The present study was carried out to evaluate efficacy of Diminazene aceturate along with erythropoietin to ameliorate trypanosome induced anaemia and osmotic fragility of erythrocytes (EOF). Twenty four adult mice were randomly divided into four groups of six animals each. All animals except those in uninfected control group were infected with Trypanosoma evansi and treated with Diminazene aceturate and/or human recombinant erythropoietin. At the end of three weeks whole blood was collected in EDTA to evaluate EOF and level of anaemia. Result of this study established a significantly higher EOF in infected controls compared to treatment group and development normocytic normochromic anaemia progressing to microcytic hypochromic anaemia.
Trypanosoma evansi infection is endemic in India affecting domestic animals (Ravindran et al., 2008). Even though limited information is available on the economic impact of surra among livestock in India, the disease can produce huge economic losses due to death, reduced reproductive performance, loss of weight and draught power of equine and bovine; and reduction in milk yield especially in cattle and buffalo having latent infection (Pholpark et al., 1999). Surra in domestic animals is characterized by haemolytic anaemia, fever, hypoglycemia and weight loss (Desquesnes et al., 2013; Rjeibi et al., 2015). Onset of anaemia is related to the appearance of parasite in the blood and the severity increases with the level and initial wave of parasitemia (Adamu et al., 2008) suggesting haemolytic anaemia a critical factor in the pathogenesis. Increased level of free fatty acids in blood and release of trypanosomal sialidase can lead to extensive extravascular erythrophagocytosis (Igbokwe and Mohammed, 1991). Decreased erythropoiesis in affected animals is also suggested to be a causative factor for anaemia in trypanosomosis (Omer et al., 2007). Anaemia has been implicated in the development of most tissue degenerative changes in surra affected animals (Adamu et al., 2008).
The erythrocytic membrane is highly susceptible to free radical induced damage since it is rich in polyunsaturated fatty acids. Thus, the structural and functional organization of the membrane gets altered and leads to decreased membrane fluidity and increased membrane permeability (Ambali et al., 2010). These changes in RBC membrane can increase the erythrocyte osmotic fragility (EOF) and susceptibility of erythrocytes to haemolysis (Habila et al., 2012). Even though available antitrypanosomal drugs kill the parasites, the trypanosome induced anaemia and associated tissue damages are not addressed. The present study was undertaken to evaluate the effect of recombinant human erythropoietin on trypanosome induced EOF and anaemia.
Materials and Methods
Twenty four adult swiss albino mice weighing 20-30g obtained from Laboratory Animal Resource Section, Indian Veterinary Research Institute, Izatnagar, Bareilly, were used for the experiment. The animals were allowed to get acclimatize for 10 days in experimental animal house, Division of Medicine, IVRI, Izatnagar. The mice were maintained at temperature between 25-30˚C and a relative humidity of 70-80% with 12h light and dark period respectively. All experiments were conducted as per CPCSEA guidelines.
Trypanosoma evansi isolate used in this study was obtained from a clinical canine trypanosomosis case presented to referral veterinary polyclinic, IVRI, Izatnagar and the parasite was cryopreserved and kept at -70˚C until use. The cryostabilites were thawed to room temperature just before inoculation to mice. The viability and motility of trypanosomes was checked under the light microscope at 400× magnification. The cryostabilites were diluted with 1X PBS to obtain a final concentration of 1×105 trypanosomes per mL.
Inoculation to Mice
Twenty four mice were randomly grouped into four groups (I, II, III, and IV) of 6 animals each. Group I was uninfected, untreated control and only distilled water was administered. Animals of group II, III and IV were inoculated with 2×104 trypanosomes intraperitoneally. Parasitemia was monitored daily in wet blood film collected by tail snip method (Baldissera et al., 2014). Group II left untreated while group III mice were treated with Diminazene aceturate 7 mg/kg bodyweight intraperitoneally on the day of appearance blood trypomastigotes (fourth or fifth day post inoculation) in wet film. Group IV mice were treated with erythropoietin 25 IU once in a week subcutaneously for 3 weeks along with a single injection of Diminazene aceturate 7 mg/kg bodyweight intraperitoneally on the day of appearance blood trypomastigotes in wet film.
