Pathology – Research and Practice 210 (2014) 812–817
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Original Article
Relationship between oxidative stress and pathological findings in abomasum of infected lambs by Haemonchus contortus Vanessa Machado a , Aleksandro S. Da Silva a,∗ , Andressa S. Schafer b , Adelina R. Aires b , Alexandre A. Tonin c , Camila B. Oliveira d , Carine L. Hermes e , Taís C. Almeida e , Rafael N. Moresco e , Lenita M. Stefani a , Sonia T.A. Lopes c , Ricardo E. Mendes f , Claiton I. Schwertz f , Marta L.R. Leal b a
Department of Animal Science, Universidade do Estado de Santa Catarina, Chapecó, SC, Brazil Laboratory of Animal Endocrinology and Metabolism, Program of Post-Graduation in Veterinary Medicine, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil c Department of Small Animal, UFSM, Santa Maria, RS, Brazil d Department of Microbiology and Parasitology, UFSM, Santa Maria, RS, Brazil e Department of Clinical and Toxicological Analysis, UFSM, Santa Maria, RS, Brazil f Laboratory of Veterinary Pathology, Instituto Federal Catarinense, Santa Maria, RS, Brazil b
a r t i c l e
i n f o
Article history: Received 11 July 2014 Received in revised form 17 August 2014 Accepted 16 September 2014 Keywords: Helminths LDH NOx FRAP AOPP
a b s t r a c t The aim of this study was to evaluate the nitric oxide (NO) levels, and oxidative and antioxidant markers of lambs experimentally and naturally infected by Haemonchus contortus, and its relation to lesions in the abomasum. For experimental study, a total of 14 healthy lambs were divided into two groups with seven animals each. Group A represented the uninfected animals (control), and Group B was formed by infected animals with 15,000 larvae of H. contortus. Blood was collected on days 15, 45, and 75 post-infection (PI) to obtain serum for biochemical analysis: lactate dehydrogenase (LDH), nitrite/nitrate (NOx ), advanced oxidation protein products (AOPP), and ferric reducing ability of plasma (FRAP). Parasitological stool examination (eggs per gram of feces – EPG) was performed on days 15, 45, and 75 PI to verify the evolution of the infection. On day 15 PI EPG was negative, but on days 45 and 75 PI the EPG was positive for animals from Group B. In the three periods evaluated it was observed an increase of LDH levels in serum of lambs infected with gastrointestinal nematodes, but on the other hand NOx levels were reduced on the same periods in infected animals. The AOPP and FRAP levels did not differ between groups on days 15 and 45 PI, but increased significantly on day 75 PI in infected lambs. The same variables were studied in 10 lambs naturally infected with helminths, where more than 97% corresponded to H. contortus (hematocrit and EPG values were 18.8 ± 2.5% and 7120 ± 2940, respectively). Similar to the experimental study, the levels of NOx reduced, and the levels of LDH, FRAP, and AOPP increased in serum of this animal associated inflammatory infiltrate in the mucosa of the abomasum. Therefore, during the infection by H. contortus it was observed alterations in oxidative markers, indicators of cell lesion confirmed by histological examination of the abomasum, and consequently there were changes in antioxidant levels, with the purpose of cell protection. We also conclude that helminth infection interferes with the nitric oxide metabolism. © 2014 Elsevier GmbH. All rights reserved.
Introduction Haemonchus contortus is known as one of the major Trichostrongylidae pathogens of small ruminants, with a worldwide
∗ Corresponding author. Tel.: +55 49 3322 4202; fax: +55 49 3322 4202. E-mail address: aleksandro [email protected] (A.S. Da Silva). http://dx.doi.org/10.1016/j.prp.2014.09.006 0344-0338/© 2014 Elsevier GmbH. All rights reserved.
distribution [9,27]. This nematode is typical of tropical and subtropical areas, where economic losses can be very high, although an increasing occurrence has been reported also in cold areas [2,32]. H. contortus penetrates the surface of the abomasal mucosa to feed on the blood of the host [20]. The systemic effects observed due to infection by this nematode are anemia and decreased levels of total plasma proteins, particularly albumin [6]. Besides the diseases directly caused by the infection, the presence of the parasite may
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induce production of potentially destructive factors [34], such as reactive oxygen species (ROS), which may damage the parasite but generate oxidative stress [24]. ROS are made up of a large number of chemically reactive molecules derived from oxygen. Among them there are superoxide radical, hydroxyl radical, and hydrogen peroxide. The superoxide radical is produced during the maximum activation of neutrophils, monocytes, macrophages and eosinophils in the presence of H. contortus infection [1,18,22]. Parameters have been used to determine oxidative stress, such as lipid peroxidation (TBARS), advanced oxidation protein products (AOPP), and nitric oxide (NO) [4,16,33]. A situation of oxidative stress causes cell death, and thus may occur an increase of lactate dehydrogenase (LDH). However, the balance between production of oxidant agents and antioxidant defense system is essential, as well as the reduction of glutathione, superoxide-dismutase, and catalase [16]. Other techniques can be used to mediate the antioxidant defenses, the ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”, this automatic method consists of a ferric to ferrous ion reduction at low pH, resulting in a colored ferrous–tripyridyltriazine complex. The FRAP provides an pool index of antioxidant with potential in biological fluids that can be evaluated within the technological reach of every laboratory, as well as the researcher interested in oxidative stress and its effects [5]. The occurrence of oxidative stress in infections by helminths has been described, but using techniques easily influenced by many factors. However, in the present study we proposed the use of automatic methods with high sensitivity. Also, the effect of infection by H. contortus on the metabolism of nitric oxide has not been elucidated. Therefore, the aim of this study was to evaluate the NO levels, and oxidative and antioxidant markers of lambs experimentally and naturally infected by H. contortus and its relation to lesions in the abomasum.
