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Full Research Article

Investigation of the Effects of Different Antiparasitic Drugs Administered Before Enterotoxemia Vaccination in Sheep on Some Blood Parameters

M
Mustafa ESER1,*
0000-0003-1542-2989
K
Kemal VAROL2
0000-0002-3057-2865
1Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Anadolu University, Eskiþehir 26470, Türkiye.
2Department of Veterinary, Burdur Food, Agriculture and Livestock Vocational College, Mehmet Akif Ersoy University, Burdur 15100, Türkiye.

ABSTRACT

Background: This study aimed to reveal the effect of antiparasitic agents with immunomodulatory or immunorestorative activity on the immune system applied before enterotoxemia vaccination.

Methods: The study was evaluated on a total of 70 merino sheep, aged between 1.5 and 4 years. The groups were composed of 7 groups in total, with 10 animals in each group. Animals in group 1 applied albendazole [15 mg/kg body weight (b/w)]; animals in group 2 applied ivermectin (0.2 mg/kg) and praziquantel (15 mg/kg b/w); animals in group 3 applied ricobendazole (7.5 mg b/w); animals in group 4 applied oxyclozanide (15 mg/kg b/w) and levamisole HCI (7.5 mg/kg b/w); animals in group 5 applied oxfendazole (5 mg/kg b/w) and oxyclozanide (15 mg/kg b/w); animals in group 6 applied rafoxanide (7.5 mg/kg b/w) and thiabendazole (100 mg b/w); animals in group 7 applied levamisole HCI (7.5 mg/kg b/w). The antiparasitic drug application determined for each group was applied one week before the enterotoxemia vaccine (Polivac CL/Vetal) application. Before antiparasitic application (0th day), 8 ml of blood was taken from the jugular veins of the animals. A total of two blood samples were collected from the jugular veins, once before the injection of the enterotoxemia vaccine (7th day) and again 14 days following administration.

Result: According to the results obtained, IL-1α and TNF-α values increased significantly in groups 3 and 4 one week after antiparasitic application. In addition, it was observed that the IgG value increased significantly in group 3 compared to the other groups 14 days after enterotoxemia vaccine administration. In conclusion, this study has shown that antiparasitic applications can stimulate immunity with or without stimulating proinflammatory response ricobendazole. The findings suggest  that the administration of ricobendazole one week before the enterotoxemia vaccine may enhance humoral response. This causes moderate oxidative stress, which leads to a significant improvement in immunity after the enterotoxemia vaccine.

INTRODUCTION

C. perfringens type D causes enterotoxemia, a frequent illness in sheep and goats. C. perfringens type D is a bacterium that lives in the intestines of a limited number of healthy animals. Sudden dietary changes, such as an abundance of highly fermentable carbohydrates, promote the growth of C. perfringens type D.  Rapid C. perfringens type D growth results in the synthesis of significant levels of epsilon prototoxin, which is activated by intestinal trypsin and proteases and is responsible for the clinical and pathological signs of enterotoxemia (Rahimoon et al., 2020; Hakeem et al., 2024; Rai et al., 2026). The use of various immunomodulators has been suggested in previous studies for use in domestic farm animals to relieve these negative effects (Jacobs et al., 1987; Movsesyan et al., 1988; Sanders et al., 1991; Alex et al., 1995; Mojzisova et al., 2004; Pekmezci and Cakiroglu, 2009). Certain antiparasitic agents have also been reported to posses immunomodulatory properties. Based on available literature information, in addition to antiparasitic activity, levamisole (Pekmezci and Cakiroglu, 2009) thiabendazole (Movsesyan et al., 1988), oxfendazole, fenbendazole (Jacobs et al., 1987) and ivermectin (Rao et al., 1987), have immunomodulatory or immune restorative activity. This study aimed to reveal the effect of antiparasitic agents with immunomodulatory or immunorestorative activity on the immune system applied before enterotoxemia vaccination.

MATERIALS AND METHODS

Parasitological methods
 
The feces taken from the rectum of each sheep were placed in numbered, transparent nylon bags. Information about each animal was recorded. The collected feces were brought to the laboratory without delay and examined macroscopically for cestode segments and microscopically for helminth eggs and protozoan agents. Fülleborn-saturated saltwater flotation and Benedek sedimentation methods were used in microscopic examination. After the parasitic examination, antiparasitic drugs were administered according to the species of parasites diagnosed in animals. To treat helminth infections, an antiparasitic drug with the active ingredient triclabendazol-levamizole (10 mg/kg, orally) was chosen (Tabari, et al., 2022). Toltrazuril was administered to animals orally at a dose of 20 mg/kg, which was shown to be effective in a single application to eliminate protozoa agents (Ok et al., 2019; De Souza Rodrigues et al., 2020). This antiparasitic practice was carried out two months before the study began. The fecal examinations were repeated on the 7th and 14th days after the drug administration. The drugs used in antiparasitic therapy were applied to all the animals involved in the study to ensure standardization.
 
Serological methods
 
This study was evaluated on a total of 70 merino sheep, aged between 1.5 and 4 years. Sheep are fed ad libitum in standard enclosed sheep pens. The groups were composed of 7 groups, with 10 animals in each group. Groups: Group 1 (n =10) albendazole (Vetalben/Vetaþ) applied group (albendazole was applied at a dose of 15 mg/kg body weight); Group 2 (n=10) ivermectin (Vilmectin/Vilsan) and praziquantel (Tenisil/Teknovet) applied group (ivermectin was applied at a dose of 0.2 mg/kg  (b/w), praziquantel was applied at a dose of 15 mg/kg b/w); Group 3 (n=10) ricobendazole (Rizal/Verano) applied group (ricabenzadol was applied at a dose of 7.5 mg/kg b/w); Group 4 (n=10) oxyclozanide and levamisole HCI (Nilzan/CEVA/DIF) administered group (levamizole HCI was administered at a dose of 7.5 mg/kg b/w and oxyclozanide was administered at a dose of 15 mg/kg b/w); Group 5 (n=10) oxfendazole and oxyclozanide (Oksavet/Vilsan) applied group (oxyclosanide was administered at a dose of 15 mg/kg b/w, oxfendazole was administered at a dose of 5 mg/kg b/w); Group 6 (n=10) rafoxanide and thiabendazole (Rabenzole/Topkim) applied group (rafoxanide was administered at a dose of 7.5 mg/kg b/w, thiabendazole was administered at a dose of 100 mg/kg b/w); Group 7 (n=10) levamisole HCI (Levatek 10%/Teknovet) applied group (levamizole HCI was applied at a dose of 7.5 mg/kg b/w). The antiparasitic drug application determined for each group was applied one week before the enterotoxemia vaccine (Polivac CL/Vetal) application.
       
