Copper oxide wire particles alone or associated with closantel: increase in the immune and antioxidant response in lambs experimentally infected with Haemonchus contortus

doi:10.21203/rs.3.rs-2634692/v1

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Copper oxide wire particles alone or associated with closantel: increase in the immune and antioxidant response in lambs experimentally infected with Haemonchus contortus

https://doi.org/10.21203/rs.3.rs-2634692/v1

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The aims of this study were to measure oxidative stress, immunological responses, parasite counts and copper concentrations in plasma and livers of lambs experimentally infected with Haemonchus contortus and treated with copper oxide wire particles (COWP), either isolated or associated with the anthelmintic agent closantel. We randomly divided 30 Corriedale lambs were into five groups (six animals each): uninfected animals (Control); animals infected with H. contortus (HC); infected and treated with closantel (HC + CL); infected and treated with COWP (HC + Cu); and infected and treated with closantel plus COWP (HC + CL + Cu). All animals in the experimental groups were infected orally with H. contortus (5,000 larvae - L3). On day 14 post-infection, the treatments were initiated. The animals infected with H. contortus showed reductions in levels of non-protein thiols and glutathione as well as lower catalase and superoxide dismutase activities in serum and tissues compared to the control group. The COWP treatment reversed these alterations and significantly increased antioxidant parameters. Treatments reduced levels of thiobarbituric acid reactive substances (TBARS) in serum and tissues. The lambs supplemented with COWP had higher levels of IgG and IgE than did the other groups, suggesting an immune response to the infection. Copper concentrations in plasma accorded with the physiological values and the animals did not present any laboratory alterations related to copper intoxication. All treatments showed anthelmintic effects manifested as lower parasite counts in infected and treated animals. These findings suggest that COWP supplementation stimulated antioxidant effects and increased immune responses in lambs with haemonchosis. COWP associated with closantel may effective for lambs infected with H. contortus, considering reasonable synergism of the commercial product with the mineral, resulting in a reduction of 100% in parasite counts.

Ovine

COWP

oxidative stress

immunoglobulins

hepatic copper

Haemonchus contortus is the major gastrointestinal nematode affecting sheep worldwide, especially in hot and humid regions [1]. This nematode infects the abomasum and is essentially hematophagous, i.e. during its parasitic life cycle, it feeds on host blood. Consequently, animals with high parasitic loads may present with anemia and marked hypoproteinemia in addition to high mortality rates [2]. The inflammatory process mobilizes macrophages and neutrophils [3] that produce reactive oxygen species (ROS) that in turn damage cells, with consequent death of the parasite [4]. Reactive oxygen species have no specific action against pathogens and, depending on their levels, can cause lesions in the cells of abomasal mucosa, as described in lambs infected with H. contortus [5]. The enzymatic and non-enzymatic antioxidant system act to minimize the harmful effects of ROS and is composed mainly of superoxide dismutase (SOD), catalase (CAT), non-protein thiols (TSH), and glutathione (GSH) [6].

Parasite resistance is the main sanitary problem in the ovine industry, being an obstacle to efficient control of gastrointestinal helminths [7–8]. H. contortus demonstrates a remarkable propensity for developing resistance to all drugs used to treat it [9]. Therefore, studies have been carried out aiming at the development of alternatives such as minerals to reduce the use of anthelmintic agents [10–14]. Microminerals such as selenium may act by reducing oxidative stress caused by H. contortus. Copper administered subcutaneously (3.5 mg/kg) increases weight gain of infected animals in relation to those not supplemented [12–13]. Copper also modulates the immune response of lambs infected by H. contortus when used in association with zinc [15]. When administered orally, copper exerts direct effects against H. contortus [16–17].

Capsules with copper oxide wire particles (COWP) were initially developed to overcome copper deficiency in ruminants. After oral administration, COWP pass through the rumen and adhere to the mucosa of the abomasum, releasing particles and increasing soluble copper levels, where levels are maintained for at least 32 days [18]. Studies have been conducted to determine the prophylactic effect of COWP on the establishment of infective H. contortus larvae; however, varied results were observed regarding the effectiveness of the treatment [18–20].

In this context, the aim of this study was to determine whether the slow-release capsule of copper oxide reduced parasitic infection and improved the antiparasitic activity of closantel through possible synergistic effects, as well as whether the treatment modulates immune and inflammatory responses. Finally, we studied the effect of copper on oxidative stress markers in lambs experimentally infected with H. contortus.

