INTRODUCTION
Trypanosoma evansi (T. evansi) is the most widely distributed pathogenic trypanosome, affecting domesticated animals (Joshi et al. Reference Joshi, Shegoka, Powar, Herder, Katti, Salkar, Dani, Bhargava, Jannin and Truc2005) and the principal host species varies geographically. Although other animals, including wildlife, are susceptible to T. evansi infection, buffaloes, cattle, camels and horses are particularly affected (World Organization for Animal Health, 2008). Trypanosoma evansi disease (Surra) occurred in acute and chronic forms, however, chronic form is the most common one and characterized by anaemia, emaciation, lachrymation, lymphadenitis and sometimes abortions (Gutierrez et al. Reference Gutierrez, Corbera, Juste, Doreste and Morales2005). Many investigators have shown alterations in haematology of camels infected with trypanosomosis (Chaudhary and Iqbal, Reference Chaudhary and Iqbal2000; Saleh et al. Reference Saleh, Al-Salahy and Sanousi2009). Recently, chronic camel trypanosomosis was associated with oxidative stress and lipid peroxidation (Saleh et al. Reference Saleh, Al-Salahy and Sanousi2009; El-Deeb and Elmoslemany Reference El-Deeb and Elmoslemany2015). Trypanosomosis in camels resulted in significant increase of malondialdehyde, significant reduction of superoxide dismutase and non-significant increase in catalase compared with the control values (Saleh et al. Reference Saleh, Al-Salahy and Sanousi2009). Upon infection, neutrophils responded to a series of reactions (Soethout et al. Reference Soethout, Muller and Rutten2002) which involved phagocytosis, elevating antibodies titre, complement fixation and stimulation of reactive oxygen and nitrogen free radicals release (Segal, Reference Segal2005). In addition, proinflammatory cytokines (tumour necrosis factor-α, interleukins 1β, IL-12 and interferon gamma) produced from monocytes and macrophages mediated the effect of acute phase proteins and favoured T-helper cell differentiation. Acute phase proteins (APP) either positive (upregulated; haptoglobin, serum amyloid A, fibrinogen) or negative (downregulated; albumin, transferring and α-fetoproteins) released in response to the challenge (Eckersall and Bell, Reference Eckersall and Bell2010). Induction of positive APP in hepatocytes by cytokines was accompanied by changes in lipid profile in the form of higher serum triacylglycerol and lower high density lipoprotein cholesterol levels (Cabana et al. Reference Cabana, Siegel and Sabesin1989). The publications regarding the estimation of the oxidative stress status in T. evansi infection in camels are lack (Saleh et al. Reference Saleh, Al-Salahy and Sanousi2009) and additional studies are required. In addition, data concerning acute phase proteins, lipid profile and proinflammatory cytokines in dromedary camels infected with trypanosomosis are scarce yet. Therefore, the present study aimed to determine the role of lipid profiles, oxidative stress biomarkers, acute phase proteins and proinflammatory cytokines as biomarkers for T. evansi in dromedary camels.
MATERIALS AND METHODS
Animals and sampling protocol
Sixty dromedary camels were assigned equally into two groups. Camels in the first group were healthy and free of trypanosomosis and served as control. Camels in the second group were infected naturally with T. evansi. Diagnosis of T. evansi infection was based on the presence of the parasite in the fresh heparinized blood using wet-blood film and micro-Haematocrit technique (Woo, Reference Woo1969). Positive camels were also examined by stained blood smears (Diff-Quick®). Moreover, latex agglutination test was applied on all examined camels according to the method described by (Olaho-Mukani et al. Reference Olaho-Mukani, Nyanga and Ouma1996). Blood samples were collected from the jugular vein of both groups into plain and ethylenediaminetetraacetic acid (EDTA) vacutainers. Plasma was used for measurement of fibrinogen. Sera were harvested and stored at −20 °C until assayed for triacylglycerol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, total cholesterol, beta hydroxyl butyric acids, non-esterified fatty acids, superoxide dismutase, catalase, reduced glutathione, malondialdehyde, haptoglobin, serum amyloid A, ceruloplasmin, tumour necrosis factor-α, interleukins-1α, 1β, 6, 10 and interferon gamma.
Analysis of samples
Serum triacylglycerol, low density lipoprotein cholesterol, high density lipoprotein cholesterol and total cholesterol were measured on a Beckman CX-7 autoanalyser using commercial kits (Sigma Chemical Co. Ltd., Poole, Dorset, UK). Serum concentration of beta hydroxyl butyric acids was determined by using a commercial kit (Randox, UK). Serum concentration of non-esterified fatty acids was carried out using commercial kits (Randox, UK). Fibrinogen concentration in plasma was measured with a commercial enzyme-linked immunosorbent assay (ELISA) kit (USCA, Life Science) according to the manufacturer's instructions. Serum haptoglobin was measured with a commercial ELISA kit (Phase SAA kit, Tridelta Ltd., Ireland). Serum amyloid A was measured with a commercial ELISA kit (Phase SAA kit, Tridelta Ltd., Ireland), according to the manufacturer's instructions. Ceruloplasmin activity was measured by using commercial kit (MyBiosource, USA). Levels of malondialdehyde, reduced glutathione, catalase and superoxide dismutase activities were determined using commercial ELISA Kits (Cayman, USA). Interleukins-1α, 1β, 6, 10, tumour necrosis factor-α and interferon gamma levels were determined from undiluted serum samples using commercial ELISA Kits (Biosource, Belgium).