Before antiparasitic application (0th day, 1st measurement), 8 ml of blood was taken from the jugular veins of the animals. A total of two blood samples were collected from the jugular vein, once before the injection of the enterotoxemia vaccine (7th day, 2nd measurement) and again 14 days (3rd measurement) following administration. Blood samples taken before antiparasitic application constituted the control of each group. The blood taken from the sheep was spun at 5000 RPM for 10 minutes and then their serum was removed. Serums were stored at -20°C until used. When the analysis began, the serums were thawed. Among the pro-inflammatory cytokines, interleukin 1 alpha (IL-1α) (ELK Biotechnology/Cat No. ELK8101), interleukin 1 beta (IL-1β), (ELK Biotechnology/Cat No: ELK8198), interleukin 6 (IL-6) (ELK Biotechnology/Cat No: ELK8039) and tumor necrosis factor-alpha (TNF-α) levels were determined using the Elisa assay. Among acute-phase proteins (APPS), haptoglobin (Hp) (Sunred/Cat No:01-07-0074) and ceruloplasmin (Cp) (Sunred/Cat No. 201-07-1007) levels were determined by the Elisa test. Among serum immunoglobulins, immunoglobulin G (IgG) (Sunred/Cat No. 201-07-2304) and immunoglobulin A (IgA) (Sunred 201-07-3102) levels were determined by the Elisa test. Among the biochemical parameters, serum cortisol (Cort) (ELK Biotechnology/Cat No. ELK8817) and serum total protein (Tp) (Elabscience/Cat No. E-BC-K318-M) levels were determined by the Elisa test. Serum total oxidant (TAS) (Erel 2004), total antioxidant (TOS) (Erel 2005) and oxidative stress index (OSI) (μmol H2O2 equivalent/L)/10 x TAS (mmol Trolox equivalent/L) levels were determined colorimetrically with the MINDRAY-BS400 device.
 
Statistical analysis
 
The findings obtained in this study were analyzed using IBM SPSS 22.0 for Windows software. Data normality was confirmed using the shapiro-wilk test. Based on the normal distribution of the data, a two-way analysis of variance was used to determine intra-group and inter-group comparisons in repeated measurements. A multiple comparison test called the Bonferroni test was utilized. An analysis of Pearson correlation was used to determine the correlation between variables. The statistical significance was deemed acceptable with a p-value of less than 0.05.

RESULTS AND DISCUSSION

Parasitological findings
 
The fecal examination showed that 54.2% of animals were infected with at least one species of parasite. The parasite species observed in animals were Dicrocoelium dentriticum (24.28%), Fasciola sp. (5.71%), Moniezia sp. (34.28%), Trichostrongylidae sp. (42.85%), Trichuris sp. (4.28%) and Eimeria spp. (25.71%). Examinations of feces on the 7th and 14th days after therapeutic antiparasitic doses showed no developmental period of the parasite.
 
Serological finding
 
In the comparison of serological finding values between measurements within groups are given in Table 1. In the comparison of serological finding values between measurements of inter group are given in Table 1. Correlations between parameters are given in Table 2.

Table 1: Biochemical analysis findings.



Table 2: Correlation findings.


       
Immunity may not develop in every vaccinated animal. Because the immune response to the vaccine is individual. In addition, environmental factors are also extremely important. Especially in animals exposed to stressful environmental factors such as fatigue, malnutrition, extreme cold and heat, the level of immune response to the vaccine decreases due to the increase in steroid levels in their bodies (Yıldız, 2021; Pulendran and Ahmed, 2011; Tizzard, 2021). In our study, a low-level, highly significant positive correlation (r = 0.234; p = 0.001) was found between serum cortisol value and IgA value. A low-level positive correlation between serum cortisol value and IgG was found (r = 0.186; p = 0.007) (Table 2). These findings show just the opposite. These finding suggest that factors other than stress hormones may also influence vaccine-induced immunity. Additionally, serum cortisol values increased (0th day: 72,13 mg/ml; 7th day: 82,07 mg/ml) in group 1 after albendazole application. A decrease in cortisol levels was observed 14 days (55,32 mg/ml) after vaccination. In group 7, the serum cortisol value decreased (0th day: 76,45 mg/ml; 7th day: 60,35 mg/ml) after levamisole application. An increase in cortisol levels was observed 14 days (75,05 mg/ml) after vaccination. More comprehensive studies should be conducted to determine the cause and mechanism of the increases and decreases in serum cortisol levels caused by albendazole and levamisole (Table 1).
       
Cytokines are multifunctional proteins that have important roles in intercellular communication and cellular activation and are classified as pro-inflammatory (Th1 type, stimulatory) and anti-inflammatory (Th2 type, inhibitory) according to their functions in the immune response (Akdoğan and Yöntem, 2018). The main proinflammatory cytokines consist of interferons, tumor necrosis factor (TNF), IL-1 and IL-6 (Yi-June et al., 1999). Evaluation of immunological effects is possible by measuring the level of pro-inflammatory cytokines (Elsabahy and Wooley, 2013). In this study, it was observed that changes were observed in the levels of pro-inflammatory cytokines in all groups after antiparasitic and vaccine applications. It is seen that immunological effects change the levels of pro-inflammatory cytokines. Although there is no statistical difference in IgA values, the differences in IgG levels between measurements and between groups support this. Interleukin-1 (IL-1) is a pro-inflammatory cytokine (Yi-June et al., 1999). IL-1 is composed of two distinct proteins, IL-1α and IL-1β (Leonides et al., 1990). IL-1α is produced by various cells of the immune system and other types of cells in normal conditions and its production can be increased by a wide range of inflammatory triggers. In general, IL-1 is responsible for increasing fever and sepsis, as well as causing inflammation. IL-1α inhibitors are being developed to treat diseases by interrupting these processes. IL-1α is primarily generated by activated macrophages, along with neutrophils, epithelial cells and endothelial cells. It has metabolic, physiological and hematopoietic functions and plays a pivotal role in regulating immunological responses. TNF-α is activated by this mechanism (Dinarello 1997; Lopez-Castejon and  Brough, 2011; Malik and Kanneganti, 2018; Yiş and Buğdaycı, 2019; Cavalli et al., 2021). In our study, it was observed that the IL-1α value increased significantly in groups 3 (0th day: 38,04 pg/ml; 7th day: 127,06 pg/ml) and groups 4 (0th day: 28,11 pg/ml; 7th day: 100,11 pg/ml) one week after antiparasitic application and there was a statistically significant difference (p<0.05) (Table 2). IL-1α value decreased in groups 5 (0th day: 44,85 pg/ml; 7th day: 34,64 pg/ml)  and groups 6 (0th day: 26,05 pg/ml; 7th day: 23,79 pg/ml) one week after antiparasitic application. However, IL-1α value increased in groups 5 (14th day: 76,22 pg/ml) and groups 6 (14th day: 64,15 pg/ml) after vaccine administration. A statistically significant (p<0.05) difference was determined between group 5 and the other groups (Table 1).
       
As can be seen, the IL-1α value increased in all groups 14 days after vaccine administration. The increase in production by neutrophils, epithelial cells and endothelial cells, as well as macrophages, after vaccine administration, clearly shows that an immune response has developed. This has shown us that IL-1α may be an important marker in determining the immune reaction in sheep. In addition, the bioactivity of IL-1α is not reliant on proteolytic processing. Therefore, cell death caused by injury from either sterile or infectious sources results in the release of bioactive IL-1α. This IL-1α then signals through the IL-1 receptor (IL-1R) to trigger inflammatory responses. IL-1R is constitutively expressed by a wide variety of cell types. It results in the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) downstream of IL-1R. This process stimulates the production of pro-inflammatory mediators like cyclooxygenase type-2 (COX-2), IL-6 and TNF. These mediators then contribute to the production of IL-1α and IL-1β, intensifying the inflammatory response triggered by the initial release of IL-1α (Malik and Kanneganti, 2018; Yiş and Buğdaycı, 2019; Cavalli et al., 2021). When looking at the correlation findings of our study in parallel with the information above, there was a low, significant positive correlation between IL-1α and IL-6 values (r = 0.171; p = 0.013) and a highly significant positive correlation between IL-1α and TNF-α values (r = 0.928; p<0.001) supporting this information (Table 2).
       