Materials and methods Experimental infection with H. contortus Animals and experimental design Banked serum and plasma samples from a previous study [9] were used in the current study. Our experiment used 14 male lambs crossbred (Corriedale-Texel), five months old and weighing in average 23 kg each. They were kept in holding pens (one pen/group) at the Veterinary Hospital of Universidade Federal de Santa Maria (UFSM) during 30 days under a diet [base of 10.7% protein (commercial feed and ryegrass hay)] for the adaptation in the experimental environment. In this period, the animals received anthelmintic treatment based on monepantel (Zolvix® ). The same diet was provided during the first 20 days of the experiment (postinfection), but after this period it was necessary to change the diet as a consequence of the severe evolution of the disease. From this period onward, the animals were fed with a mixture of hay ground ryegrass (70%), commercial feed with 20% crude protein (CP), and soybean meal with 10% (CP) [totaling a diet with 13% CP]. Each animal consumed 1 kg of dry matter/day. Hematological (erythrogram and leukogram) and biochemical (hepatic and renal function) evaluations were performed three times at 15-day intervals. After 30 days (day 0 of the experiment), the evaluated patterns showed normal values, according to Feldman et al. [13]. Apparently the animals were healthy, and they had negative fecal exam for eggs, cysts and oocysts of parasites. The animals were divided into two groups with seven animals each: Group A was composed by of healthy animals (uninfected) and it was used as a negative control group; Group B, the animals were infected by H. contortus, representing the positive control.
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Each animal from Group B was infected orally with a total of 15,000 larvae (L3), divided in three moments of infection of 5000 larvae each time, at intervals of 3 days between them. The larvae were obtained from the Laboratory of Animal Endocrinology and Metabolism of the Federal University of Santa Maria. They were obtained by coproculture technique [28]. Collection of samples Blood was drawn through Vacutainer® system on the days 15, 45, and 75 post-infection (PI). To measure the levels of NOx (nitrite/nitrate), blood samples were stored in tubes without anticoagulant. To measure the levels of AOPP and FRAP in plasma, blood samples were stored in tubes with anticoagulant (EDTA). To measure levels of LDH in the plasma, blood samples were stored in tubes containing EDTA and fluoride. To obtain the serum or plasma, blood samples were centrifuged (3500 g for 10 min). The serum/plasma was stored at −20 ◦ C and the plasma fluoride stored in the refrigerator (4 ◦ C) until analysis. Stool testing Fecal samples for quantification of eggs per gram (EPG) were collected on days 15, 45, and 75 PI, and processed according to the technique described by Gordon and Whitlock [15]. Five days after the end of the experiment (day 80 PI), five animals from each group were euthanized (10 mg intravenous (IV) of acepromazine; 2 g IV of sodium thiopental; 100 ml IV of potassium chloride), and their parasite loads were determined [31]. The euthanasia was necessary to confirm that the animals were infected (Group B) or negative (Group A), since the EPG by itself may not be suffice. Naturally infection with H. contortus In order to verify the experimental infection reflect a natural conditions, another study was conducted. For this, 10 lambs naturally infected with helminths (identified as group C), where more than 97% corresponded to H. contortus (result obtained by culture of larvae) were used. This lambs presents in mean hematocrit values [13] and EPG [15] the 18.8 ± 2.5% and 7120 ± 2940, respectively. Blood was drawn through Vacutainer® system similar experimental study to measure the levels of LDH, NOx AOPP and FRAP, according describe in “Collection of samples” this study. The results of biochemical analyzes in naturally infected animals (Group C) were compared with non-infected lambs, which corresponds to day 75 PI in group A. Necropsy of lambs that died was performed and observed that this abomasum filled with H. contortus (adults and larvae) (Fig. 1A), with fragments of the abomasum collected for histological analysis (fixed in buffered formalin 10% and stained with hematoxylin and eosin). Biochemical analyzes LDH, NOx , AOPP, and FRAP assays were performed using the automated Cobas Mira® . The LDH in plasma fluoride was measured within 5 days of blood collection using a commercial kit Bioclin® (kinetic method), and results were expressed as mg dL−1 . NO levels in the serum of lambs infected by H. contortus were analyzed indirectly by quantifying serum NOx according to the technique described in detail by Tatsch et al. [30], and results were expressed as mol L−1 . AOPP concentration (protein oxidation) was determined using the semiautomated method described by Hanasand et al. [17], and results were expressed as mol L−1 . Levels of FRAP was measured according to the technique described by automatically Benzie and Strain [5] that uses plasma. The results of FRAP were expressed as mol L−1 .