2.1 Animals and groups

Thirty male lambs (Corriedale, eight-months old, 30 kg on average), were maintained in collective pens until group allocation. The animals underwent a period of environmental adaptation of 30 days. During this period, the lambs were treated once with an anthelmintic agent (2 ml of monepantel, 2 ml of albendazole and 4 ml of Fortgal® (sulfadoxine, trimethoprim and piroxicam) to eliminate possible gastrointestinal parasitosis before the beginning of the experiment. The parasitological analyses were conducted once before the anthelmintic treatment. After the treatment, seven parasitological analyses were performed to ensure the absence of parasites before the experiment starts.

The lambs were randomly divided in five groups with six animals each. The groups were separated as follow: Control - uninfected lambs; HC – lambs infected with H. contortus; HC + CL – infected with H. contortus and treated with closantel; HC + Cu – infected with H. contortus and treated with copper oxide wire particles; and HC + CL + Cu – infected with H. contortus and treated with closantel plus copper oxide wire particles. Each group was maintained in a separate box and the control group was kept isolated from the other groups. The boxes were cleaned daily and all procedures in the control group were performed first and with separate equipment in order to avoid the infection of control group. All lambs were certified as parasite negative by egg per gram of feces (EPG) prior to experimental infection.

The experimental protocol was approved by the Committee of Ethics in the Use of Animals (CEUA) of the Universidade Federal de Santa Maria, registration number 3748011116.

2.2 Diet

Each lamb received 1,800 g of ryegrass hay, 250 g of Puro Trato® brand feed with 14% crude protein, 240 g of soybean meal and 12 g of calcitic limestone per day. The feed was divided and offered twice a day, at 07:30 and 18:00.

2.3 Experimental infection

The H. contortus larvae used for the experimental infection were obtained by coproculture of feces collected from sheep donors with haemonchosis from Embrapa Gado de Corte (Bagé-RS), using the method described by Roberts and O'Sullivan [21]. The experimental infection was performed with a saline suspension containing 5,000 infective larvae (L3) of H. contortus and administered orally to the lambs of infected groups. Control group received only saline by the same route. The day of the experimental infection was considered as day 0 (D0).

2.4 Treatments

At 14 days post-infection (p.i.), when an increase in the parasitic load was observed in the EPG count, lambs were treated according to each experimental group as a single dose. The capsules with 2 g of copper oxide wire particles (Copasure®) were administered orally to the groups HC + Cu and HC + CL + Cu; and 1 mL/kg of the anthelmintic closantel (Diantel®) was administered orally to the HC + CL and HC + CL + Cu groups.

2.5 Sample collection

On days 0, 21, 35 and 49 p.i., blood samples were collected from the jugular vein of the lambs with vacutainer system and were stored in tubes with anticoagulant ethylenediaminetetraacetic acid 10% (EDTA) and without anticoagulant. The blood tubes without anticoagulant were centrifuged for 5 minutes at 3,200 rpm, in order to obtain serum samples. The serum was divided in three polypropylene tubes and stored at − 80°C for further analysis.

In the same experimental periods, fecal samples were collected directly from the rectal ampulla for egg counts per gram of feces (EPG) by the McMaster method [22]. After the final collection (day 49 p.i.) and according to animal experimentation standards [23] the animals were euthanized in a slaughterhouse and the abomasum, liver, and kidneys were collected. Pieces of these organs (the same region of each tissue was collected to avoid differences associated with tissue anatomy and physiology) were used to perform various analyses; therefore, the sampling procedures used are described for each item below. To quantitatively monitor the parasitic load, the abomasal contents were transferred to graduated containers and 10% of the content was preserved in Railliet-Henry solution for nematode counting [24].

2.6. Parameters evaluated

2.6.1. Copper concentration in the liver

The liver samples were weighed individually and transferred to tubes containing 6 ml of reversed regal water for tissue digestion (3:1 HNO₃:HCl) and covered with plastic lids to reduce evaporation of the acid mixture during the heating step. The tubes were heated for 1 h at 120°C in order to the acid mixture oxidized the organic part of the samples and the analytes were solubilized. The digested samples were diluted to 25 ml and stored in polypropylene tubes until the determination of copper (Cu) using flame atomic absorption spectrometry (FAAS).