IL-1β plays a crucial role in the inflammatory response and is implicated in several cellular processes, such as cell growth, specialization and programmed cell death (Yaseen et al., 2022). Maqbool et al., (2021) reported that there was no increase in IL-1β value in vaccinated goats compared to unvaccinated goats during the experimental infection of Mannheimia (M.) haemolytica A2 under tropical conditions. Arteche-Villasol et al. (2021) conducted a comparison of the early response of monocyte-derived macrophages from vaccinated and unvaccinated goats when infected with Mycobacterium avium subsp. paratuberculosis in vitro; their findings indicated that the vaccinated control group did not show an increase in IL-1β value.
       
In our study, an increase in IL-1β value was observed in all groups except group 7 after antiparasitic application (Table 2). This increase has been associated with the stimulation of macrophages (Yaseen et al., 2022). The decrease in group 7 may be related to the dose of levamisole (Table 1). Because levamisole also causes a cell-based immune response (Spector et al., 1998). Therefore, the increase in Group 7 may be related to the dose of levamisole because there is a relationship between levamisole and dosing (Undiandeye et al., 2014; El-shahedy et al., 2015; Aydin 2020; Aydın and Aktaş, 2023).
       
However, it was observed that the release of IL-1β decreased after enterotoxemia vaccine administration. This decrease may be related to multiple previous enterotoxemia vaccinations (Table 1). Because IL-1β is the main regulator of natural immunity (Lopez-Castejon and, Brough 2011).
       
Vaccines aim to efficiently and promptly activate a cell-mediated immune response. Nevertheless, the precise processes elucidating the process of this modulation remain unknown. The emergence of a T1-type immune response, made possible by CD4+ cells and the typical activation of macrophages (M1), has been associated with the ability to control the disease (Arteche-Villasol et al., 2021).
       
TNF-α is released by various cells in the body, including keratinocytes, dermal dendrocytes, monocytes, macrophages, mast cells and activated B and T cells. TNF-α plays a crucial role in activating macrophages and increasing the production of nitric oxide, along with other proinflammatory cytokines and chemokines. TNF-α is also among the pro-inflammatory cytokines. TNF-α released by Th1-type cells activates macrophages, causing a cell-mediated immune response (Atmaca and Kul, 2012). Arteche-Villasol et al. (2021) conducted a comparison of the early response of monocyte-derived macrophages from vaccinated and unvaccinated goats when infected with Mycobacterium avium subsp. paratuberculosis in vitro; they reported that there was no increase in TNF-α values in the vaccinated control group.
       
In parallel with this information, in our study, TNF-α values increased in group 3 (ricabendazole administered group) (0th day: 38,04 pg/ml; 7th day: 128,05 pg/ml) and group 4 (oxyclozanide and levamisole HCI administered group) (0th day: 28,10 pg/ml; 7th day: 100,11 pg/ml). After vaccination, TNF-α value increased in all groups and the most significant increase was seen in group 5 (Table 1). The increase in IL-1α value in all groups 14 days after vaccine administration indicates that TNF-α released by Th1-type cells activates macrophages, causing a cell-mediated immune response (Table 1). This has shown us that TNF-α may be an important marker in the formation of a mediated immune response in sheep.
       
IL-6 is also a pro-inflammatory cytokine. Th2 cells are responsible for its production and they play a role as mediators in the process of forming the acute phase response (APR) Additionally, it speeds up the infiltration of inflammatory cells (Sevgisunar and Şahinduran, 2021).
       
IL-6 is one of the most important cytokines in B lymphocyte proliferation-differentiation, Ig production, fever and acute phase protein production (Akdoğan and Yöntem, 2018). However, in our study, no relationship was observed between acute-phase proteins and IL-6.
       
IL-6 acts synergistically with IL-1 to increase bone resorption (Erdemir et al., 2007). In our study, there were low positive and significant correlations between the IL-6 value and IL-1α (r = 0.171; p = 0.013), IL-1β (r = 0.214; p = 0.002) and TNF-α  (r = 0.161; p = 0.020) values, which supports this (Table 2).
       
IL-6 released from T cells, macrophages, fibroblasts and endothelial cells plays a very effective role in the host defense mechanism by stimulating B cell proliferation and differentiation. IL-6, which is an important cytokine in the diagnosis of periodontal diseases, has been found to be significantly higher in inflamed tissues (Erdemir et al., 2007).
       
Together with this information in our study, one week after the antiparasitic application, it was observed that the IL-6 value increased in group 4 (0th day: 19,51 pg/ml; 7th day: 38,97 pg/ml) and showed a statistically significant difference (p <0.05) with the other groups except group 5 (0th day: 20,78 pg/ml; 7th day: 28,77 pg/ml). Although an increase was also seen in group 5, no statistical difference was observed. However, 14 days after the vaccine application, there was a decrease in groups 4 (14th day: 17,58 pg/ml).  and groups 5 (14th day: 17,10 pg/ml) compared  to the first measurement, while an increase was observed in the other groups. No statistically significant difference was observed between groups after vaccine administration (Table 1).
       
Contrary to this situation, Arteche-Villasol et al. (2021) conducted a comparison of the early response of monocyte-derived macrophages from vaccinated and unvaccinated goats when infected with Mycobacterium avium subsp. paratuberculosis in vitro; they reported an increase in IL-6 value in the vaccinated control group.
       
Likewise, Maqbool et al., (2021) reported in their study that during the experimental infection of M. haemolytica A2 under tropical conditions, there was an increase in the IL-6 value in vaccinated goats compared to unvaccinated goats.
       
Contrary to these studies, the reason for the excessive increase after antiparasitic application in groups 4 and 5 compared to the first measurement and the decrease after enterotoxemia vaccination should be investigated in more comprehensive studies (Table 1).
       
Considering the statement that it is possible to evaluate immunological effects by measuring the level of circulating proinflammatory cytokines (Elsabahy and Wooley, 2013). The findings obtained in our study indicate that antiparasitic applications applied before enterotoxemia vaccination in sheep provide greater activation of the immune system in sheep. It is also suggested that IL-1β can lead to metabolic, hormonal and cellular changes that are crucial in initiating an immune response (Eryavuz et al., 2022).
       
IgG has an important role in the defense mechanisms of animals. IgG constitutes 70-75% of the immunoglobulins in circulation. It is the only antibody with antitoxin activity and the main antibody generated during a secondary immune response. It plays a role in several autoimmune diseases and humoral deficiencies. It is stated that it is synthesised between 7 and 28 days following immune stimulation in adult and newborn animals. IgA is found primarily in secretions lining mucosal surfaces and relatively small amounts can be found in circulation (Başbuğan et al., 2021). With a primary immune response, IgM appears first as the main Ig. Although IgM also occurs in a secondary response, it does not show as high levels as IgG (Tizzard 2021). Maqbool et al. (2021) reported that there was an increase in IgG value in vaccinated goats compared to unvaccinated goats during the experimental infection of M. haemolytica A2 under tropical conditions.
       
In our study, it was observed that the IgG value increased significantly in group 3 compared to the other groups 14 days after enterotoxemia vaccine administration and there was a statistically significant (p<0.05) difference (Table 1).
       
It was observed that the IgA value increased in group 3 compared to the other groups 14 days after enterotoxemia vaccine administration and there was no statistically significant (p>0.05) difference (Table 1).
       
The application time and dose of levamisole are extremely important in stimulating the responsiveness of T and B lymphocytes (Undiandeye et al., 2014).
       
Aydın and Aktaş (2023) reported that 5 mg/kg for 4 days of levamisole administered together with (peste des petits ruminants) PPR vaccine in Morkaraman sheep stimulated IgG production and increased IgG at a statistically significant level.
       