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Results Experimental infection with H. contortus Course of infection The animals used as control (Group A) did not show any clinical signs of the disease. However, infected lambs (Group B) showed pale-pink mucosa when checked on days 45 and 75 PI. Despite the fact that the animals were not weighted, weight loss was also observed on infected animals. Biochemical analyzes The results of LDH, NOx , AOPP, and FRAP are presented in Table 1. In the three periods evaluated it was observed a LDH increase in the serum of lambs infected with gastrointestinal nematodes, however NOx levels were reduced at the same periods in infected animals. The AOPP and FRAP levels did not differ between groups on days 15 and 45 PI, but increased significantly in lambs infected on day 75 PI when compared to uninfected animals. Eggs per gram of feces Animals from the control (Group A) showed negative EPG throughout the experiment. Examination at the necropsy confirmed that the lambs were negative for helminths. The animals in Group B showed negative EPG on day 15 PI, but in the other two analyzes EPG was positive for eggs of H. contortus at days 45 PI (9828 ± 5426 EPG) and 75 PI (4100 ± 2277 EPG). The mean parasite load in animals of Group B was of 2404 of H. contortus. Other helminths were not found during the necropsy of these animals. Naturally infection with H. contortus Histology abomasum of lambs infected with H. contortus was observed moderate focal lymphocytic inflammatory infiltrate in the mucosa (Fig. 1B) and moderate focal mixed inflammatory cell infiltrate in the mucosa (Fig. 1C). The results of the oxidant and antioxidant markers were shown in Fig. 2. An increase in the levels of LDH, AOPP (Fig. 2A), and FRAP (Fig. 2B) was observed in lambs naturally infected with H. contortus compared uninfected. Already, the levels of NOx significantly reduced (P < 0.01) in infected animals (Fig. 2B). Apparently these animals showed diarrhea and edema. But the ocular mucosa was pale, indicating anemia, confirmed by hematocrit. Discussion
Fig. 1. Histology abomasum of lambs infected by Haemonchus contortus (A) was observed moderate focal lymphocytic inflammatory infiltrate in the mucosa (B) and moderate focal mixed inflammatory cell infiltrate in the mucosa (C).
Statistical analysis All data were initially tested for normality, however EPG data did not show to be normal distributed, it was logarithm (log 10) transformed previously to statistical analysis. Then, the NOx , LDH, AOPP, FRAP, and EPG results were subjected to the Student test. The analyses were performed using SAS statistical package (SAS Institute, Cary, NC, USA) with a significance level of 5% (P < 0.05).
The hematological variables did not differ between groups on day 15 PI, different from the days 45 and 75 PI, when it was observed a reduction significant of hematocrit, total erythrocytes count, hemoglobin concentration, and total leukocytes in lambs infected when compared to not-infected the according a previous study [9]. Therefore, in the current study H. contortus infected lambs developed a clinical condition of anemia and leukopenia, associated with altered levels of antioxidants and oxidative markers, from the NO metabolism. The occurrence of oxidative stress in lambs experimentally infected by H. contortus has been reported, mainly due to the detection of lipid peroxidation and increased glutathione reductase activity [24,26]. This oxidative imbalance has been associated with severity of pathological conditions, as well as anemia, due to the ROS damage caused to the erythrocyte membrane (Wolkmer et al., 2009). Gastrointestinal nematode infection in H. contortus, anemia is caused by blood loss since this is a blood sucking parasite. However we cannot disregard the possibility that oxidative stress is contributing to the pathogenesis of this disease, similarly to what occurs in other parasitic diseases [29,35].
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Table 1 Experimental infection by Haemonchus contortus in lambs: mean and standard deviation of hematocrit, lactate dehydrogenase (LDH), nitrite/nitrate (NOx ), advanced oxidation protein products (AOPP), and ferric reducing ability of plasma (FRAP) on days 15, 45 and 75 post-infection. Parameters
Day −1
LDH (mg dL
)
15 45 75
Group A: Control 8.6 ± 3.3 9.0 ± 3.0 9.9 ± 3.4
Group B: Infected
Probability (P)#
17.4 ± 2.8 14.7 ± 4.0 15.9 ± 4.2
P < 0.01# P < 0.01# P < 0.05#
52.8 ± 14.5 49.6 ± 22.3 55.5 ± 30.6
P < 0.05# P < 0.01# P < 0.01#
NOx (mol L−1 )
15 45 75
141.0 ± 75.5 103.6 ± 38.2 116.1 ± 40.2
AOPP (mol L−1 )
15 45 75
14.5 ± 1.1 14.3 ± 3.7 15.3 ± 4.6
16.1 ± 2.3 13.6 ± 3.3 24.0 ± 3.6
P > 0.05 P > 0.05 P < 0.05
FRAP (mol L−1 )
15 45 75
295.9 ± 36.3 298.6 ± 42.0 283.0 ± 44.2
265.1 ± 28.9 262.6 ± 86.6 339.2 ± 61.6
P > 0.05 P > 0.05 P < 0.05#
Hematocrit (%)*
15 45 75
30.7 ± 2.2 30.6 ± 5.1 37.5 ± 2.8
28.5 ± 2.8 22.0 ± 4.7 24.8 ± 5.3
P > 0.01 P < 0.01# P < 0.01#
# *
In the same line, was considered significant difference between groups in the Student test, when presenting P < 0.05 or P < 0.01. Results hematocrit was presented in a previous study [9].