Statistical analysis
All the data were presented as mean ± standard error of mean by using one way analysis of variance (ANOVA). All tests were performed using computer package of the statistical analysis system (SAS, 2002).
Ethics statement
All the animal procedures were performed according to the guidelines of the Animal Ethics Committee of College of Veterinary Medicine and Animal Resources, King Faisal University, Saudi Arabia (committee protocol number: 42/1943).
RESULTS
Clinical examination
The diseased camels showed progressive anaemia, emaciation marked depression, recurrent fever, atrophy of the thigh muscles dullness, corneal opacity, diarrhoea, oedema of the dependent parts, loss of condition and some cases showed nervous signs like trembling and unusual aggressiveness.
Lipid profiles
The data shown in the same table (Table 1) revealed a significant reduction (P ⩽ 0·05) in the values of triacylglycerol, cholesterol and high density lipoprotein cholesterol with significant elevation (P ⩽ 0·05) of low density lipoprotein cholesterol, beta hydroxyl butyric acids and non-esterified fatty acids concentrations in serum of infected camels compared with the control.
Lipids profiles in control (n = 30), and Trypanosoma evansi infested camels (n = 30)
* Means are significantly different at the level (P ⩽ 0·05) when compared with the control.
Oxidative stress, acute phase proteins and cytokines analysis
The results shown in Table 2 indicated that, the activities of superoxide dismutase, catalase and reduced glutathione level were reduced significantly (P ⩽ 0·05) in infected camels compared with the control whereas, lipid peroxidation was increased significantly (P ⩽ 0·05) as reflected on higher malondialdehyde value in the serum of infected camels compared with the control. The present study showed also that acute phase proteins namely haptoglobin, serum amyloid A, ceruloplasmin and fibrinogen were significantly (P ⩽ 0·05) increased in the serum of infected camel compared with the control (Table 3). The data shown in Table 4 indicated that, the values of all proinflammatory cytokines namely, interleukins-1α, 1β, 6, 10, tumour necrosis factor-α and interferon gamma were increased significantly (P ⩽ 0·05) in T. evansi infected camels compared with the control.
The levels of oxidative stress biomarkers and lipid peroxidation in control (n = 30), and Trypanosoma evansi infested camels (n = 30)
* Means are significantly different at the level (P ⩽ 0·05) when compared with the control.
Acute phase proteins in control (n = 30), and Trypanosoma evansi infested camels (n = 30)
* Means are significantly different at the level (P ⩽ 0·05) when compared with the control.
Proinflamatory cytokines in control (n = 30), and Trypanosoma evansi infested camels (n = 30)
* Means are significantly different at the level (P ⩽ 0·05) when compared with the control.
DISCUSSION
The significant decrease of triacylglycerol, total cholesterol and high density lipoprotein cholesterol in trypanosome infected camels compared with the control agrees with that reported in Trypanosoma congolense and Trypanosoma brucei infection of goats (Biryomumaisho et al. Reference Biryomumaisho, Katunguka-Rwakishaya and Rubaire-Akiiki2003), in T. congolense infection of sheep (Adamu et al. Reference Adamu, Ige, Jatau, Neils, Useh, Bisalla, Ibrahim, Nok and Esievo2008), in Trypanosoma rhodesiense infections of cattle (Wellde et al. Reference Wellde, Rearson, Kovatch, Chumo and Wykoff1989). As trypanosomes require lipoproteins for multiplication in vitro (Black and Vanderweed, Reference Black and Vanderweed1989), the hypolipidemia and hypocholesterolemia observed in the present study might be due to the utilization of the lipid molecules by trypanosome (Admau et al. Reference Adamu, Barde, Abenga, Useh, Ibrahim and Esievo2009). Blood-stream forms of trypanosomes require cholesterol, phospholipids and total lipids for membrane biosynthesis and maintaining growth (Nok et al. Reference Nok, Nock and Bonire2003). However, the parasites are not able to biosynthesize such molecules and depend upon the host to fulfil this task. During the journey of trypanosomes in the blood stream, they utilize blood glucose as a source of energy (Chaudhuri et al. Reference Chaudhuri, Ott and Hill2006), which could ultimately lead to hypoglycemia in the trypanosome-infected animal. Because of depletion of glucose caused by trypanosome, the body of the infected animal switches on lipid catabolism as an obligation mechanism for energy supply. In the present study, lipid catabolism leads to an increase in the level of free fatty acids that undergoes β-oxidation and gives rise to tremendous amount of ketone bodies (beta hydroxyl butyric acids) as noticed in the serum of infected camels. The significant decrease in serum cholesterol of infected camels may attribute to impaired cholesterol biosynthesis as a result of mechanical liver injury by trypanosome (Logan-Henfrey et al. Reference Logan-Henfrey, Gardiner, Mahmoud, Kreier and Baker1992) or insufficient respiration of liver cells due to hypoxia caused by anaemia in infected camels as discussed above. As cholesterol is vital in cell signalling in neuronal synapses formation, its decrease along with lipids could induce the observed neurological disorders.