Abdullah and Başbuğan (2020) reported that the application of levamisole tablets (5 mg/kg per oral) together with the foot and mouth disease (FMD) vaccine stimulated IgG production and increased IgG at a statistically significant level.
       
Rashid and Yüksek (2019) reported that the application of levamisole together with the enterotoxemia vaccine in sheep stimulated the production of IgG and IgA levels, although this was not statistically significant.
       
In our study, it was observed that levamisole application one week before the enterotoxemia vaccine increased the IgG value (0th day: 3.54 mg/ml; 7th day: 4.92 mg/ml). to a statistically significant level. On the contrary, a decrease was observed in the IgA value. When comparing the first measurement to 14 days after (14th day: 4.13 mg/ml).  The enterotoxemia vaccine administration, the IgG value increased. However, when comparing it to 1 week after the levamisole application, it decreased (Table 1).
       
Rashid and Yüksek (2019) reported that levamisole may enhance the immune response to the enterotoxemia vaccine, particularly in cases of immunosuppression. They also noted that immunostimulants can be beneficial in improving the immune response to vaccines. Simply getting vaccinated may not effectively boost the body’s immune response to a specific pathogen. Thus, it is advisable to combine these immune-stimulating agents with vaccines to enhance and prolong immunity (Rashid and Yüksek, 2019).
       
In our study, the increase in IgG value after levamisole application and the decrease after the enterotoxemia vaccine support this (Table 1). For this reason, it has been shown that if the immune response is to be strengthened with levamisole, it would be more beneficial to administer levamisole together with the vaccine rather than before the vaccine.
       
APPs are highly sensitive biomarkers, although their specificity in detecting different infectious agents is limited. It is commonly used in the diagnosis, prognosis and health status screening of animals (Murata et al., 2004).  The APR is a typical reaction that takes place soon after tissue damage as a result of various factors like infection, immune response, cancer, trauma, parasites, or other causes. The most important structures secreted as a response of the organism to acute inflammation are cytokines, APPs and leukocytes. The initiation of the APR is triggered by proinflammatory cytokines, including IL-1, IL-6 and TNF-α, which are secreted by activated leukocytes near the location of tissue injury. Cytokines coordinate the first reaction to infectious illnesses and inflammation and trigger various systemic responses. Within the liver, these cytokines induce the synthesis and subsequent secretion of glycoproteins referred to as APP (Gökçe and Bozukluhan, 2009; Taşçene 2017).
       
Plasma levels of ceruloplasmin increase in cases of infection, malignancy, trauma, obstruction and infection of the biliary tract; this increase is more evident in RES diseases such as Hodgkin’s. It has been shown that endogenous mediator substances called leukocytes mediate the synthesis of ceruloplasmin. It is known that the increase in ceruloplasmin concentration in diseases enables the release of copper from the liver stores into the plasma (Taşçene 2017).
       
In cattle, Hp functions in the regulation of lipid metabolism (Nakagawa et al., 1997) and as an immunomodulator in lymphocyte functions (Murata and Miyamoto, 1993). Due to this feature, it is reported that Hp concentration can be used to monitor the immune functions of calves (Gökçe and Bozukluhan, 2009). Maqbool et al., (2021) reported that there was an increase in Hp and serum amyloid A (SAA) values in vaccinated goats compared to unvaccinated goats during the experimental infection of M. haemolytica A2 under tropical conditions.
       
In our study, Hp values increased in all groups 14 days after the enterotoxemia vaccine, but no statistical difference (p>0.05)  was found in the comparison between groups (Table 1).
       
Cp increased in group 3 and decreased in the other groups 14 days after enterotoxemia vaccination and no statistical differences (p>0.05) were detected (Table 2). Oxidative stress occurs as a result of disruption of the balance between oxidant and antioxidant mechanisms (Akşit et al., 2014). TOS serves as a reliable measure to assess the oxidants generated by the body and absorbed by ambient elements (Ertaş and Kırmızıgül, 2021). The levels of TOS will increase in inflammatory conditions (Bozukluhan et al., 2016). The OSI plays a crucial role in assessing oxidative stress levels. Several research investigations have been carried out to assess the effectiveness of OSI in measuring oxidative stress in farm animals. These investigations have shown that OSI levels dramatically increase in disorders characterized by oxidative stress (Hanedan et al., 2015; Gürgöze et al., 2024). In parallel with this information, in our study, it was observed that TOS (0th day: 3,94 µmol/L; 7th day: 4,54 µmol/L; 14th day: 11,24 µmol/L) and OSI (0th day: 0,24 arbitrary unit; 7th day: 0,28 arbitrary unit; 14th day: 0,70 arbitrary unit) values in group 4 increased significantly compared to the other groups 14 days after enterotoxemia vaccine administration and a statistically significant difference (p<0.05) was observed (Table 1). This showed us that oxidative stress was stimulated following vaccine administration in group 4 (Table 1).
       
Antioxidants play a crucial role in safeguarding cells against the harmful impacts of oxidative stress. Given the wide range of antioxidant substances and pathways, it is crucial to measure the quantitative antioxidant power, or TAS level, in biological samples. This is an important step in preventing oxidative stress and assessing the overall antioxidant capacity (Akdoğan et al., 2018; Ertaş and Kırmızıgül, 2021; Bilmez and Özyurtlu, 2023). In our study, we detected that the TAS value (0th day: 1,68 µmol/L; 7th day: 1,85 µmol/L) increased in group 6 after antiparasitic application and there was a statistically significant difference (p<0.05) between group 6 and the other groups, except for group 5. In group 5 (0th day: 1,70 µmol/L; 7th day: 1,79 µmol/L), the TAS value increased compared to groups 1 (0th day: 1,62 µmol/L; 7th day: 1,45 µmol/L), groups 2 (0th day: 1,70 µmol/L; 7th day: 1,52 µmol/L) and groups 3 (0th day: 1,65 µmol/L; 7th day: 1,54 µmol/L) after antiparasitic application and a statistically significant difference (p<0.05) was determined (Table 1).
       
In group 7 (0th day: 1,59 µmol/L; 7th day: 1,70 µmol/L), the TAS value increased compared to group 1 after antiparasitic application and a statistically significant difference was detected. This has clearly shown us that the application of the antiparasitic drugs rafoxanide and thiabendazole, oxfendazole and oxyclozanide and levamisole can be useful in protecting from the destructive effects of oxidative stress, which increases TAS (Table 1).

CONCLUSION

In conclusion, this study has shown that antiparasitic applications can stimulate immunity with or without stimulating proinflammatory stocks.
       
In particular, it has been demonstrated that the administration of ricobendazole 1 week before the enterotoxemia vaccine stimulates the immune system by stimulating the proinflammatory stocks without creating an acute phase response and increasing the IgG value by creating moderate oxidative stress, resulting in a significant improvement in immunity after the enterotoxemia vaccine.
       
The findings indicate that ricobendazole may have immunomodulatory potential that warrants further investigation. For this reason, new clinical and experimental studies will contribute to science by conducting more detailed research on ricabendazole.

ACKNOWLEDGEMENT

Anadolu University Scientific Projects Fund (Project No. 2107S112) has financially supported this study.
 
Disclaimers
 
The data of this study are available upon request from the corresponding author.
 