Experimentally and naturally infected lambs showed increased levels of LDH in this study, an enzyme involved in the metabolism of carbohydrates and generally altered in cardiac, hepatic and parasitic disease [23], a parameter indicator of hypoxia, ischemia, and cellular injury. All these changes could be related to increased LDH, because the animals were anemic, probably having lower tissue oxygenation as well as could be occurring lesions of blood cells as mentioned
earlier, due to the condition of oxidative imbalance installed in lambs. The animals in experimental study had a classic case of acute infection, with great loss of blood, which made it necessary to increase energy and protein in the diet of these animals on day 20 PI. Despite the anemia and loss of body condition observed, other differences between the groups regarding the markers of oxidative imbalance in the days 15 and 45 PI were not seen. Already on day
Fig. 2. Levels of lactate dehydrogenase (LDH), advanced oxidation protein products (AOPP), nitrite/nitrate (NOx ), and ferric reducing ability of plasma (FRAP) in lambs naturally infected with Haemonchus contortus when compared uninfected.
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75 PI, there was an increase in the AOPP levels, an indicator of protein oxidation, and it might be considered as true mediator of the pro-inflammatory effects of oxidative stress [33], similar naturally infected lambs. The protein oxidation was probably associated with the increase in FRAP levels, an indicator of total antioxidant capacity of plasma, as a result of the increase of antioxidant enzymes to minimize the effects of ROS produced during infection by H. contortus. Therefore, there is a situation of oxidative imbalance which can influence various physiological mechanisms as well as the actual reduction in the metabolism of nitric oxide seen in lambs. As stated in the literature, in the state of oxidative stress, the production of ROS exceeds the available antioxidant defense systems, and consequence the bioactivity of NO is reduced [14]. A dominant mechanism reducing bioavailability of vascular NO relates to its rapid oxidative inactivation by the ROS superoxide (O2 − ), and there is evidence that persisting oxidative stress will render eNOS dysfunctional such that it no longer produces NO, but O2 − [14]. As already mentioned, the serum levels of NOx reduced in lambs infected by H. contortus, a fact that may be associated with factors other than those mentioned above, such as the reduction of hemoglobin, albumin, iron, as well as its own oxidative stress, the findings described in lambs infected with H. contortus [13]. Studies have established a relation between the occurrence of anemia associated with reduced nitric oxide [11], situation observed in the present study. According to literature, the nitrite ion is a direct causative agent for methemoglobinemia, that is the oxidation of hemoglobin to methemoglobin under aerobic conditions is induced by nitrite [10]. Furthermore, the nitrite production is dependent on the ratio of nitrous oxide and oxygen-hemoglobin [10]. Therefore, the anemia seen in infections by H. contortus may decrease hemoglobin, which can probably interfere with the synthesis of nitrite, resulting in reduced nitrite/nitrate (NOx ) levels like in the present study. According to researchers, nitric oxide interactions with iron are the most important biological reactions in which NO participates, thus many iron proteins may have their activity modulated by NO in vivo [7], but the infection by H. contortus would reduce iron levels [3], due to blood loss, so that could interfere with the NOx levels as observed in the current study. Because NO is involved in various physiological mechanisms, other explanations for NOx reduction can be considered. A typical pathological finding in H. contortus infection is reduced albumin [26], and this hypoalbuminemia may affect the viscosity of blood and/or the function of endothelial cells [8,12,21], cells where is present the isoforms of NO, also called endothelial NOS (eNOS), involved in several vascular events. According to researchers, albumin serves as a reservoir for nitric oxide [25], therefore the hypoalbuminemia frequently observed in lambs infected by H. contortus can justify the reduction of NO. Another important point that may have interfered with the synthesis of NO was the of leukocyte reduction detected in the animals infected by H. contortus. According to the literature, decreased levels of NO production by inflammatory cells may result in lower ONOO− formation and consequently lower nitrotyrosine levels [19]. Briefly, this study aimed to detect NO levels, and oxidative markers and antioxidants in serum of lambs experimentally and naturally infected by H. contortus associated with disorders of abomasum. This study allowed us to conclude that during the infection by H. contortus there are alterations in oxidative markers (AOPP), indicators of cell lesion, and consequently occurs changes in antioxidant levels (FRAP), with the purpose of cellular protection. It is possible to conclude that infection by H. contortus caused injury or cell death, due to an increase in LDH in the serum, confirmed by histological examination of the abomasum by inflammatory infiltrate. We also conclude that helminth infection interferes with the metabolism of nitric oxide, which had reduced levels.
Ethics committee The procedure was approved by the Comissão de Ética no Uso de Animais (CEUA) from the Universidade Federal de Santa Maria (UFSM), under the number 012/2011.
