INTRODUCTION
Haemonchus contortus is the main helminth of sheep in Brazil (Cavalcante et al. Reference Cavalcante, Vieira, Chagas and Molento2009), and is also considered endemic in Australia, South Africa and South America, as well as other countries (Waller et al. Reference Waller, Dash, Barger, Le Jambre and Plant1995). Animals parasitized may exhibit weight loss, dehydration, diarrhoea and anaemia during the acute phase. In hyperacute infections it is possible to observe pale mucous membranes and even the death of some animals. In the chronic phase the clinical signs usually intensify, often allowing the observation of ventral and submandibular swelling, weakness and apathy. Infected animals can present with mucosa of abomasums swollen, oedematous, anaemic and bright; as well as small ulcers at the site of attachment of H. contortus (Cavalcante et al. Reference Cavalcante, Vieira, Chagas and Molento2009).
Several factors are involved in the pathogenesis of infection by H. contortus. For the development of the disease, pathogenicity of the parasite and host response are among the most important factors. The main pathogenic mechanisms of H. contortus are the direct injury on the gastric mucosa and the haematophagia. The immune response is complex through cellular and humoral mechanisms (McClure et al. Reference McClure, Davey, Emery, Colditz and Lloyd1996; Meeusen, Reference Meeusen1999), which may vary depending on the stage of the parasite (Balic et al. Reference Balic, Bowles and Meeusen2000).
Among the several inflammatory mechanisms, purinergic signalling is well documented (Atkinson et al. Reference Atkinson, Dwyer, Enjyoji and Robson2006; Bours et al. Reference Bours, Swennen, Di Virgilio, Cronstein and Dagnelie2006; Yegutkin, Reference Yegutkin2008). It represents a common route of cell-to-cell communication involved in many physiological functions, such as immune response, inflammation, pain, platelet aggregation, vasodilation, proliferation and cellular death (Burnstock and Knight, Reference Burnstock and Knight2004). This system consists of nucleotides (ATP, ADP and AMP) and nucleosides (adenosine and inosine), representing signalling molecules that are involved in activating the immune response (Atkinson et al. Reference Atkinson, Dwyer, Enjyoji and Robson2006; Yegutkin, Reference Yegutkin2008). The ATP has proinflammatory functions, since it controls the stimulation and proliferation of lymphocytes, cells involved in cytokine release (Bours et al. Reference Bours, Swennen, Di Virgilio, Cronstein and Dagnelie2006). However, the endogenous nucleoside ‘adenosine’ is formed extracellularly (Ralevic and Burnstock, Reference Ralevic and Burnstock1998) and presents itself as an anti-inflammatory molecule (Gessi et al. Reference Gessi, Merighi, Varani, Cattabriga, Benini, Mirandola, Leung, Mac Lennan, Feo, Baraldi and Borea2007).
The activity of ecto-adenosine deaminase (E-ADA) has been recently investigated in lambs infected with H. contortus. This enzyme is responsible for the deamination of adenosine, and when measured in erythrocytes, it was found a co-relation between the enzyme activity and anaemia (Da Silva et al. Reference Da Silva, Schafer, Aires, Tonin, Pimentel, Oliveira, Zanini, Schetinger, Lopes and Leal2013b ). Therefore, this study aimed to assess the purines levels in serum of lambs experimentally infected with H. contortus.
MATERIALS AND METHODS
Animals
Our experiment used 12 male lambs crossbred Corriedale×Texel, 5 months old and weighing on average 23 kg each. They were kept in holding pens (one pen/group) during 30 days under a diet [base of 10·7% protein (commercial feed and ryegrass hay)] for adaptation to 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 (post-infection), 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) [totalling a diet with 13% CP]. Each animal consumed 1 kg of dry matter/day. Haematological (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. (Reference Feldman, Zinkl and Jain2000). The animals were apparently healthy, and they had negative fecal exam for eggs, cysts and oocysts of parasites.
Experimental design
The animals were divided into two groups with 6 animals each: Group A was composed of healthy animals (uninfected) and was used as a negative control group; Group B comprised the animals infected by H. contortus, representing the positive control. Each animal from group B was infected orally with a total of 15 000 larvae (L3), divided in three episodes of infection of 5000 larvae each time, at intervals of 3 days between them. The larvae were obtained by coproculture technique (Roberts and O'Sullivan, Reference Roberts and O'Sullivan1950).