Informed consent
 
This study was conducted by the Animal Experiments Local Ethics Board on 09.11.2020 and 50/51. It was carried out with approval no.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

REFERENCES


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    Full Research Article

    Investigation of the Effects of Different Antiparasitic Drugs Administered Before Enterotoxemia Vaccination in Sheep on Some Blood Parameters

    M
    Mustafa ESER1,*
    0000-0003-1542-2989
    K
    Kemal VAROL2
    0000-0002-3057-2865
    1Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Anadolu University, Eskiþehir 26470, Türkiye.
    2Department of Veterinary, Burdur Food, Agriculture and Livestock Vocational College, Mehmet Akif Ersoy University, Burdur 15100, Türkiye.

    ABSTRACT

    Background: This study aimed to reveal the effect of antiparasitic agents with immunomodulatory or immunorestorative activity on the immune system applied before enterotoxemia vaccination.

    Methods: The study was evaluated on a total of 70 merino sheep, aged between 1.5 and 4 years. The groups were composed of 7 groups in total, with 10 animals in each group. Animals in group 1 applied albendazole [15 mg/kg body weight (b/w)]; animals in group 2 applied ivermectin (0.2 mg/kg) and praziquantel (15 mg/kg b/w); animals in group 3 applied ricobendazole (7.5 mg b/w); animals in group 4 applied oxyclozanide (15 mg/kg b/w) and levamisole HCI (7.5 mg/kg b/w); animals in group 5 applied oxfendazole (5 mg/kg b/w) and oxyclozanide (15 mg/kg b/w); animals in group 6 applied rafoxanide (7.5 mg/kg b/w) and thiabendazole (100 mg b/w); animals in group 7 applied levamisole HCI (7.5 mg/kg b/w). The antiparasitic drug application determined for each group was applied one week before the enterotoxemia vaccine (Polivac CL/Vetal) application. Before antiparasitic application (0th day), 8 ml of blood was taken from the jugular veins of the animals. A total of two blood samples were collected from the jugular veins, once before the injection of the enterotoxemia vaccine (7th day) and again 14 days following administration.

    Result: According to the results obtained, IL-1α and TNF-α values increased significantly in groups 3 and 4 one week after antiparasitic application. In addition, it was observed that the IgG value increased significantly in group 3 compared to the other groups 14 days after enterotoxemia vaccine administration. In conclusion, this study has shown that antiparasitic applications can stimulate immunity with or without stimulating proinflammatory response ricobendazole. The findings suggest  that the administration of ricobendazole one week before the enterotoxemia vaccine may enhance humoral response. This causes moderate oxidative stress, which leads to a significant improvement in immunity after the enterotoxemia vaccine.

    INTRODUCTION

    C. perfringens type D causes enterotoxemia, a frequent illness in sheep and goats. C. perfringens type D is a bacterium that lives in the intestines of a limited number of healthy animals. Sudden dietary changes, such as an abundance of highly fermentable carbohydrates, promote the growth of C. perfringens type D.  Rapid C. perfringens type D growth results in the synthesis of significant levels of epsilon prototoxin, which is activated by intestinal trypsin and proteases and is responsible for the clinical and pathological signs of enterotoxemia (Rahimoon et al., 2020; Hakeem et al., 2024; Rai et al., 2026). The use of various immunomodulators has been suggested in previous studies for use in domestic farm animals to relieve these negative effects (Jacobs et al., 1987; Movsesyan et al., 1988; Sanders et al., 1991; Alex et al., 1995; Mojzisova et al., 2004; Pekmezci and Cakiroglu, 2009). Certain antiparasitic agents have also been reported to posses immunomodulatory properties. Based on available literature information, in addition to antiparasitic activity, levamisole (Pekmezci and Cakiroglu, 2009) thiabendazole (Movsesyan et al., 1988), oxfendazole, fenbendazole (Jacobs et al., 1987) and ivermectin (Rao et al., 1987), have immunomodulatory or immune restorative activity. This study aimed to reveal the effect of antiparasitic agents with immunomodulatory or immunorestorative activity on the immune system applied before enterotoxemia vaccination.

    MATERIALS AND METHODS

    Parasitological methods
     
    The feces taken from the rectum of each sheep were placed in numbered, transparent nylon bags. Information about each animal was recorded. The collected feces were brought to the laboratory without delay and examined macroscopically for cestode segments and microscopically for helminth eggs and protozoan agents. Fülleborn-saturated saltwater flotation and Benedek sedimentation methods were used in microscopic examination. After the parasitic examination, antiparasitic drugs were administered according to the species of parasites diagnosed in animals. To treat helminth infections, an antiparasitic drug with the active ingredient triclabendazol-levamizole (10 mg/kg, orally) was chosen (Tabari, et al., 2022). Toltrazuril was administered to animals orally at a dose of 20 mg/kg, which was shown to be effective in a single application to eliminate protozoa agents (Ok et al., 2019; De Souza Rodrigues et al., 2020). This antiparasitic practice was carried out two months before the study began. The fecal examinations were repeated on the 7th and 14th days after the drug administration. The drugs used in antiparasitic therapy were applied to all the animals involved in the study to ensure standardization.
     
    Serological methods
     
    This study was evaluated on a total of 70 merino sheep, aged between 1.5 and 4 years. Sheep are fed ad libitum in standard enclosed sheep pens. The groups were composed of 7 groups, with 10 animals in each group. Groups: Group 1 (n =10) albendazole (Vetalben/Vetaþ) applied group (albendazole was applied at a dose of 15 mg/kg body weight); Group 2 (n=10) ivermectin (Vilmectin/Vilsan) and praziquantel (Tenisil/Teknovet) applied group (ivermectin was applied at a dose of 0.2 mg/kg  (b/w), praziquantel was applied at a dose of 15 mg/kg b/w); Group 3 (n=10) ricobendazole (Rizal/Verano) applied group (ricabenzadol was applied at a dose of 7.5 mg/kg b/w); Group 4 (n=10) oxyclozanide and levamisole HCI (Nilzan/CEVA/DIF) administered group (levamizole HCI was administered at a dose of 7.5 mg/kg b/w and oxyclozanide was administered at a dose of 15 mg/kg b/w); Group 5 (n=10) oxfendazole and oxyclozanide (Oksavet/Vilsan) applied group (oxyclosanide was administered at a dose of 15 mg/kg b/w, oxfendazole was administered at a dose of 5 mg/kg b/w); Group 6 (n=10) rafoxanide and thiabendazole (Rabenzole/Topkim) applied group (rafoxanide was administered at a dose of 7.5 mg/kg b/w, thiabendazole was administered at a dose of 100 mg/kg b/w); Group 7 (n=10) levamisole HCI (Levatek 10%/Teknovet) applied group (levamizole HCI was applied at a dose of 7.5 mg/kg b/w). The antiparasitic drug application determined for each group was applied one week before the enterotoxemia vaccine (Polivac CL/Vetal) application.
           