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Original Article
Relationship between oxidative stress and pathological findings in abomasum of infected lambs by Haemonchus contortus Vanessa Machado a , Aleksandro S. Da Silva a,∗ , Andressa S. Schafer b , Adelina R. Aires b , Alexandre A. Tonin c , Camila B. Oliveira d , Carine L. Hermes e , Taís C. Almeida e , Rafael N. Moresco e , Lenita M. Stefani a , Sonia T.A. Lopes c , Ricardo E. Mendes f , Claiton I. Schwertz f , Marta L.R. Leal b a
Department of Animal Science, Universidade do Estado de Santa Catarina, Chapecó, SC, Brazil Laboratory of Animal Endocrinology and Metabolism, Program of Post-Graduation in Veterinary Medicine, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil c Department of Small Animal, UFSM, Santa Maria, RS, Brazil d Department of Microbiology and Parasitology, UFSM, Santa Maria, RS, Brazil e Department of Clinical and Toxicological Analysis, UFSM, Santa Maria, RS, Brazil f Laboratory of Veterinary Pathology, Instituto Federal Catarinense, Santa Maria, RS, Brazil b
a r t i c l e
i n f o
Article history: Received 11 July 2014 Received in revised form 17 August 2014 Accepted 16 September 2014 Keywords: Helminths LDH NOx FRAP AOPP
a b s t r a c t The aim of this study was to evaluate the nitric oxide (NO) levels, and oxidative and antioxidant markers of lambs experimentally and naturally infected by Haemonchus contortus, and its relation to lesions in the abomasum. For experimental study, a total of 14 healthy lambs were divided into two groups with seven animals each. Group A represented the uninfected animals (control), and Group B was formed by infected animals with 15,000 larvae of H. contortus. Blood was collected on days 15, 45, and 75 post-infection (PI) to obtain serum for biochemical analysis: lactate dehydrogenase (LDH), nitrite/nitrate (NOx ), advanced oxidation protein products (AOPP), and ferric reducing ability of plasma (FRAP). Parasitological stool examination (eggs per gram of feces – EPG) was performed on days 15, 45, and 75 PI to verify the evolution of the infection. On day 15 PI EPG was negative, but on days 45 and 75 PI the EPG was positive for animals from Group B. In the three periods evaluated it was observed an increase of LDH levels in serum of lambs infected with gastrointestinal nematodes, but on the other hand NOx levels were reduced on the same periods in infected animals. The AOPP and FRAP levels did not differ between groups on days 15 and 45 PI, but increased significantly on day 75 PI in infected lambs. The same variables were studied in 10 lambs naturally infected with helminths, where more than 97% corresponded to H. contortus (hematocrit and EPG values were 18.8 ± 2.5% and 7120 ± 2940, respectively). Similar to the experimental study, the levels of NOx reduced, and the levels of LDH, FRAP, and AOPP increased in serum of this animal associated inflammatory infiltrate in the mucosa of the abomasum. Therefore, during the infection by H. contortus it was observed alterations in oxidative markers, indicators of cell lesion confirmed by histological examination of the abomasum, and consequently there were changes in antioxidant levels, with the purpose of cell protection. We also conclude that helminth infection interferes with the nitric oxide metabolism. © 2014 Elsevier GmbH. All rights reserved.
Introduction Haemonchus contortus is known as one of the major Trichostrongylidae pathogens of small ruminants, with a worldwide
∗ Corresponding author. Tel.: +55 49 3322 4202; fax: +55 49 3322 4202. E-mail address: aleksandro [email protected] (A.S. Da Silva). http://dx.doi.org/10.1016/j.prp.2014.09.006 0344-0338/© 2014 Elsevier GmbH. All rights reserved.
distribution [9,27]. This nematode is typical of tropical and subtropical areas, where economic losses can be very high, although an increasing occurrence has been reported also in cold areas [2,32]. H. contortus penetrates the surface of the abomasal mucosa to feed on the blood of the host [20]. The systemic effects observed due to infection by this nematode are anemia and decreased levels of total plasma proteins, particularly albumin [6]. Besides the diseases directly caused by the infection, the presence of the parasite may
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induce production of potentially destructive factors [34], such as reactive oxygen species (ROS), which may damage the parasite but generate oxidative stress [24]. ROS are made up of a large number of chemically reactive molecules derived from oxygen. Among them there are superoxide radical, hydroxyl radical, and hydrogen peroxide. The superoxide radical is produced during the maximum activation of neutrophils, monocytes, macrophages and eosinophils in the presence of H. contortus infection [1,18,22]. Parameters have been used to determine oxidative stress, such as lipid peroxidation (TBARS), advanced oxidation protein products (AOPP), and nitric oxide (NO) [4,16,33]. A situation of oxidative stress causes cell death, and thus may occur an increase of lactate dehydrogenase (LDH). However, the balance between production of oxidant agents and antioxidant defense system is essential, as well as the reduction of glutathione, superoxide-dismutase, and catalase [16]. Other techniques can be used to mediate the antioxidant defenses, the ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”, this automatic method consists of a ferric to ferrous ion reduction at low pH, resulting in a colored ferrous–tripyridyltriazine complex. The FRAP provides an pool index of antioxidant with potential in biological fluids that can be evaluated within the technological reach of every laboratory, as well as the researcher interested in oxidative stress and its effects [5]. The occurrence of oxidative stress in infections by helminths has been described, but using techniques easily influenced by many factors. However, in the present study we proposed the use of automatic methods with high sensitivity. Also, the effect of infection by H. contortus on the metabolism of nitric oxide has not been elucidated. Therefore, the aim of this study was to evaluate the NO levels, and oxidative and antioxidant markers of lambs experimentally and naturally infected by H. contortus and its relation to lesions in the abomasum.