Parallel to the present study, earlier researches demonstrated higher level of malondialdehyde in naturally infected camel with T. evansi (Saleh et al. Reference Saleh, Al-Salahy and Sanousi2009). The decrease in superoxide dismutase and catalase activities may postulate to the consumption of antioxidants as free radical scavengers during the oxidative stress in the naturally chronic T. evansi infected camels (Saleh et al. Reference Saleh, Al-Salahy and Sanousi2009). Consistent with the present results, chronic T. evansi infection in camels resulted in inhibition of the antioxidants (albumin, ascorbic acid, reduced glutathione and superoxide dismutase) (Saleh et al. Reference Saleh, Al-Salahy and Sanousi2009). The decrease in glutathione level in the serum of infected camels was reflected on the observed reduction in the activities of superoxide dismutase and catalase enzymes as discussed above. The significant increase in haptoglobin, serum amyloid A, ceruloplasmin and fibrinogen levels in T. evansi infected dromedary camels is consistent with previous result (Eckersall et al. Reference Eckersall, Gow, McComb, Bradley, Rodgers, Murray and Kennedy2001) in mice experimentally infected with T. brucei brucei.
The significant elevation of acute phase proteins in infected dromedary camels may attribute to the initial secretion of pro-inflammatory cytokines by macrophages at infection site. The increased levels of selected cytokines may attribute to liver necrosis, spleen necrosis, generalized lymphoid tissue hyperplasia (Enwezor and Sackey, Reference Enwezor and Sackey2005). These lesions induce the release of cytokines such as interleukins-1, interleukins-6 and tumour necrosis factor-α and therefore, acute phase proteins are biosynthesized in liver (Radostits et al. Reference Radostits, Gay, Hinchcliff and Constable2007). Similar results were observed in mice experimentally infected with T. brucei brucei (Eckersall, et al. Reference Eckersall, Gow, McComb, Bradley, Rodgers, Murray and Kennedy2001). Recently, it has been suggested that, elevated levels of interleukins-1, tumour necrosis factor-α and interferon gamma in T. evansi infected rats participated in the development of anaemia (Paim et al. Reference Paim, Duarte, Costa, Da Silva, Wolkmer, Silva, Paim, França, Mazzanti, Monteiro and Krause2011). This increase was associated with the regulation of immune responses against the parasite. In addition, pro-inflammatory cytokines particularly tumour necrosis factor-α have been shared in mediating anaemia associated with tropical theileriosis (Graham et al. Reference Graham, Brown, Vatansever, Waddington, Taylor, Nichani, Campbell, Adamson, Glass and Spooner2001).
The present study concluded that, T. evansi natural infection reflected on the infected camels in the form of several clinical signs are represented by depression, recurrent fever, corneal opacity, diarrhoea and loss of condition. These clinical signs resulted from a series of biochemical reactions inside the body of the infected camels. The parasitaemia induced a state of oxidative stress in infected camels as reflected on significant elevation of measured oxidative stress biomarkers. The body of infected camels responds to this stressful situation by three ways. Firstly, triacylglycerol has been mobilized and the produced fatty acids have been oxidized to provide an extra energy to the body of infected camel as reflected on significant decrease of triacylglycerol, cholesterol and high density lipoprotein cholesterol with significant increase of free fatty acids and ketone bodies. Secondly, the activities of enzymatic (superoxide dismutase and catalase) and non-enzymatic (reduced glutathione) antioxidants levels have been elevated in serum of infected camels. Finally, significant increase in proinflammatory cytokines that mediate the effect of acute phase proteins has been conducted. Therefore, the antioxidant therapy may be useful in the treatment of T. evansi infection in camels. In addition, lipid profile, acute phase proteins, proinflammatory cytokines and oxidative stress parameters could be used as biomarkers of T. evansi infection in dromedary camel.
ACKNOWLEDGEMENTS
The authors thank the Deanship of Scientific Research in King Faisal University, Saudi Arabia for supporting this study. The authors thank camel owners for their cooperation during sampling procedures
FINANCIAL SUPPORT
This study was supported by Deanship of Scientific Research in King Faisal University, Saudi Arabia (Project No. DSR130031; both authors benefitted from this grant).