For the analysis of the samples using the FAAS technique, a calibration curve was prepared in acidic medium. For each polypropylene tube we prepared 6 ml of inverted regal water and diluted to 25 ml, in order to simulate the same conditions of the sample. Concentration standards of 0.1, 0.25, 0.5, 1.0. and 2.5 mg/L were prepared for spectrometer calibration. The determinations were made at 324.7 nm. Due to the high concentrations of Cu in the samples, these were diluted at least twice before determination. This dilution factor was considered for the concentration calculations. To determine the detection limits, 17 control solutions (acid solution subjected to the same steps as the samples) determinations were made, considering the determined average concentration (mg/g) and the standard deviation (LOD = 3*SD + control solution value). All liver samples could be quantified by the FAAS technique and were analyzed in duplicate.

2.6.2. Copper concentration in plasma

The samples were pipetted (200 µl) individually and transferred to tubes containing 6 ml of a mixture of nitric acid distilled for sample digestion (14.4 mol/L). The tubes were covered with plastic lids to reduce evaporation of the acid mixture during the heating step (1 h at 120°C). The digested samples were diluted to 25 ml and stored in polypropylene tubes until copper determination by inductively coupled plasma optical emission spectrometry (ICP-OES).

For determination of the samples by the ICP-OES technique, a calibration curve was prepared in 5% nitric acid medium. Concentrations of 1, 2.5, 5, 10, 20, 40, 60, 80, and 100 µg/L were prepared for the calibration of the spectrometer. The determinations were made 324.7 nm. Despite the low concentration of Cu in the plasma samples (µg/mL), the samples were diluted at least 4 times before determination, so as to avoid matrix interference by ICP-OES. The dilution factor was considered in the concentration calculations. All samples were analyzed in duplicate.

2.6.3. Catalase in liver, kidneys, and abomasum samples

For the catalase (CAT) activity assay, fractions of liver, kidney, and abomasum were homogenized separately in 50 mM potassium phosphate buffer (pH 7.5). The homogenate was centrifuged at 5,000 rpm for 10 min to obtain supernatants that were used for the enzyme assay. Catalase activity was measured using the method of Nelson and Kiesow [25]. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.5), 10 mM H₂O₂ and 20 µl of the homogenate supernatant sample. The reaction rate of H₂O₂ was monitored at 240 nm for 2 min at room temperature. The enzymatic activity was expressed as pmol/mg protein/minute.

2.6.4. Total thiols and glutathione in serum, liver, kidneys, and abomasum samples

Total thiols groups (TSH) were analyzed spectrophotometrically using the method of Boyne and Ellman [26], using a standard curve with cysteine to calculate the thiol group content in samples, and the results were expressed as µmol SH/ml protein.

For the determination of glutathione (GSH), a spectrophotometer (412 nm) was used as described by Ellman [27], using a standard curve with GSH to calculate the content of non-protein thiol groups in samples. The values were expressed as nmol of GSH/ml.

2.6.5. Superoxide dismutase in liver, kidneys, and abomasum samples

Superoxide dismutase activity (SOD) was measured according to McCord and Fridovich [28], with modifications. The assay was performed in a total volume of 1 ml containing 50 mmol of glycine buffer (pH 10), 60 mmol of epinephrine and supernatants from liver, kidney, or abomasum samples, previously homogenized separately in phosphate buffer and centrifuged. Epinephrine was added and the formation of adrenochrome was measured at 480 nm with an ultraviolet-visible (UV-VIS) spectrophotometer for 4 min. The results were expressed as SOD IU/mg of protein.

2.6.6. Thiobarbituric acid reactive substances (TBARS) in serum, liver, kidneys and abomasum samples

Serum TBARS levels were determined according to Jentzsch et al. [29] by measuring the concentration of malondialdehyde (MDA) as a product of lipid peroxidation by reaction with thiobarbituric acid (TBA). The absorbance was measured at 532 nm, and results were expressed in nmols of MDA/mg of protein. For the determination of TBARS levels in samples of liver, kidney or abomasum homogenates the method described by Ohkawa et al. [30], and three absorbance was measured at 532 nm. The TBARS levels in tissue samples were expressed in nmol MDA/mg of protein.

2.6.7. Immunoglobulins

The concentrations of IgG and IgE in serum samples were determined according to the manufacturer's specifications using the ELISA method with kits from Bethyl Laboratories.