Materials and methods Experimental infection with H. contortus Animals and experimental design Banked serum and plasma samples from a previous study [9] were used in the current study. Our experiment used 14 male lambs crossbred (Corriedale-Texel), five months old and weighing in average 23 kg each. They were kept in holding pens (one pen/group) at the Veterinary Hospital of Universidade Federal de Santa Maria (UFSM) during 30 days under a diet [base of 10.7% protein (commercial feed and ryegrass hay)] for the adaptation in the experimental environment. In this period, the animals received anthelmintic treatment based on monepantel (Zolvix® ). The same diet was provided during the first 20 days of the experiment (postinfection), but after this period it was necessary to change the diet as a consequence of the severe evolution of the disease. From this period onward, the animals were fed with a mixture of hay ground ryegrass (70%), commercial feed with 20% crude protein (CP), and soybean meal with 10% (CP) [totaling a diet with 13% CP]. Each animal consumed 1 kg of dry matter/day. Hematological (erythrogram and leukogram) and biochemical (hepatic and renal function) evaluations were performed three times at 15-day intervals. After 30 days (day 0 of the experiment), the evaluated patterns showed normal values, according to Feldman et al. [13]. Apparently the animals were healthy, and they had negative fecal exam for eggs, cysts and oocysts of parasites. The animals were divided into two groups with seven animals each: Group A was composed by of healthy animals (uninfected) and it was used as a negative control group; Group B, the animals were infected by H. contortus, representing the positive control.
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Each animal from Group B was infected orally with a total of 15,000 larvae (L3), divided in three moments of infection of 5000 larvae each time, at intervals of 3 days between them. The larvae were obtained from the Laboratory of Animal Endocrinology and Metabolism of the Federal University of Santa Maria. They were obtained by coproculture technique [28]. Collection of samples Blood was drawn through Vacutainer® system on the days 15, 45, and 75 post-infection (PI). To measure the levels of NOx (nitrite/nitrate), blood samples were stored in tubes without anticoagulant. To measure the levels of AOPP and FRAP in plasma, blood samples were stored in tubes with anticoagulant (EDTA). To measure levels of LDH in the plasma, blood samples were stored in tubes containing EDTA and fluoride. To obtain the serum or plasma, blood samples were centrifuged (3500 g for 10 min). The serum/plasma was stored at −20 ◦ C and the plasma fluoride stored in the refrigerator (4 ◦ C) until analysis. Stool testing Fecal samples for quantification of eggs per gram (EPG) were collected on days 15, 45, and 75 PI, and processed according to the technique described by Gordon and Whitlock [15]. Five days after the end of the experiment (day 80 PI), five animals from each group were euthanized (10 mg intravenous (IV) of acepromazine; 2 g IV of sodium thiopental; 100 ml IV of potassium chloride), and their parasite loads were determined [31]. The euthanasia was necessary to confirm that the animals were infected (Group B) or negative (Group A), since the EPG by itself may not be suffice. Naturally infection with H. contortus In order to verify the experimental infection reflect a natural conditions, another study was conducted. For this, 10 lambs naturally infected with helminths (identified as group C), where more than 97% corresponded to H. contortus (result obtained by culture of larvae) were used. This lambs presents in mean hematocrit values [13] and EPG [15] the 18.8 ± 2.5% and 7120 ± 2940, respectively. Blood was drawn through Vacutainer® system similar experimental study to measure the levels of LDH, NOx AOPP and FRAP, according describe in “Collection of samples” this study. The results of biochemical analyzes in naturally infected animals (Group C) were compared with non-infected lambs, which corresponds to day 75 PI in group A. Necropsy of lambs that died was performed and observed that this abomasum filled with H. contortus (adults and larvae) (Fig. 1A), with fragments of the abomasum collected for histological analysis (fixed in buffered formalin 10% and stained with hematoxylin and eosin). Biochemical analyzes LDH, NOx , AOPP, and FRAP assays were performed using the automated Cobas Mira® . The LDH in plasma fluoride was measured within 5 days of blood collection using a commercial kit Bioclin® (kinetic method), and results were expressed as mg dL−1 . NO levels in the serum of lambs infected by H. contortus were analyzed indirectly by quantifying serum NOx according to the technique described in detail by Tatsch et al. [30], and results were expressed as mol L−1 . AOPP concentration (protein oxidation) was determined using the semiautomated method described by Hanasand et al. [17], and results were expressed as mol L−1 . Levels of FRAP was measured according to the technique described by automatically Benzie and Strain [5] that uses plasma. The results of FRAP were expressed as mol L−1 .