Collection of samples
Blood was drawn through a Vacutainer® system on days 15, 45 and 75 PI. To measure the levels of purines, blood samples were stored in tubes without anticoagulant; and to hematocrit the samples was collected and stored in tubes with anticoagulant (EDTA). The determination of microhaematocrit was performed according to the technique described by Feldman et al. (Reference Feldman, Zinkl and Jain2000). To obtain the serum, blood samples were centrifuged (5000 g for 5 min at 37 °C). The serum was stored at −20 °C until analysis.
Analysis of purine levels in serum by high pressure liquid chromatography (HPLC)
The denaturation of sample proteins was performed using 0·6 mol L−1 perchloric acid. All samples were then centrifuged (14 000 g for 10 min at 4 °C) and the supernatants were neutralized with 4·0 N KOH and clarified with a second centrifugation (14 000 g for 15 min at 4 °C). Aliquots of 20 μL were applied to a reversed-phase HPLC system (Shimadzu, Japan) using a C18 column (Ultra C18, 25 cm × 4·6 mm×5 μ m, Restek – USA). The elution was carried out applying a linear gradient from 100% of solvent A (60 mm KH2PO4 and 5 mm of tetrabutylammonium phosphate, pH 6·0) to 100% of solvent B (solvent A plus 30% methanol) over a 30 min period (flow rate at 1·4 mL min−1) according to a method previously described (Voelter et al. Reference Voelter, Zech, Arnold and Ludwig1980) with minor modifications. The amounts of purines were measured by absorption at 260 nm. The retention time of standards was used as parameter for identification and quantification. Purine concentrations are expressed as nmol of the different compounds per mL of serum.
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 (Reference Gordon and Whitlock1939). Five days after the end of the experiment (day 80 PI), 5 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 (Ueno and Gonçalves, Reference Ueno and Gonçalves1998). 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 sufficient.
Statistical analysis
EPG data were initially tested for normality, however they did not present a normal distribution and they were converted to logarithm before statistical analysis. Then, the purines and hematocrit results were subjected to the Student's t-test. Values with probability (P) less than 5% were considered statistically different.
RESULTS
Infection course
The animals of Group A (negative control) showed negative EPG throughout the experiment; necropsy confirmed that the lambs were negative for helminths. The animals in group B (positive control) showed negative EPG on day 15 PI, but in the other two analyses EPG was positive for eggs of H. contortus, with a mean (s.d.) of 9828 EPG (±5426) at day 45 PI and 4100 EPG (±2277) at day 75 PI. Other helminths were not found during the necropsy of these animals.
The hematocrit was assessed to check the course of the disease in animals, and the results are shown in Fig. 1. On day 15 PI there were no significant (P>0·05) differences between groups in hematocrit. However, a significant (P<0·01) reduction in hematocrit were observed in the infected lambs on days 45 and 75 PI.
Fig. 1. Haematocrit of lambs experimentally infected with Haemonchus contortus assessed on days 15, 45 and 75 post-infection (*P<0·01).
Purine levels in serum
Results of the purine levels are shown in Table 1. On day 15 PI a significant (P<0·05) increase in the levels of ATP and inosine was observed, concomitant with reduced levels of ADP, adenosine, xanthine and uric acid levels in the infected animals when compared with the negative control group. However, on day 45 PI group B showed a significant (P<0·05) increase in the levels of ATP and xanthine, together with reduced levels of ADP and uric acid. Finally, on day 75 PI there was an increase in the levels of ATP, adenosine and hypoxanthine, as well as reduction in levels of ADP and uric acid (P<0·05) in experimentally infected lambs. AMP levels did not differ between groups throughout the experiment.
Table 1. Mean and s.d. of purine levels in serum of lambs uninfected and infected with gastrointestinal nematode (H. contortus) on days 15, 45 and 75 post-infection
#In the same line, indicates a significant difference between groups in the Student's t-test, when presenting P<0·05 or P<0·01.