2.7. Statistical analysis

Statistical analysis was performed using a two-way ANOVA for independent samples, followed by Tukey's post hoc analysis. Significance was set at p < 0.05 and the results were expressed as mean ± standard error of the mean (SEM).

3.1 Experimental infection and EPG

The use of 5,000 H. contortus larvae (L3) was able to establish a very mild infection in the animals, mimicking a natural low-grade infection acquired in a pasture (Table 1). The control group remained free of infection during the entire experimental period. All animals in the HC group eliminated eggs from the parasite in the feces on all evaluated days (21, 35, and 49 days p.i.). Regarding the treated groups, we observed a reduction of 2%1, 98% and 97% in the average of EPG in the HC + CL group when compared to the HC group on days 21, 35, and 49 p.i., respectively. On day 21 p.i., the HC + Cu group showed a reduction of 93% in the average EPG compared to the HC group, and one animal had negative EPG count. On day 35 p.i., there was a reduction of 100% in the average EPG and four animals were negative. On day 49 p.i., there was a reduction of 98% in the average EPG and three negative lambs. When closantel was used in association with COWP (HC + CL + Cu), only two animals presented eggs of H. contortus in the feces on day 21 p.i., with a reduction of 99% in the average EPG compared to the HC group. On days 35 and 49 p.i., all animals had negative EPG counts.

Table 1

Egg per gram of feces (EPG) count of lambs experimentally infected with *Haemonchus contortus*, treated or not with closantel, copper or closantel plus copper. Day 21 is the seventh day after treatment.
EPG
Days		Control	HC	HC + CL	HC + Cu	HC + CL + Cu
0	Mean	0.0	0.0	0.0	0.0	0.0
0	S.D.	0.0	0.0	0.0	0.0	0.0
14	Mean	0.0^a	262.5^a	233.3^a	65.0^a	191.7^a
14	S.D.	0.0	505.2	522.7	80.2	237.0
Reduction*		0.0	-	11%	75%	27%
21	Mean	0.0^a	1,445.8^b	1,137.5^b	104.2^c	12.5^c
21	S.D.	0.0	745.2	656.8	135.5	20.9
Reduction*		0.0	-	21%	93%	99%
35	Mean	0.0^a	3,900.0^b	66.7^a,c	12.5^a,c	0.0^a,c
35	S.D.	0.0	2,167.0	80.1	20.9	0.0
Reduction*		0.0	-	98%	100%	100%
49	Mean	0.0^a	3,162.5^b	95.8^a,c	62.5^a,c	0.0^a,c
49	S.D.	0.0	995.5	64.1	108.1	0.0
Reduction*		0.0	-	97%	98%	100%
Control - uninfected; HC – infected and untreated; HC + CL - infected and treated with closantel; HC + Cu - infected and treated with copper oxide wire particle capsule; HC + CL + Cu - infected and treated with closantel associated with the capsule of copper oxide wire particles. Closantel − 1 ml/10 kg single oral dose; Copper oxide particle capsule − 2g single oral dose. *Percentage of reduction in the EPG compared to the HC group of the respective day. Different letters in the same row means statistical difference (p < 0.05; n = 6 per group). Results are expressed as mean ± standard error.

At the end of the experiment, lambs were euthanized and the abomasum was collected to perform the count of adult H. contortus (Table 2), in order to confirm the success of the experimental infection and the EPG count. We observed that the animals in the control group did not present the parasite in the abomasum, whereas the infected and untreated animals (HC) had the highest parasitic load (154.20 ± 41.29). Treatment with closantel plus copper (HC + CL + Cu) was able to eliminate all abomasal parasites from the lambs. Comparing the treatment with closantel alone (3.40 ± 3.21) and treatment with copper alone (11.2 ± 11.25), closantel was more effective in reducing parasite count in the abomasum of the lambs.