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Results Experimental infection with H. contortus Course of infection The animals used as control (Group A) did not show any clinical signs of the disease. However, infected lambs (Group B) showed pale-pink mucosa when checked on days 45 and 75 PI. Despite the fact that the animals were not weighted, weight loss was also observed on infected animals. Biochemical analyzes The results of LDH, NOx , AOPP, and FRAP are presented in Table 1. In the three periods evaluated it was observed a LDH increase in the serum of lambs infected with gastrointestinal nematodes, however NOx levels were reduced at the same periods in infected animals. The AOPP and FRAP levels did not differ between groups on days 15 and 45 PI, but increased significantly in lambs infected on day 75 PI when compared to uninfected animals. Eggs per gram of feces Animals from the control (Group A) showed negative EPG throughout the experiment. Examination at the necropsy confirmed that the lambs were negative for helminths. The animals in Group B showed negative EPG on day 15 PI, but in the other two analyzes EPG was positive for eggs of H. contortus at days 45 PI (9828 ± 5426 EPG) and 75 PI (4100 ± 2277 EPG). The mean parasite load in animals of Group B was of 2404 of H. contortus. Other helminths were not found during the necropsy of these animals. Naturally infection with H. contortus Histology abomasum of lambs infected with H. contortus was observed moderate focal lymphocytic inflammatory infiltrate in the mucosa (Fig. 1B) and moderate focal mixed inflammatory cell infiltrate in the mucosa (Fig. 1C). The results of the oxidant and antioxidant markers were shown in Fig. 2. An increase in the levels of LDH, AOPP (Fig. 2A), and FRAP (Fig. 2B) was observed in lambs naturally infected with H. contortus compared uninfected. Already, the levels of NOx significantly reduced (P < 0.01) in infected animals (Fig. 2B). Apparently these animals showed diarrhea and edema. But the ocular mucosa was pale, indicating anemia, confirmed by hematocrit. Discussion
Fig. 1. Histology abomasum of lambs infected by Haemonchus contortus (A) was observed moderate focal lymphocytic inflammatory infiltrate in the mucosa (B) and moderate focal mixed inflammatory cell infiltrate in the mucosa (C).
Statistical analysis All data were initially tested for normality, however EPG data did not show to be normal distributed, it was logarithm (log 10) transformed previously to statistical analysis. Then, the NOx , LDH, AOPP, FRAP, and EPG results were subjected to the Student test. The analyses were performed using SAS statistical package (SAS Institute, Cary, NC, USA) with a significance level of 5% (P < 0.05).
The hematological variables did not differ between groups on day 15 PI, different from the days 45 and 75 PI, when it was observed a reduction significant of hematocrit, total erythrocytes count, hemoglobin concentration, and total leukocytes in lambs infected when compared to not-infected the according a previous study [9]. Therefore, in the current study H. contortus infected lambs developed a clinical condition of anemia and leukopenia, associated with altered levels of antioxidants and oxidative markers, from the NO metabolism. The occurrence of oxidative stress in lambs experimentally infected by H. contortus has been reported, mainly due to the detection of lipid peroxidation and increased glutathione reductase activity [24,26]. This oxidative imbalance has been associated with severity of pathological conditions, as well as anemia, due to the ROS damage caused to the erythrocyte membrane (Wolkmer et al., 2009). Gastrointestinal nematode infection in H. contortus, anemia is caused by blood loss since this is a blood sucking parasite. However we cannot disregard the possibility that oxidative stress is contributing to the pathogenesis of this disease, similarly to what occurs in other parasitic diseases [29,35].
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Table 1 Experimental infection by Haemonchus contortus in lambs: mean and standard deviation of hematocrit, lactate dehydrogenase (LDH), nitrite/nitrate (NOx ), advanced oxidation protein products (AOPP), and ferric reducing ability of plasma (FRAP) on days 15, 45 and 75 post-infection. Parameters
Day −1
LDH (mg dL
)
15 45 75
Group A: Control 8.6 ± 3.3 9.0 ± 3.0 9.9 ± 3.4
Group B: Infected
Probability (P)#
17.4 ± 2.8 14.7 ± 4.0 15.9 ± 4.2
P < 0.01# P < 0.01# P < 0.05#
52.8 ± 14.5 49.6 ± 22.3 55.5 ± 30.6
P < 0.05# P < 0.01# P < 0.01#
NOx (mol L−1 )
15 45 75
141.0 ± 75.5 103.6 ± 38.2 116.1 ± 40.2
AOPP (mol L−1 )
15 45 75
14.5 ± 1.1 14.3 ± 3.7 15.3 ± 4.6
16.1 ± 2.3 13.6 ± 3.3 24.0 ± 3.6
P > 0.05 P > 0.05 P < 0.05
FRAP (mol L−1 )
15 45 75
295.9 ± 36.3 298.6 ± 42.0 283.0 ± 44.2
265.1 ± 28.9 262.6 ± 86.6 339.2 ± 61.6
P > 0.05 P > 0.05 P < 0.05#
Hematocrit (%)*
15 45 75
30.7 ± 2.2 30.6 ± 5.1 37.5 ± 2.8
28.5 ± 2.8 22.0 ± 4.7 24.8 ± 5.3
P > 0.01 P < 0.01# P < 0.01#
# *
In the same line, was considered significant difference between groups in the Student test, when presenting P < 0.05 or P < 0.01. Results hematocrit was presented in a previous study [9].