Collection of Blood and Complete Blood Count
At the end of the experiment ( 21 days post infection) the mice were sacrificed by chloroform anesthesia except group II and blood was collected from heart into tri-potassium ethylene diamine tetra acetic acid vacutainer (BD Franklin Lakes, NJ, U. S. A.) and immediately subjected to complete blood count (Jain, 1986) to evaluate the severity of anaemia. The animals of group II were sacrificed when the heavy parasitemia of approximately 109 parasites per ml of blood i.e., on sixth day post infection. Blood smear was prepared and stained with Giemsa stain and examined under 1000× magnification of light microscope to confirm the presence/absence of the parasite in blood.
In vitro Determination of Erythrocyte Osmotic Fragility
Erythrocyte osmotic fragility was evaluated as described by Oyewale (1991). Briefly, sodium chloride solution (pH=7.4) at varying concentration 0.1%, 0.5% and 0.9% was prepared. Whole blood (200µL) was added to 5ml of each sodium chloride concentration and to 5 ml distilled water and gently mixed and incubated at room temperature for 30 min. The samples were centrifuged at 2500 rpm for 5min to obtain supernatant and the absorbance of supernatant was measured spectrophotometrically at 540 nm. Haemolysis in distilled water was considered as 100% and percentage of haemolysis with sodium chloride solutions were evaluated by comparing with that of distilled water.
Data obtained were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison post-hoc test using IBM SPSS Statistics 20 soft ware for windows (IBM Corporation, New York, U.S.A.) and expressed as mean± SE. Values of P ≤ 0.05 were considered to be significantly different.
The parasites were pathogenic to mice. Prepatent period varied between four to five days i.e., circulating blood trypomastigotes could be observed in peripheral blood film from fourth/fifth day after infection in all infected groups. The blood smear examination of group II animals revealed presence of haemoflagellates (Fig.1) where as those of group III and IV at the end of the experiments showed absence of the parasites.
Fig 1: Blood smear of group II showing Trypanosoma evansi
The effect of treatment on haematological parameters is shown in Table 1.
Table 1: Effect of treatment of diminiazene aceturate alone and along with human recombinant erythropoietin on haematology of mice experimentally infected with Trypanosoma evansi
Different superscript letters indicates means showed statistically significant difference (P<0.05); Group I: uninfected untreated, Group II: infected untreated, Group III: infected and treated with Diminazene aceturate, Group IV: infected and treated with Diminazene aceturate and human recombinant erythropoietin
There was significant increase in total erythrocyte count (TEC) in treatment groups (group III and IV) as compared to infected untreated mice (group II). Although there is no statistically significant difference in TEC between uninfected (group I) and treatment groups, group III i.e., infected and treated with Diminazene aceturate showed slightly lower TEC (6.89±0.30) than the reference range of 7.5-11×106/µL (Bolliger et al., 2010). Group IV animals showed normal TEC values similar to uninfected group. Haemoglobin levels were significantly lower in group II compared to other three groups. Animals in group III (Diminazene aceturate alone treatment) showed a reduction in haemoglobin level (9.76±0.46) as compared with group I and IV, but the difference is not statistically significant (P>0.05). Mean corpuscular volume of the treatment groups III and IV showed no significant difference as compared to group I (P>0.05). Significant reduction in MCV could be noted in Group II when compared group III (P<0.05) and, the value (44.04±1.10 fL) was slightly lower than reference range (47-59 fL) reported for mice earlier (Bolliger et al., 2010). There was a significant difference in MCH between uninfected untreated groups and infected untreated group (P<0.005) and MCH level was slightly lower in infected untreated group (group II). Mean corpuscular haemoglobin concentration showed no significant difference between the groups and were within the normal range (27-34g/dL). Total platelet count was higher in group IV (infected and treated with Diminazene aceturate and erythropoietin) when compared with group II (P<0.01). Group II showed a lower platelet count (174.25±40.30×103/µL) than normal reference value for mice (321-1866×103/µL). The platelet distribution width was statistically similar in all experimental groups. The red blood cell distribution widths (RDW) of all the experimental groups were within the normal range (11.6-16%) although there was significant reduction in group II in comparison with group I and III.
Erythrocyte Osmotic Fragility
At 0.9% sodium chloride concentration the percentage haemolysis of infected untreated mice (group II) were significantly (P<0.001) higher (14.22±1.11) than that of group I (uninfected untreated), III (infected and treated with Diminazene aceturate) and IV (infected and treated with Diminazene aceturate and human recombinant erythropoietin) (Fig.2).