Table 2

Count of adult *H. contortus* in the abomasum of lambs experimentally infected with *Haemonchus contortus*, treated or not with closantel, copper or closantel plus copper, on day 50 p.i.
Number of H. contortus in abomasum
	Control	HC	HC + CL	HC + Cu	HC + CL + Cu
Mean	0.00^a	154.20^b	3.40^a,c	11.2^a,c	0.00^a,c
S.D.	0.00	41.29	3.21	11.25	0.00
Control - uninfected; HC – infected and untreated; HC + CL - infected and treated with closantel; HC + Cu - infected and treated with copper oxide wire particle capsule; HC + CL + Cu - infected and treated with closantel associated with the capsule of copper oxide wire particles. Closantel − 1 ml/10 kg single oral dose; Copper oxide particle capsule – 2 g single oral dose. Different letters in the same row means statistical difference (p < 0.05; n = 6 per group). Results are expressed as mean ± standard error.

3.2 Determination of copper in plasma and liver

The values obtained for plasma copper (Table 3) were on average 0.74 to 1.41 µg/mL. There was no significant difference between the treatments. Regarding the concentration of copper in the liver (Fig. 1), the animals that received copper (HC + Cu and HC + CL + Cu) presented higher levels of this mineral in the liver when compared to the other groups.

Table 3

Plasma copper concentration of lambs experimentally infected with *Haemonchus contortus*, treated or not with closantel, copper or closantel plus copper.
Plasma copper concentration
		Control	HC	HC + CL	HC + Cu	HC + CL + Cu
0	Mean	1.41^a	1.21^a	1.07ª	0.96^a	1.33^a
0	S.D.	0.36	0.14	0.15	0.17	0.25
21	Mean	1.00^a	1.22^a,b	0.79 ^b	1.18^a,b	0.98^a,b
21	S.D.	0.23	0.12	0.17	0.20	0.27
35	Mean	0.90^a	0.74^a	0.81^a	0.76^a	0.91^a
35	S.D.	0.14	0.09	0.08	0.11	0.17
49	Mean	1.19^a	0.92^a	0.93^a	0.81^a	0.93^a
49	S.D.	0.27	0.15	0.10	0.26	0.11
Control - uninfected; HC – infected and untreated; HC + CL - infected and treated with closantel; HC + Cu - infected and treated with copper oxide wire particle capsule; HC + CL + Cu - infected and treated with closantel associated with the capsule of copper oxide wire particles. Closantel − 1 ml/10 kg single oral dose; Copper oxide particle capsule – 2 g single oral dose. Different letters in the same row means statistical difference (p < 0.05; n = 6 per group). Results are expressed as mean ± standard error.

3.3 Antioxidants in liver, kidneys and abomasum

3.3.1 Abomasum

The results of antioxidants in the abomasum are presented in Fig. 2. There was a decrease in the activity of CAT and SOD and in levels of GSH and TSH in the HC group when compared to the control. Comparing the HC group with the groups that received treatment, we observed an increase in enzymatic activity and antioxidant levels. The lambs that received copper (HC + Cu and HC + CL + Cu) showed an increase in CAT activity and in GSH levels compared to those treated with closantel alone (HC + CL). For SOD activity and TSH levels, we observed that the treatment with copper alone (HC + Cu) increased these antioxidants in relation to the HC + CL group; however, there was no significant difference. Furthermore, TSH levels were higher in the HC + CL + Cu group than in the HC + Cu group.

3.3.2 Liver

Haemonchus contortus infection (HC group) led to decreased CAT and SOD activity as well as GSH and TSH levels (Fig. 3). In the HC + CL group, there were no significant differences in CAT activity or GSH levels in relation to the HC group; however, there was an increase in TSH levels and in SOD activity. In the groups that received COWP (HC + Cu and HC + CL + Cu), there was an increase in the enzymatic activity and in the levels of the antioxidants evaluated when compared to the HC group. Comparing the various treatments, we observed that the lambs that received COWP (HC + Cu and HC + CL + Cu) showed an increase in CAT and GSH compared to those treated only with closantel (HC + CL) (Fig. 3).

3.3.3 Kidneys

The results of CAT, SOD, GSH and TSH in kidneys are presented in Fig. 4. The HC group showed less CAT and SOD activity and lower GSH and TSH levels than in the control group. The treatment with closantel (HC + CL) increased SOD activity and TSH levels when compared to HC group; however, for CAT activity and GSH levels, no significant differences were observed. The treatment with COWP (HC + Cu) and the treatment with closantel plus COWP (HC + CL + Cu) increased the enzymatic activity and the levels of all tested antioxidants when compared to the HC group.

