 41.78 LLOG FECR % = 74.22 D: Day, EPG: Eggs per gram of feces, FLOG: Free levamizole group, LLOG: Liposomal levamizole group, FECR: Fecal egg count reducon 4 of 8

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico In both the FLOG and LLOG treatment groups, the total number of EPGs decreased from d 0 to d 28. In the study, the 0-28 d FECR value was 52.78% in the Nematodirus FLOG group and 71.85% in the LLOG group (TABLE III). TABLE III. The EPG loads and treatment eﬃciencies of Nematodirus infecon at days 0, 7, 14, 21, and 28 Days FLOG EPG Loads LLOG EPG Loads D 0 65.550 72.850 D 7 60.250 58.350 D 14 53.450 42.750 D 21 40.100 28.350 D 28 30.950 20.500 Days Treatment Groups’ Eﬃcacies Determined According to the Days 0–7 FLOG FECR % = 8.08 LLOG FECR % = 19.90 0–14 FLOG FECR % = 18.45 LLOG FECR % = 41.31 0–21 FLOG FECR % = 38.82 LLOG FECR % = 61.08 0–28 FLOG FECR % = 52.78 LLOG FECR % = 71.85 D: Day, EPG: Eggs per gram of feces, FLOG: Free levamizole group, LLOG: Liposomal levamizole group, FECR: Fecal egg count reducon In the negave control group, no parasic agent was detected on d 0 and 7, but the parasite load increased from d 14 to d 28. In the posive control group, parasite load increased steadily from d 0 to d 28 (TABLE IV). In this study, the parasite load in the posive control group showed a connuous increase during the study period, while the negave group became parasically posive from the d 14 and the parasic load gradually increased unl the end of the study (TABLE IV). The consumpon of food and water contaminated with feces, licking of wool and feathers play an important role in the transmission of parasites between animals on the farm, increasing the risk of contaminaon [40]. The reason for this situaon may be the contaminaon in the farm. TABLE IV. Posive and negave control groups total (Strongylids, Trichuris, and Nematodirus) EPG loads Days Posive Group EPG Loads Negave Group EPG Loads D 0 45.550 0 D 7 56.750 0 D 14 60.150 12.600 D 21 65.600 14.900 D 28 78.900 21.800 D: Day, EPG: Eggs per gram of feces Strongylids EPG analyses on d 7 indicated a stascally signiﬁcant diﬀerence between groups (P<0.05). No stascally signiﬁcant diﬀerence was observed between the groups on the remaining days (P>0.05). In the LLOG, there was a stascally signiﬁcant diﬀerence between the ﬁrst Strongylids values and those taken on d 7, 14, 21, and 28 (P<0.001). A decrease was observed from the inial mean to the ﬁnal mean on the 28th d. A stascally signiﬁcant diﬀerence was observed in the Strongylids values of the animals in the FLOG on the inial, 7th, 14th, 21st, and 28th d (P<0.001). For FLOG, the inial mean for Strongylids was 6525.00±2138.13, and the ﬁnal mean (28th d) was 1945.83±826.12. A reducon was noted from the inial mean to the ﬁnal mean (TABLE V). TABLE V. Intragroup and intergroup comparisons of Strongylids EPG value Liposomal Levamizole Free Levamizole Mean±SD Median (Min-Max) Mean±SD Median (Min-Max) U p Strongylids D 0 7079.17±2164.11 7575 (1250-9500) 6525.00±2138.13 6925 (1000-9300) 1.126 0.260 D 7 5637.50±1895.46 6100 (800-7600) 4470.83±1646.27 4900 (500-6800) 2.196 0.028 D 14 4745.83±1778.46 4800 (600-6900) 3708.33±1502.85 3800 (300-5800) 1.848 0.065 D 21 3662.50±1443.34 3750 (350-5500) 2979.17±1135.47 3075 (250-4300) 1.647 0.100 D 28 1600.00±809.09 1500 (200-3500) 1945.83±826.12 2050 (150-3000) 1.396 0.163 F=48.000 P<0.001 F=48.000 P<0.001 D: Day, F: Firedman Test, U: Mann Whitney U Test, P<0.05, SD: Standart deviaon, Min: Minumum, Max: Maximum The Trichuris values on the 21st d indicated a stascally signiﬁcant diﬀerence between the groups (P<0.05). The average Trichuris count on d 7 was markedly reduced in the LLOG (2404.17±1120.97) compared to the FLOG (3962.50±1502.14). On the remaining d, no stascally signiﬁcant diﬀerence was observed between the groups (P>0.05). A stascally signiﬁcant diﬀerence was observed in the Trichuris values of the animals in the LLOG on the inial d, as well as on the 7th, 14th, 21st, and 28th d (P<0.001). The mean values for the inial d (6870.83±2116.76) and the 28th d (1787.50±775.22) were calculated. A reducon was noted from day 0 to d 28. A stascally signiﬁcant diﬀerence was observed in the Trichuris values of the animals in the FLOG on the inial, 7th, 14th, 21st, and 28th d (P<0.001). The mean value for the inial d was 6087.50±2118.33, and the mean value for d 28 was 2275.00±1124.22. A reducon was noted from day 0 to d 28 (TABLE VI). 5 of 8