DISCUSSION
According to the results found in our study, at 15 days PI the animals tested negative for the presence of eggs in their feces. This can be attributed to the pre-patent period of H. contortus, which is on average 14–21 days (Cavalcante et al. Reference Cavalcante, Vieira, Chagas and Molento2009). The literature explains that the penetration of the fourth stage larvae (L4) in the abomasum of the host occurs during the first week of infection, followed by subsequent changes to the fifth stage larvae (adults) (Monteiro, Reference Monteiro2010). During this period a response of the host against the infection can be observed, since the L4 perform haematophagy, allowing rapid growth and metabolic activity. Consequently, L4 excrete immunogenic products, as well as proteases and digestive enzymes, besides causing direct damage to the abomasum (Gamble and Mansfield, Reference Gamble and Mansfield1996), leading to an immune response. A Th2-type response to infection by helminths such as H. contortus is generally stimulated (Miller and Horohov, Reference Miller and Horohov2006).
In our study, during the three evaluated periods there was an increase in the serum levels of ATP. It is well known that ATP is an important molecule for the functioning of the cells, mainly related to energy storage for basic life activity of the cells. In the event of cellular injury or cell stimulation by pathogens, the concentration of extracellular ATP increases, initiating an inflammatory response characterized by the stimulation of leucocytes and secretion of various inflammatory mediators such as cytokines (Langston et al. Reference Langston, Ke, Gewirtz, Dombrowski and Kapp2003; La Sala et al. Reference La Sala, Ferrari, Di Virgilio, Idzko, Norgauer and Girolomoni2003). Therefore, the immune response against infection with H. contortus in sheep is mediated by ATP, a molecule that whose function is well documented (Langston et al. Reference Langston, Ke, Gewirtz, Dombrowski and Kapp2003; La Sala et al. Reference La Sala, Ferrari, Di Virgilio, Idzko, Norgauer and Girolomoni2003; Bours et al. Reference Bours, Swennen, Di Virgilio, Cronstein and Dagnelie2006). The higher ATP levels on day 15 PI compared with the days 45 and 75 PI in infected animals can be related to an overreaction of the host against the infection, which occurs during the parasitism by larvae in the abomasal mucosa of the host, reflecting an acute inflammatory response (ATP molecule acute phase; Bours et al. Reference Bours, Swennen, Di Virgilio, Cronstein and Dagnelie2006).
Similar to the ATP pattern, on day 15 PI there was an increase in the levels of inosine, unlike what happened with the levels of ADP and adenosine which were reduced in the serum of infected lambs. The findings may be related to reduction of NTPDase activity, which consequently causes a reduction in ATP hydrolysis to ADP, and an increase in the activity of the E-ADA, leading to an increase in the adenosine deamination to inosine (Yegutkin, Reference Yegutkin2008). However, in a study with a similar design, a reduction in E-ADA in serum of lambs infected with H. contortus was observed (Da Silva et al. Reference Da Silva, Fausto, Grando, Cadore, Pimentel, Jaques, Schetinger, Monteiro and Leal2013a ). In another study, a negative correlation was verified between E-ADA activity in erythrocytes of lambs infected by H. contortus, and therefore, the researchers showed that E-ADA had participated in the pathogenesis of anaemia (Da Silva et al. Reference Da Silva, Schafer, Aires, Tonin, Pimentel, Oliveira, Zanini, Schetinger, Lopes and Leal2013b ). In our study, adenosine levels fluctuated during infection, reducing on day 15 PI, increasing on day 45 PI (but did not differ from the control group), and on day 75 PI there was a large increase of adenosine in serum. The high concentration of adenosine probably acted as an anti-inflammatory mechanism, providing an immunomodulatory effect against the chronicity of the disease (Sala-Newby et al. Reference Sala-Newby, Skladanowski and Newby1999; Sawynok and Liu, Reference Sawynok and Liu2003). Deamination of adenosine to inosine is favourable for the maintenance and survival of invading organisms, since adenosine promotes chemotaxis, activation and degranulation of mast cells (Jin et al. Reference Jin, Shepherd, Duling and Linden1997; Gounaris and Selkirk, Reference Gounaris and Selkirk2005). Studies have shown that mast cells are mucosal effectors against various intestinal nematodes such as Trichinella spiralis, and thus assist in eliminating the parasite (Knight et al. Reference Knight, Wright, Lawrence, Paterson and Miller2000). It is important to emphasize that adenosine inhibits platelet aggregation and it has vasodilator activity, and these events are favourable to the survival of blood-sucking parasites. Researchers have already shown that adenosine is involved in a response to the stimulation of cellular and tissue damage, protecting against organ damage (Newby, Reference Newby1984). A recent study reported that adenosine assists in the maintenance of tissue integrity by modulating immune system function (Haskó and Cronstein, Reference Haskó and Cronstein2004). Different types of cells are able to produce extracellular adenosine, some examples being endothelial cells and neutrophils which are constantly co-related to high levels of adenosine in areas of inflammation (Cronstein, Reference Cronstein1994).