3.4 TBARS in liver, kidneys and abomasum

The levels of thiobarbituric acid reactive substances (TBARS) behaved similarly in the organs evaluated in this study, being higher for the HC group than in the control group (Fig. 5). All treatments reduced lipid peroxidation compared to untreated animals (HC). In the abomasum and kidneys, COWP (HC + Cu and HC + CL + Cu) reduced levels of TBARS when compared to the group that received only closantel (HC + CL). There was no difference between the treatments with respect to levels of TBARS in the liver.

3.5 TBARS, TSH, and GSH in serum

On days 21, 35, and 49 p.i. there was an increase in TBARS levels in the HC group compared to the control group; the treatments (HC + CL, HC + Cu, HC + CL + Cu) reduced lipid peroxidation in relation to the HC group, except for the HC + CL group at 21 and 35 days p.i. (Fig. 6).

On days 21, 35, and 49 p.i., the animals in the HC group presented lower levels of GSH and TSH in relation to the control. The treatment with closantel was not able to reverse this decrease, except for day 35 p.i., when GSH levels were higher in the HC + CL group than in the HC. The animals that received COWP (HC + Cu and HC + CL + Cu) showed an increase in the levels of these antioxidants compared to the infected group (HC); however, the treatment with COWP alone (HC + Cu) did not differ from the HC group in terms of TSH levels on day 21 p.i. Comparing the treatments, we observed that copper (HC + Cu and HC + CL + Cu) increased GSH and TSH levels in relation to the group treated only with closantel (HC + CL), with the exception of TSH levels in the HC + Cu group at 21 days p.i. (Fig. 6).

3.6 IgG and IgE

The results for IgG and IgE levels are presented in Fig. 7. On days 21, 35, and 49 p.i., an increase in IgG and IgE levels was observed in all infected groups (HC, HC + CL, HC + Cu, and HC + CL + Cu) when compared to the control group. IgG and IgE levels were increased in the lambs that received the combination of treatments (HC + CL + Cu) when compared to the groups HC, HC + CL and HC + Cu; the exception was IgG levels on day 21 p.i. in the HC + Cu group, in which no significant difference was observed.

On days 35 and 49 p.i., the levels of IgG were higher in the HC + Cu group than in the HC and HC + CL groups. The levels of IgE were higher in the HC + Cu group at 21, 35, and 49 days p.i. when compared to the HC group; and when compared to HC + CL on day 49 p.i. Animals treated with closantel alone (HC + CL) presented higher levels of IgG, when compared to the HC group, only on day 49 p.i. No difference was observed between the HC + CL and HC groups for IgE levels.

This study demonstrates, for the first time, the increase in the immune and antioxidant responses of lambs infected with H. contortus and treated with COWP in association with closantel. The results of the present study demonstrated the occurrence of oxidative stress during H. contortus infection, because all experimentally infected animals showed higher TBARS levels in various tissues than were measured in uninfected animals. H. contortus infection decreased levels of GSH and TSH in serum and in the evaluated organs, and lowered activities of SOD and CAT in liver, kidney, and abomasum when compared to uninfected animals. All treatments reduced TBARS levels and increased the antioxidant capacity of the lambs when compared to the infected and untreated group, obtaining better results for those supplemented with COWP (HC + Cu and HC + CL + Cu).

Higher EPG values in the HC group on days 21, 35, and 49 p.i. compared with day 0 suggest the efficacy of the experimental infection. Moreover, we observed that the groups treated with copper and those treated with closantel plus copper presented higher percentages of EPG reductions on the evaluated days compared to the lambs treated only with closantel. Thus, in addition to the beneficial effects of the use of copper on oxidative stress, mineral supplementation helped to reduce EPG, as reported by other researchers [13, 31]. Regarding to the count of H. contortus in the abomasum, we observed that the treatment with closantel plus copper eliminated the parasites of the abomasum, whereas the treatment only with copper showed a higher mean number of parasites in the abomasum (11.2 ± 11.25) than the group treated only with closantel (3.4 ± 3.21). Nevertheless, all treatments were effective when compared to untreated animals (154.2 ± 41.29).