Anparasic Acvity of Levamisole with diﬀerent structures in Sheep / Özüiçli et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico TABLE VI. Intragroup and intergroup comparisons of Trichuris EPG value Liposomal Levamizole Free Levamizole Mean±SD Media (Min-Max) Mean±SD Media (Min-Max) Trichuris D 0 6870.83±2116.76 7225 (1200-9400) 6087.50±2118.33 6475 (800-8500) 1.271 0.204 D 7 5479.17±1833.09 5825 (800-7500) 5537.50±1998.76 5800 (700-8000) 0.087 0.931 D 14 4170.83±1533.63 4400 (700-5900) 4937.50±1854.62 5250 (600-7500) 1.184 0.236 D 21 2404.17±1120.97 2250 (500-4600) 3962.50±1502.14 4150 (450-5800) 2.600 0.009 D 28 1787.50±775.22 1900 (300-3000) 2275.00±1124.22 2000 (300-4500) 0.961 0.336 F=47.267 p<0.001 F=48.000 p<0.001 D: Day, F: Firedman Test, U: Mann Whitney U Test, P<0.05, SD: Standart deviation, Min: Minumum, Max: Maximum The d 14 Nematodirus value exhibited a stascally signiﬁcant diﬀerence between the groups (P<0.05). The mean Nematodirus count on d 14 for the animals in the LLOG (3608.33±1284.49) was signiﬁcantly lower than that of the animals in the FLOG (4712.50±1750.60). Analysis of Nematodirus on d 21 revealed a stascally signiﬁcant diﬀerence between the groups (P<0.05). The average Nematodirus count in the LLOG on d 21 (2695.83±989.60) was signiﬁcantly lower than that in the FLOG (3550.00±1346.21). No stascally signiﬁcant diﬀerence was observed between the groups on the other d (P>0.05). A stascally signiﬁcant diﬀerence in Nematodirus values was noted in the animals of the LLOG on the inial, 7th, 14th, 21st, and 28th d (P<0.001). The means of Nematodirus on d 0 (6350.00±2132.22) and d 28 (2000.00±921.71) were calculated. A reducon was noted from day 0 to d 28 (TABLE VII). The reason why these diﬀerences are evident on the d 14 and d 21 that there is eﬃcacy against adults and not against larvae in the mucosa, as indicated by Muñoz et al. [41]. TABLE VII. Intragroup and intergroup comparisons of Nematodirus EPG value Liposomal Levamizole Free Levamizole Mean±SD Media (Min-Max) Mean±SD Media (Min-Max) Nematodirus D 0 6350.00±2132.22 6500 (1000-9000) 5787.50±2144.56 6250 (600-8800) 0.866 0.386 D 7 5320.83±1806.23 5650 (900-7000) 5312.50±1922.37 5775 (700-7100) 0.260 0.795 D 14 3608.33±1284.49 3900 (800-4900) 4712.50±1750.60 5175 (400-6500) 2.398 0.016 D 21 2695.83±989.60 2950 (500-4000) 3550.00±1346.21 3725 (300-5000) 2.026 0.043 D 28 2000.00±921.71 1975 (400-3500) 2370.83±902.64 2500 (150-3500) 1.157 0.247 F=48.000 p<0.001 F=46.467 p<0.001 D: Day, F: Firedman Test, U: Mann Whitney U Test, P<0.05, SD: Standart deviaon, Min: Minumum, Max: Maximum CONCLUSION In this study, the eﬃcacies of FLOG and LLOG treatment groups against adults of Strongylids, Trichuris, and Nematodirus parasites were evaluated, and the results were obtained (65.36%-70.67%, 41.78%-74.22%, 52.78%-71.85%), respecvely. The study demonstrated that liposomal levamisole exhibited greater therapeuc eﬃcacy compared to free levamisole. The increase in the eﬃcacy of levamisole is aributed to the encapsulaon process with liposomes. This study aimed to examine the parasicidal eﬀects of free levamisole versus liposomal levamisole. According to the data we obtained, treatment eﬃcacy was lower in FLOG treatment groups compared to LLOG treatment groups. This is an indicaon that the parasites developed resistance to free levamisole. This study showed that liposomal drugs were more eﬀecve on parasites than their free forms. However, the fact that the study was carried out only on sheep is the limitaon of the study; therefore, research should be carried out in diﬀerent animal species in order to beer reveal the eﬀect of liposomal drugs on parasites. This study will shed light on future research in this respect. Funding This research was conducted with the support of the Balıkesir University Scienﬁc Research Coordinaon Oﬃce (BAP Project No: 2022 / 084). Conﬂict of Interest The authors have stated that they do not have any compeng interests. BIBLIOGRAPHIC REFERENCES [1] Aydin KB, Ye B, Brito LF, Ulutaş Z, Morota G. Review of sheep breeding and genec research in Türkiye. Front. Genet. [Internet]. 2024; 15:1308113. doi: hps://doi. org/pcsx [2] Jacobson C, Larsen JW, Besier RB, Lloyd JB, Kahn LP. Diarrhoea associated with gastrointesnal parasites in grazing sheep. Vet. Parasitol. [Internet]. 2020; 282:109139. doi: hps://doi.org/pcsz 6 of 8

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