    Before antiparasitic application (0th day, 1st measurement), 8 ml of blood was taken from the jugular veins of the animals. A total of two blood samples were collected from the jugular vein, once before the injection of the enterotoxemia vaccine (7th day, 2nd measurement) and again 14 days (3rd measurement) following administration. Blood samples taken before antiparasitic application constituted the control of each group. The blood taken from the sheep was spun at 5000 RPM for 10 minutes and then their serum was removed. Serums were stored at -20°C until used. When the analysis began, the serums were thawed. Among the pro-inflammatory cytokines, interleukin 1 alpha (IL-1α) (ELK Biotechnology/Cat No. ELK8101), interleukin 1 beta (IL-1β), (ELK Biotechnology/Cat No: ELK8198), interleukin 6 (IL-6) (ELK Biotechnology/Cat No: ELK8039) and tumor necrosis factor-alpha (TNF-α) levels were determined using the Elisa assay. Among acute-phase proteins (APPS), haptoglobin (Hp) (Sunred/Cat No:01-07-0074) and ceruloplasmin (Cp) (Sunred/Cat No. 201-07-1007) levels were determined by the Elisa test. Among serum immunoglobulins, immunoglobulin G (IgG) (Sunred/Cat No. 201-07-2304) and immunoglobulin A (IgA) (Sunred 201-07-3102) levels were determined by the Elisa test. Among the biochemical parameters, serum cortisol (Cort) (ELK Biotechnology/Cat No. ELK8817) and serum total protein (Tp) (Elabscience/Cat No. E-BC-K318-M) levels were determined by the Elisa test. Serum total oxidant (TAS) (Erel 2004), total antioxidant (TOS) (Erel 2005) and oxidative stress index (OSI) (μmol H2O2 equivalent/L)/10 x TAS (mmol Trolox equivalent/L) levels were determined colorimetrically with the MINDRAY-BS400 device.
     
    Statistical analysis
     
    The findings obtained in this study were analyzed using IBM SPSS 22.0 for Windows software. Data normality was confirmed using the shapiro-wilk test. Based on the normal distribution of the data, a two-way analysis of variance was used to determine intra-group and inter-group comparisons in repeated measurements. A multiple comparison test called the Bonferroni test was utilized. An analysis of Pearson correlation was used to determine the correlation between variables. The statistical significance was deemed acceptable with a p-value of less than 0.05.

    RESULTS AND DISCUSSION

    Parasitological findings
     
    The fecal examination showed that 54.2% of animals were infected with at least one species of parasite. The parasite species observed in animals were Dicrocoelium dentriticum (24.28%), Fasciola sp. (5.71%), Moniezia sp. (34.28%), Trichostrongylidae sp. (42.85%), Trichuris sp. (4.28%) and Eimeria spp. (25.71%). Examinations of feces on the 7th and 14th days after therapeutic antiparasitic doses showed no developmental period of the parasite.
     
    Serological finding
     
    In the comparison of serological finding values between measurements within groups are given in Table 1. In the comparison of serological finding values between measurements of inter group are given in Table 1. Correlations between parameters are given in Table 2.

    Table 1: Biochemical analysis findings.



    Table 2: Correlation findings.


           
    Immunity may not develop in every vaccinated animal. Because the immune response to the vaccine is individual. In addition, environmental factors are also extremely important. Especially in animals exposed to stressful environmental factors such as fatigue, malnutrition, extreme cold and heat, the level of immune response to the vaccine decreases due to the increase in steroid levels in their bodies (Yıldız, 2021; Pulendran and Ahmed, 2011; Tizzard, 2021). In our study, a low-level, highly significant positive correlation (r = 0.234; p = 0.001) was found between serum cortisol value and IgA value. A low-level positive correlation between serum cortisol value and IgG was found (r = 0.186; p = 0.007) (Table 2). These findings show just the opposite. These finding suggest that factors other than stress hormones may also influence vaccine-induced immunity. Additionally, serum cortisol values increased (0th day: 72,13 mg/ml; 7th day: 82,07 mg/ml) in group 1 after albendazole application. A decrease in cortisol levels was observed 14 days (55,32 mg/ml) after vaccination. In group 7, the serum cortisol value decreased (0th day: 76,45 mg/ml; 7th day: 60,35 mg/ml) after levamisole application. An increase in cortisol levels was observed 14 days (75,05 mg/ml) after vaccination. More comprehensive studies should be conducted to determine the cause and mechanism of the increases and decreases in serum cortisol levels caused by albendazole and levamisole (Table 1).
           
    Cytokines are multifunctional proteins that have important roles in intercellular communication and cellular activation and are classified as pro-inflammatory (Th1 type, stimulatory) and anti-inflammatory (Th2 type, inhibitory) according to their functions in the immune response (Akdoğan and Yöntem, 2018). The main proinflammatory cytokines consist of interferons, tumor necrosis factor (TNF), IL-1 and IL-6 (Yi-June et al., 1999). Evaluation of immunological effects is possible by measuring the level of pro-inflammatory cytokines (Elsabahy and Wooley, 2013). In this study, it was observed that changes were observed in the levels of pro-inflammatory cytokines in all groups after antiparasitic and vaccine applications. It is seen that immunological effects change the levels of pro-inflammatory cytokines. Although there is no statistical difference in IgA values, the differences in IgG levels between measurements and between groups support this. Interleukin-1 (IL-1) is a pro-inflammatory cytokine (Yi-June et al., 1999). IL-1 is composed of two distinct proteins, IL-1α and IL-1β (Leonides et al., 1990). IL-1α is produced by various cells of the immune system and other types of cells in normal conditions and its production can be increased by a wide range of inflammatory triggers. In general, IL-1 is responsible for increasing fever and sepsis, as well as causing inflammation. IL-1α inhibitors are being developed to treat diseases by interrupting these processes. IL-1α is primarily generated by activated macrophages, along with neutrophils, epithelial cells and endothelial cells. It has metabolic, physiological and hematopoietic functions and plays a pivotal role in regulating immunological responses. TNF-α is activated by this mechanism (Dinarello 1997; Lopez-Castejon and  Brough, 2011; Malik and Kanneganti, 2018; Yiş and Buğdaycı, 2019; Cavalli et al., 2021). In our study, it was observed that the IL-1α value increased significantly in groups 3 (0th day: 38,04 pg/ml; 7th day: 127,06 pg/ml) and groups 4 (0th day: 28,11 pg/ml; 7th day: 100,11 pg/ml) one week after antiparasitic application and there was a statistically significant difference (p<0.05) (Table 2). IL-1α value decreased in groups 5 (0th day: 44,85 pg/ml; 7th day: 34,64 pg/ml)  and groups 6 (0th day: 26,05 pg/ml; 7th day: 23,79 pg/ml) one week after antiparasitic application. However, IL-1α value increased in groups 5 (14th day: 76,22 pg/ml) and groups 6 (14th day: 64,15 pg/ml) after vaccine administration. A statistically significant (p<0.05) difference was determined between group 5 and the other groups (Table 1).
           
    As can be seen, the IL-1α value increased in all groups 14 days after vaccine administration. The increase in production by neutrophils, epithelial cells and endothelial cells, as well as macrophages, after vaccine administration, clearly shows that an immune response has developed. This has shown us that IL-1α may be an important marker in determining the immune reaction in sheep. In addition, the bioactivity of IL-1α is not reliant on proteolytic processing. Therefore, cell death caused by injury from either sterile or infectious sources results in the release of bioactive IL-1α. This IL-1α then signals through the IL-1 receptor (IL-1R) to trigger inflammatory responses. IL-1R is constitutively expressed by a wide variety of cell types. It results in the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) downstream of IL-1R. This process stimulates the production of pro-inflammatory mediators like cyclooxygenase type-2 (COX-2), IL-6 and TNF. These mediators then contribute to the production of IL-1α and IL-1β, intensifying the inflammatory response triggered by the initial release of IL-1α (Malik and Kanneganti, 2018; Yiş and Buğdaycı, 2019; Cavalli et al., 2021). When looking at the correlation findings of our study in parallel with the information above, there was a low, significant positive correlation between IL-1α and IL-6 values (r = 0.171; p = 0.013) and a highly significant positive correlation between IL-1α and TNF-α values (r = 0.928; p<0.001) supporting this information (Table 2).
           