The occurrence of oxidative stress in lambs with haemonchosis has been reported; the infection may lead to increased levels of TBARS [11–13, 32–33] as observed in the present study, which is an indication of lipid peroxidation, a common finding during haemonchosis [11–13, 32–33]. In addition to the damage caused directly by the parasitic disease, the organism develops a defense mechanism in which local influx of eosinophils, monocytes, macrophages, and neutrophils occurs [34–36]. The phagocytic cells are the main defense mechanism of the organism against the infection by H. contortus [37–38], when activated, these cells produce ROS that damage lipid membranes, proteins, and nucleic acids of the parasite [39]. However, despite destabilizing and killing the parasite, the excess reactive species may also damage host tissues and generate oxidative stress, as observed in the present study, in which increased lipid peroxidation of infected lambs was manifested by the increase of TBARS levels.

The various treatments reduced lipid peroxidation when compared to the infected and untreated group. Lambs receiving copper supplementation, either alone or in combination with closantel, presented lower levels of TBARS in serum, abomasum, and kidneys when compared to conventional treatment only with closantel. This may occur because of the action of copper as an adjuvant enhancing the immune response and as a cofactor for several components/enzymes in the organism, including ceruloplasmin and the antioxidant enzyme Cu, Zn-SOD [40–42]. These results are similar to those observed in other study [13], who found treatment with copper plus selenium reduced lipid peroxidation in animals with haemonchosis.

Reactive oxygen species and antioxidant production are important for host resistance to parasitic infections [39, 43–44]. During H. contortus infection in resistant sheep, there is a specific inflammatory response characterized by increased production of oxidants and antioxidants [45]. The animals infected by this parasite and left untreated have low levels/activity of antioxidants when compared to healthy animals. However, using the treatment with closantel, copper or their combination, there was an increase in the antioxidant response, which is necessary for the development of an adequate immune response against infection [46–48]. The increase in antioxidant defenses observed in treated lambs may occur due to the reduction in parasite load; however, it is also suggested that this alteration in antioxidants occurs as a response to increased oxidative stress caused by the infection [49]. The antioxidant system does not act directly on the parasite; rather, it occurs by the augmentation of the antioxidant defense system through improvement of animal health status. The improved capacity to remove free radicals produced by infection is the action mode of antioxidant defense system, i.e., to exert indirect effects on parasitic loads. Supplementation with copper appears to potentiate antioxidant defenses during H. contortus infection. When comparing the various treatments, we observed that COWP (isolated or in combination with closantel) improved CAT and SOD activity, as well as GSH and TSH levels when compared to the use of closantel alone. This difference may occur because copper is essential for antioxidant defense. In this manner, COWP presents advantages when compared to the conventional treatment, because copper is necessary for the catalytic function of the enzyme Cu, Zn-SOD [42] and this enzyme is the first line of defense for the organism when activated phagocytes cells produce superoxide radical in response to H. contortus infection [37–39]. Low levels of copper impair the function of this enzyme [50–52] accompanied with decrease catalase transcription rates [53].

Supplementation with minerals can improve host immune responses against infection [17]. In fact, lambs supplemented with copper had higher levels of IgE and IgG when compared to those treated only with closantel. Moreover, treatment only with closantel did not differ significantly or showed a slight difference in immunoglobulin levels compared to infected and untreated animals, demonstrating the potential of copper oxide particles to increase the immune response. The increase in immunoglobulin levels is essential in helminth load control, because increased IgE levels are seen in the self-healing reaction in sheep infected with H. contortus. The combination of antigens of this parasite with IgE-bound mast cells triggers degranulation and release of vasoactive molecules and proteases. These molecules stimulate smooth muscle contractions and increase vascular permeability, causing the detachment of the parasite from the abomasal mucosa [54]. In relation to IgG, this immunoglobulin has been shown to act in various parasite infections, inducing the production of antibodies in the abomasum and intestine, which act against some carbohydrates present on the surface of the parasite [55].

Increased lipid peroxidation in infected animals, as observed by increased TBARS levels in the present study, may contribute to the pathogenesis of haemonchosis, similar to what occurs in other parasitic infections [56–57]. Thus, ROS can lead to a reduction in the host's ability to handle infection [6] related to a dysfunction of the immune response [58], characterized by decreased IgE and IgG levels in lambs with haemonchosis and left untreated. However, because of the treatments used in the present study, we observed that, with an increase in the activity of SOD, CAT and in the levels of GSH and TSH, there was a reduction in TBARS levels, as well as an increase in IgE and IgG levels, with the purpose of cell protection and infection control. In particular, COWP presented better results in the variables analyzed when compared to animals treated only with closantel.