Haemonchus contortus is a blood-sucking parasite, with mechanisms to neutralize the haemostatic system of the host, such as anti-platelet and anticoagulant proteins (Seymour et al. Reference Seymour, Henzel, Nevins, Stults and Lazarus1990; Stanssens et al. Reference Stanssens, Bergum, Gansemans, Jespers, Laroche, Huang, Maki, Messens, Lauwereys, Cappello, Hotez, Lasters and Vlasuk1996; Depraetere et al. Reference Depraetere, Kerekes and Deckmyn1999; Francischetti et al. Reference Francischetti, Ribeiro, Champagne and Andersen2000; Crab et al. Reference Crab, Noppe, Pelicaen, Hoorelbeke and Deckmyn2002). In this study, we observed a reduction in the level of ADP in all analysed periods in the infected group; however this reduction was more pronounced on days 45 and 75 PI, coinciding with a large number of parasite eggs in the feces. The reduction in ATP hydrolysis (involved in the inflammatory response) can be the main cause of reduction in serum ADP in lambs infected with H. contortus. This reduction in ADP probably favours the parasite, because it hinders platelet aggregation and formation of a homeostatic plug, and thus facilitates the feeding of the parasite.
Hypoxanthine, xanthine and uric acid are products of catabolism of purines (Chen and Gomes, Reference Chen and Gomes1992). In ruminants most of the uric acid comes from the diet and in extension, from the breakdown of endogenous nucleic acid (González and Silva, Reference González and Sílva2006). Thus, the a lower intake and/or malabsorption of nutrients (proteins, vitamins and minerals), a situation previously reported due to infection by H. contortus, can result in lower blood levels of uric acid. Our results showed reduction of uric acid in all evaluated periods. Researchers have emphasized that infected animals require higher protein intake compared with healthy animals, mainly due to the loss of endogenous nitrogen into the intestine and the low degree of protein synthesis in muscle (Veloso et al. Reference Veloso, Louvandini, Kimura, Azevedo, Enoki, França, McManus, Dell'porto and Santana2004). Thus, the ingested protein promotes the restoration of tissue loss, repairing and replacing damaged tissues.
The chronicity of infection by H. contortus in the animals of group B led to an increase in the levels of xanthine and hypoxanthine. These results may be related to a possible reduction in the activity of enzymes such as xanthine oxidase and xanthine oxidoreductase, responsible for the catabolism of hypoxanthine to xanthine and xanthine to uric acid, the latter a potent antioxidant (Haskó et al. Reference Haskó, Sitkovsky and Szabó2004). It is important to highlight that increased levels of hypoxanthine may be due to cell damage during ischaemia (Shahbazian et al. Reference Shahbazian, Mombini, Moghaddam, Jasemi, Hosseini and Vaziri2006). It is worth reporting that anaemia is the main pathological symptom observed in infections by H. contortus, and when the parasite is ingesting blood, it can cause ischaemia or bleeding after its detachment from the abomasal mucosa. Consequently, it may be related to the increase of hypoxanthine and xanthine observed in this study.
We conclude that infection by H. contortus causes changes in purine levels in lambs. Due to all the functions of the purines aforementioned, we suggest that these changes in concentrations of ATP, ADP, adenosine, inosine, hypoxanthine, xanthine and uric acid may influence the pathophysiology of disease and the host immune response against the parasite. It is believed that the increase in serum levels of ATP and adenosine may be related to the inflammatory response, influencing the release of pro-inflammatory and anti-inflammatory molecules, respectively. Changes in the levels of certain nucleotides such as ADP appear to be linked to homeostatic factors, resulting in massive blood loss, leading to anaemia, a major clinical sign of disease.
ETHICAL PROCEDURES
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.