Experimentally and naturally infected lambs showed increased levels of LDH in this study, an enzyme involved in the metabolism of carbohydrates and generally altered in cardiac, hepatic and parasitic disease [23], a parameter indicator of hypoxia, ischemia, and cellular injury. All these changes could be related to increased LDH, because the animals were anemic, probably having lower tissue oxygenation as well as could be occurring lesions of blood cells as mentioned
earlier, due to the condition of oxidative imbalance installed in lambs. The animals in experimental study had a classic case of acute infection, with great loss of blood, which made it necessary to increase energy and protein in the diet of these animals on day 20 PI. Despite the anemia and loss of body condition observed, other differences between the groups regarding the markers of oxidative imbalance in the days 15 and 45 PI were not seen. Already on day
Fig. 2. Levels of lactate dehydrogenase (LDH), advanced oxidation protein products (AOPP), nitrite/nitrate (NOx ), and ferric reducing ability of plasma (FRAP) in lambs naturally infected with Haemonchus contortus when compared uninfected.
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75 PI, there was an increase in the AOPP levels, an indicator of protein oxidation, and it might be considered as true mediator of the pro-inflammatory effects of oxidative stress [33], similar naturally infected lambs. The protein oxidation was probably associated with the increase in FRAP levels, an indicator of total antioxidant capacity of plasma, as a result of the increase of antioxidant enzymes to minimize the effects of ROS produced during infection by H. contortus. Therefore, there is a situation of oxidative imbalance which can influence various physiological mechanisms as well as the actual reduction in the metabolism of nitric oxide seen in lambs. As stated in the literature, in the state of oxidative stress, the production of ROS exceeds the available antioxidant defense systems, and consequence the bioactivity of NO is reduced [14]. A dominant mechanism reducing bioavailability of vascular NO relates to its rapid oxidative inactivation by the ROS superoxide (O2 − ), and there is evidence that persisting oxidative stress will render eNOS dysfunctional such that it no longer produces NO, but O2 − [14]. As already mentioned, the serum levels of NOx reduced in lambs infected by H. contortus, a fact that may be associated with factors other than those mentioned above, such as the reduction of hemoglobin, albumin, iron, as well as its own oxidative stress, the findings described in lambs infected with H. contortus [13]. Studies have established a relation between the occurrence of anemia associated with reduced nitric oxide [11], situation observed in the present study. According to literature, the nitrite ion is a direct causative agent for methemoglobinemia, that is the oxidation of hemoglobin to methemoglobin under aerobic conditions is induced by nitrite [10]. Furthermore, the nitrite production is dependent on the ratio of nitrous oxide and oxygen-hemoglobin [10]. Therefore, the anemia seen in infections by H. contortus may decrease hemoglobin, which can probably interfere with the synthesis of nitrite, resulting in reduced nitrite/nitrate (NOx ) levels like in the present study. According to researchers, nitric oxide interactions with iron are the most important biological reactions in which NO participates, thus many iron proteins may have their activity modulated by NO in vivo [7], but the infection by H. contortus would reduce iron levels [3], due to blood loss, so that could interfere with the NOx levels as observed in the current study. Because NO is involved in various physiological mechanisms, other explanations for NOx reduction can be considered. A typical pathological finding in H. contortus infection is reduced albumin [26], and this hypoalbuminemia may affect the viscosity of blood and/or the function of endothelial cells [8,12,21], cells where is present the isoforms of NO, also called endothelial NOS (eNOS), involved in several vascular events. According to researchers, albumin serves as a reservoir for nitric oxide [25], therefore the hypoalbuminemia frequently observed in lambs infected by H. contortus can justify the reduction of NO. Another important point that may have interfered with the synthesis of NO was the of leukocyte reduction detected in the animals infected by H. contortus. According to the literature, decreased levels of NO production by inflammatory cells may result in lower ONOO− formation and consequently lower nitrotyrosine levels [19]. Briefly, this study aimed to detect NO levels, and oxidative markers and antioxidants in serum of lambs experimentally and naturally infected by H. contortus associated with disorders of abomasum. This study allowed us to conclude that during the infection by H. contortus there are alterations in oxidative markers (AOPP), indicators of cell lesion, and consequently occurs changes in antioxidant levels (FRAP), with the purpose of cellular protection. It is possible to conclude that infection by H. contortus caused injury or cell death, due to an increase in LDH in the serum, confirmed by histological examination of the abomasum by inflammatory infiltrate. We also conclude that helminth infection interferes with the metabolism of nitric oxide, which had reduced levels.
Ethics committee The procedure was approved by the Comissão de Ética no Uso de Animais (CEUA) from the Universidade Federal de Santa Maria (UFSM), under the number 012/2011.
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