    IL-1β plays a crucial role in the inflammatory response and is implicated in several cellular processes, such as cell growth, specialization and programmed cell death (Yaseen et al., 2022). Maqbool et al., (2021) reported that there was no increase in IL-1β value in vaccinated goats compared to unvaccinated goats during the experimental infection of Mannheimia (M.) haemolytica A2 under tropical conditions. Arteche-Villasol et al. (2021) conducted a comparison of the early response of monocyte-derived macrophages from vaccinated and unvaccinated goats when infected with Mycobacterium avium subsp. paratuberculosis in vitro; their findings indicated that the vaccinated control group did not show an increase in IL-1β value.
           
    In our study, an increase in IL-1β value was observed in all groups except group 7 after antiparasitic application (Table 2). This increase has been associated with the stimulation of macrophages (Yaseen et al., 2022). The decrease in group 7 may be related to the dose of levamisole (Table 1). Because levamisole also causes a cell-based immune response (Spector et al., 1998). Therefore, the increase in Group 7 may be related to the dose of levamisole because there is a relationship between levamisole and dosing (Undiandeye et al., 2014; El-shahedy et al., 2015; Aydin 2020; Aydın and Aktaş, 2023).
           
    However, it was observed that the release of IL-1β decreased after enterotoxemia vaccine administration. This decrease may be related to multiple previous enterotoxemia vaccinations (Table 1). Because IL-1β is the main regulator of natural immunity (Lopez-Castejon and, Brough 2011).
           
    Vaccines aim to efficiently and promptly activate a cell-mediated immune response. Nevertheless, the precise processes elucidating the process of this modulation remain unknown. The emergence of a T1-type immune response, made possible by CD4+ cells and the typical activation of macrophages (M1), has been associated with the ability to control the disease (Arteche-Villasol et al., 2021).
           
    TNF-α is released by various cells in the body, including keratinocytes, dermal dendrocytes, monocytes, macrophages, mast cells and activated B and T cells. TNF-α plays a crucial role in activating macrophages and increasing the production of nitric oxide, along with other proinflammatory cytokines and chemokines. TNF-α is also among the pro-inflammatory cytokines. TNF-α released by Th1-type cells activates macrophages, causing a cell-mediated immune response (Atmaca and Kul, 2012). Arteche-Villasol et al. (2021) conducted a comparison of the early response of monocyte-derived macrophages from vaccinated and unvaccinated goats when infected with Mycobacterium avium subsp. paratuberculosis in vitro; they reported that there was no increase in TNF-α values in the vaccinated control group.
           
    In parallel with this information, in our study, TNF-α values increased in group 3 (ricabendazole administered group) (0th day: 38,04 pg/ml; 7th day: 128,05 pg/ml) and group 4 (oxyclozanide and levamisole HCI administered group) (0th day: 28,10 pg/ml; 7th day: 100,11 pg/ml). After vaccination, TNF-α value increased in all groups and the most significant increase was seen in group 5 (Table 1). The increase in IL-1α value in all groups 14 days after vaccine administration indicates that TNF-α released by Th1-type cells activates macrophages, causing a cell-mediated immune response (Table 1). This has shown us that TNF-α may be an important marker in the formation of a mediated immune response in sheep.
           
    IL-6 is also a pro-inflammatory cytokine. Th2 cells are responsible for its production and they play a role as mediators in the process of forming the acute phase response (APR) Additionally, it speeds up the infiltration of inflammatory cells (Sevgisunar and Şahinduran, 2021).
           
    IL-6 is one of the most important cytokines in B lymphocyte proliferation-differentiation, Ig production, fever and acute phase protein production (Akdoğan and Yöntem, 2018). However, in our study, no relationship was observed between acute-phase proteins and IL-6.
           
    IL-6 acts synergistically with IL-1 to increase bone resorption (Erdemir et al., 2007). In our study, there were low positive and significant correlations between the IL-6 value and IL-1α (r = 0.171; p = 0.013), IL-1β (r = 0.214; p = 0.002) and TNF-α  (r = 0.161; p = 0.020) values, which supports this (Table 2).
           
    IL-6 released from T cells, macrophages, fibroblasts and endothelial cells plays a very effective role in the host defense mechanism by stimulating B cell proliferation and differentiation. IL-6, which is an important cytokine in the diagnosis of periodontal diseases, has been found to be significantly higher in inflamed tissues (Erdemir et al., 2007).
           
    Together with this information in our study, one week after the antiparasitic application, it was observed that the IL-6 value increased in group 4 (0th day: 19,51 pg/ml; 7th day: 38,97 pg/ml) and showed a statistically significant difference (p <0.05) with the other groups except group 5 (0th day: 20,78 pg/ml; 7th day: 28,77 pg/ml). Although an increase was also seen in group 5, no statistical difference was observed. However, 14 days after the vaccine application, there was a decrease in groups 4 (14th day: 17,58 pg/ml).  and groups 5 (14th day: 17,10 pg/ml) compared  to the first measurement, while an increase was observed in the other groups. No statistically significant difference was observed between groups after vaccine administration (Table 1).
           
    Contrary to this situation, Arteche-Villasol et al. (2021) conducted a comparison of the early response of monocyte-derived macrophages from vaccinated and unvaccinated goats when infected with Mycobacterium avium subsp. paratuberculosis in vitro; they reported an increase in IL-6 value in the vaccinated control group.
           
    Likewise, Maqbool et al., (2021) reported in their study that during the experimental infection of M. haemolytica A2 under tropical conditions, there was an increase in the IL-6 value in vaccinated goats compared to unvaccinated goats.
           
    Contrary to these studies, the reason for the excessive increase after antiparasitic application in groups 4 and 5 compared to the first measurement and the decrease after enterotoxemia vaccination should be investigated in more comprehensive studies (Table 1).
           
    Considering the statement that it is possible to evaluate immunological effects by measuring the level of circulating proinflammatory cytokines (Elsabahy and Wooley, 2013). The findings obtained in our study indicate that antiparasitic applications applied before enterotoxemia vaccination in sheep provide greater activation of the immune system in sheep. It is also suggested that IL-1β can lead to metabolic, hormonal and cellular changes that are crucial in initiating an immune response (Eryavuz et al., 2022).
           
    IgG has an important role in the defense mechanisms of animals. IgG constitutes 70-75% of the immunoglobulins in circulation. It is the only antibody with antitoxin activity and the main antibody generated during a secondary immune response. It plays a role in several autoimmune diseases and humoral deficiencies. It is stated that it is synthesised between 7 and 28 days following immune stimulation in adult and newborn animals. IgA is found primarily in secretions lining mucosal surfaces and relatively small amounts can be found in circulation (Başbuğan et al., 2021). With a primary immune response, IgM appears first as the main Ig. Although IgM also occurs in a secondary response, it does not show as high levels as IgG (Tizzard 2021). Maqbool et al. (2021) reported that there was an increase in IgG value in vaccinated goats compared to unvaccinated goats during the experimental infection of M. haemolytica A2 under tropical conditions.
           
    In our study, it was observed that the IgG value increased significantly in group 3 compared to the other groups 14 days after enterotoxemia vaccine administration and there was a statistically significant (p<0.05) difference (Table 1).
           
    It was observed that the IgA value increased in group 3 compared to the other groups 14 days after enterotoxemia vaccine administration and there was no statistically significant (p>0.05) difference (Table 1).
           
    The application time and dose of levamisole are extremely important in stimulating the responsiveness of T and B lymphocytes (Undiandeye et al., 2014).
           
    Aydın and Aktaş (2023) reported that 5 mg/kg for 4 days of levamisole administered together with (peste des petits ruminants) PPR vaccine in Morkaraman sheep stimulated IgG production and increased IgG at a statistically significant level.
           