Therapeutics is the conventional method of treatment for gastrointestinal nematodes. However, due to the wide resistance of the parasites to the commonly used anthelmintic agents [59], studies with alternative treatment methods have been developed. Among these, COWP has demonstrated substantial anthelmintic action in animals infected with H. contortus, presenting an EPG reduction rate of 93% at 21 days p.i. This effect was superior to that of closantel alone. On the other hand, the combination of the two treatments (closantel plus COWP) led to elimination of EPG in the infected animals. This anthelmintic action may be related to the composition and formulation of the copper product; a study comparing the use of particles of copper oxide and copper sulfate showed that copper oxide reduced the degree of FAMACHA and EPG of animals with haemonchosis [60].

Regarding oxidative stress and the improvement in host immune response, COWP appears to have superior action compared to other compounds reported in the literature. COWP in a single dose reduced TBARS levels, increased antioxidant activity and increased the immunity of treated animals at 21 days p.i., while the use of selenium and copper in two applications reduced TBARS levels at 40 and 80 days p.i. and increased the activity of glutathione peroxidase only at day 80 p.i. [13]. Similarly, zinc edetate in two doses decreased the oxidative stress index only at 31 days p.i. [14]. It is important to emphasize that excessive use of copper can lead to toxicity [61]. However, in the present study, treated animals did not show any signs of copper poisoning. Moreover, plasma and hepatic copper concentrations of the lambs treated with copper were within the normal range for the species [62–63]. This finding suggests that the dose used in the presented study was adequate, as already expected, because in previous studies that used higher doses of COWP, no toxic effects were observed [64–65]. Animals with copper overload have high lipid peroxidation in the liver and serum, decreased levels of hepatic GSH and low SOD activity [66–69], which was not observed in this study, further suggesting the safety profile of the dose.

In the present study, when comparing the results of hepatic copper concentration in the various groups, increased hepatic copper levels in the groups that received COWP were observed. A study observed a negative relationship between the amount of hepatic copper of the animals that received COWP and the number of parasites in the abomasal contents, suggesting that, when the capsules release more copper, fewer parasites survive in the abomasum and more copper accumulates in the liver [16]. Other studies have also associated higher levels of copper with increased clearance of parasites [70–71]. Therefore, higher levels of hepatic copper observed in animals supplemented with COWP may be related to a higher percentage of reduction in EPG and to the number of parasites in the abomasum of these animals compared to the untreated lambs or the lambs treated only with closantel. In addition, COWP has the potential to reduce the fecundity of Haemonchus spp. when it is offered for long periods, and may reduce larval grass contamination, especially in areas where anthelmintic resistance is an emerging problem [20].

The copper oxide wire particle (COWP) capsule associated with the anthelmintic closantel is an alternative treatment for lambs infected with H. contortus in view of the synergism of the commercial product with this mineral, causing a reduction of 100% in the parasite count in treated lambs. COWP also increased antioxidant actions and immune responses to the parasite without showing producing signs of toxicity. Taken together, this treatment may represent an alternative for sheep farmers who must manage resistance to multiple anthelmintic agents.

Funding: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Declarations of interest: none

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Copper oxide wire particles alone or associated with closantel: increase in the immune and antioxidant response in lambs experimentally infected with Haemonchus contortus

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Abstract

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1. Introduction

2. Material And Methods

2.1 Animals and groups

2.2 Diet

2.3 Experimental infection

2.4 Treatments

2.5 Sample collection

2.6. Parameters evaluated

2.6.1. Copper concentration in the liver

2.6.2. Copper concentration in plasma

2.6.3. Catalase in liver, kidneys, and abomasum samples

2.6.4. Total thiols and glutathione in serum, liver, kidneys, and abomasum samples

2.6.5. Superoxide dismutase in liver, kidneys, and abomasum samples

2.6.6. Thiobarbituric acid reactive substances (TBARS) in serum, liver, kidneys and abomasum samples

2.6.7. Immunoglobulins

2.7. Statistical analysis

3. Results

3.1 Experimental infection and EPG

3.2 Determination of copper in plasma and liver

3.3 Antioxidants in liver, kidneys and abomasum

3.3.1 Abomasum

3.3.2 Liver

3.3.3 Kidneys

3.4 TBARS in liver, kidneys and abomasum

3.5 TBARS, TSH, and GSH in serum

3.6 IgG and IgE

4. Discussion

Declarations

References

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