    Abdullah and Başbuğan (2020) reported that the application of levamisole tablets (5 mg/kg per oral) together with the foot and mouth disease (FMD) vaccine stimulated IgG production and increased IgG at a statistically significant level.
           
    Rashid and Yüksek (2019) reported that the application of levamisole together with the enterotoxemia vaccine in sheep stimulated the production of IgG and IgA levels, although this was not statistically significant.
           
    In our study, it was observed that levamisole application one week before the enterotoxemia vaccine increased the IgG value (0th day: 3.54 mg/ml; 7th day: 4.92 mg/ml). to a statistically significant level. On the contrary, a decrease was observed in the IgA value. When comparing the first measurement to 14 days after (14th day: 4.13 mg/ml).  The enterotoxemia vaccine administration, the IgG value increased. However, when comparing it to 1 week after the levamisole application, it decreased (Table 1).
           
    Rashid and Yüksek (2019) reported that levamisole may enhance the immune response to the enterotoxemia vaccine, particularly in cases of immunosuppression. They also noted that immunostimulants can be beneficial in improving the immune response to vaccines. Simply getting vaccinated may not effectively boost the body’s immune response to a specific pathogen. Thus, it is advisable to combine these immune-stimulating agents with vaccines to enhance and prolong immunity (Rashid and Yüksek, 2019).
           
    In our study, the increase in IgG value after levamisole application and the decrease after the enterotoxemia vaccine support this (Table 1). For this reason, it has been shown that if the immune response is to be strengthened with levamisole, it would be more beneficial to administer levamisole together with the vaccine rather than before the vaccine.
           
    APPs are highly sensitive biomarkers, although their specificity in detecting different infectious agents is limited. It is commonly used in the diagnosis, prognosis and health status screening of animals (Murata et al., 2004).  The APR is a typical reaction that takes place soon after tissue damage as a result of various factors like infection, immune response, cancer, trauma, parasites, or other causes. The most important structures secreted as a response of the organism to acute inflammation are cytokines, APPs and leukocytes. The initiation of the APR is triggered by proinflammatory cytokines, including IL-1, IL-6 and TNF-α, which are secreted by activated leukocytes near the location of tissue injury. Cytokines coordinate the first reaction to infectious illnesses and inflammation and trigger various systemic responses. Within the liver, these cytokines induce the synthesis and subsequent secretion of glycoproteins referred to as APP (Gökçe and Bozukluhan, 2009; Taşçene 2017).
           
    Plasma levels of ceruloplasmin increase in cases of infection, malignancy, trauma, obstruction and infection of the biliary tract; this increase is more evident in RES diseases such as Hodgkin’s. It has been shown that endogenous mediator substances called leukocytes mediate the synthesis of ceruloplasmin. It is known that the increase in ceruloplasmin concentration in diseases enables the release of copper from the liver stores into the plasma (Taşçene 2017).
           
    In cattle, Hp functions in the regulation of lipid metabolism (Nakagawa et al., 1997) and as an immunomodulator in lymphocyte functions (Murata and Miyamoto, 1993). Due to this feature, it is reported that Hp concentration can be used to monitor the immune functions of calves (Gökçe and Bozukluhan, 2009). Maqbool et al., (2021) reported that there was an increase in Hp and serum amyloid A (SAA) values in vaccinated goats compared to unvaccinated goats during the experimental infection of M. haemolytica A2 under tropical conditions.
           
    In our study, Hp values increased in all groups 14 days after the enterotoxemia vaccine, but no statistical difference (p>0.05)  was found in the comparison between groups (Table 1).
           
    Cp increased in group 3 and decreased in the other groups 14 days after enterotoxemia vaccination and no statistical differences (p>0.05) were detected (Table 2). Oxidative stress occurs as a result of disruption of the balance between oxidant and antioxidant mechanisms (Akşit et al., 2014). TOS serves as a reliable measure to assess the oxidants generated by the body and absorbed by ambient elements (Ertaş and Kırmızıgül, 2021). The levels of TOS will increase in inflammatory conditions (Bozukluhan et al., 2016). The OSI plays a crucial role in assessing oxidative stress levels. Several research investigations have been carried out to assess the effectiveness of OSI in measuring oxidative stress in farm animals. These investigations have shown that OSI levels dramatically increase in disorders characterized by oxidative stress (Hanedan et al., 2015; Gürgöze et al., 2024). In parallel with this information, in our study, it was observed that TOS (0th day: 3,94 µmol/L; 7th day: 4,54 µmol/L; 14th day: 11,24 µmol/L) and OSI (0th day: 0,24 arbitrary unit; 7th day: 0,28 arbitrary unit; 14th day: 0,70 arbitrary unit) values in group 4 increased significantly compared to the other groups 14 days after enterotoxemia vaccine administration and a statistically significant difference (p<0.05) was observed (Table 1). This showed us that oxidative stress was stimulated following vaccine administration in group 4 (Table 1).
           
    Antioxidants play a crucial role in safeguarding cells against the harmful impacts of oxidative stress. Given the wide range of antioxidant substances and pathways, it is crucial to measure the quantitative antioxidant power, or TAS level, in biological samples. This is an important step in preventing oxidative stress and assessing the overall antioxidant capacity (Akdoğan et al., 2018; Ertaş and Kırmızıgül, 2021; Bilmez and Özyurtlu, 2023). In our study, we detected that the TAS value (0th day: 1,68 µmol/L; 7th day: 1,85 µmol/L) increased in group 6 after antiparasitic application and there was a statistically significant difference (p<0.05) between group 6 and the other groups, except for group 5. In group 5 (0th day: 1,70 µmol/L; 7th day: 1,79 µmol/L), the TAS value increased compared to groups 1 (0th day: 1,62 µmol/L; 7th day: 1,45 µmol/L), groups 2 (0th day: 1,70 µmol/L; 7th day: 1,52 µmol/L) and groups 3 (0th day: 1,65 µmol/L; 7th day: 1,54 µmol/L) after antiparasitic application and a statistically significant difference (p<0.05) was determined (Table 1).
           
    In group 7 (0th day: 1,59 µmol/L; 7th day: 1,70 µmol/L), the TAS value increased compared to group 1 after antiparasitic application and a statistically significant difference was detected. This has clearly shown us that the application of the antiparasitic drugs rafoxanide and thiabendazole, oxfendazole and oxyclozanide and levamisole can be useful in protecting from the destructive effects of oxidative stress, which increases TAS (Table 1).

    CONCLUSION

    In conclusion, this study has shown that antiparasitic applications can stimulate immunity with or without stimulating proinflammatory stocks.
           
    In particular, it has been demonstrated that the administration of ricobendazole 1 week before the enterotoxemia vaccine stimulates the immune system by stimulating the proinflammatory stocks without creating an acute phase response and increasing the IgG value by creating moderate oxidative stress, resulting in a significant improvement in immunity after the enterotoxemia vaccine.
           
    The findings indicate that ricobendazole may have immunomodulatory potential that warrants further investigation. For this reason, new clinical and experimental studies will contribute to science by conducting more detailed research on ricabendazole.

    ACKNOWLEDGEMENT

    Anadolu University Scientific Projects Fund (Project No. 2107S112) has financially supported this study.
     
    Disclaimers
     
    The data of this study are available upon request from the corresponding author.
     
    Informed consent
     
    This study was conducted by the Animal Experiments Local Ethics Board on 09.11.2020 and 50/51. It was carried out with approval no.

    CONFLICT OF INTEREST

    The authors declare no conflict of interest.

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