There was no significant difference (P>0.05) in EOF between groups at 0.1% sodium chloride concentration haemolysis. At 0.9% concentration of NaCl, the untreated group (group II) showed a significant increase (P<0.001) in percentage of haemolysis as compared to uninfected (group I) and the treatment groups (group III and IV).
Fig 2: Effect of Diminazene aceturate alone and along with human recombinant erythropoietin on erythrocyte osmotic fragility in rats experimentally infected with T.evansi. Group I: uninfected, untreated, GroupII: infected, untreated, Group III: infected
Trypanosoma evansi is reported to be the most widely distributed pathogenic trypanosomes affecting domestic mammals and is endemic in south-east Asia (Desquesnes et al., 2013). The pathogenicity of the parasite varies with host species as it produces acute infection in equines and canines and a latent infection in ruminants. Fever, progressive anaemia, cachexia, lethargy and haemostatic abnormalities are the most frequent clinical signs. Anaemia and hypoglycemia were major factors influencing the disease progress. Anaemia in trypanosomosis is mostly haemolytic caused by the release of trypanosomal sialidase that cleaves sialic acid from erythrocytic membrane (Esievo et al., 1982). The parasite utilizes sialoglycoprotein of RBC membrane for multiplication and differentiation (Briones et al., 1994). Release of reactive oxygen species in trypanosomosis also has a major role loss of erythrocyte membrane integrity, leading to release of free fatty acids (Umar et al., 2007). Another suggested mechanism of anaemia in trypanosomosis dyserythropoiesis (Omer et al., 2007). This study showed increased osmotic fragility of erythrocytes in infected untreated group as compared to healthy and both treatment groups, suggesting loss of RBC membrane damage in these animals. Our study result was in consensus with earlier reports (Mijares et al., 2010; Habila et al., 2012). The reduced ability of erythrocytes of infected animals to prevent membrane damage and increased membrane permeability mediates increased haemolysis and anaemia in those animals. The trypanocidal drug, Diminazene aceturate used for treatment in both group III and group IV, had eliminated the parasites from blood, thus prevented the effect of trypanosomes on erythrocyte membrane such as increased permeability and loss of integrity of the membrane. Thus the percentages of haemolysis in both treatment groups were decreased and more or less similar to uninfected control group.
The haematological findings in infected untreated group showed reduced total erythrocytes, haemoglobin and haematocrit (HCT) which is consistent with anaemia associated with Trypanosoma evansi infection. The animals of group III i.e., treated with Diminazene aceturate alone also showed a slight reduction in total erythrocytes, haemoglobin and haematocrit than the reference range reported earlier (Bolliger et al., 2010). The mean corpuscular volume (MCV) of the infected untreated group was slightly lower the normal values but not significantly different from the uninfected control animals. The mean corpuscular haemoglobin (MCH) values of group II were lower than the normal values and were significantly lower than the uninfected control whereas mean corpuscular haemoglobin concentration (MCHC) values of all groups were within the normal range (Bolliger et al., 2010). Since the MCV and MCH were slightly lower than the normal reference range (MCV: 47-59 fL, MCH: 14-17pg) in Group II, anaemia in this group can be classified as normocytic normochromic anaemia progressing to microcytic hypochromic form. The reduction in MCV and MCH may be due to drainage of iron reserve for erythropoiesis since there is a high demand and finally leads to unavailability of iron for haemoglobin synthesis. Thus, in later stages of infection hypochromic anaemia is a consistent finding. The total platelet count of the untreated infected control group of this study was lower than the reference range of the species (total platelet count for mice: 326-1866 ×103/µL) (Bollinger et al., 2010). Even though reduced platelet count was observed in untreated mice, no overt clinical symptoms were observed throughout the experiment. Similar reduction in platelet count were reported earlier in dogs, cats, bovine and rats (Damayanti et al., 1994; De La Rue et al., 1997; Da Silva et al., 2010; Da Silva et al., 2011)
Since, the animals of group III i.e., treated with diminazene aceturate alone showed a reduction RBC count, haemoglobin and HCT but normal MCV, MCH and MCHC suggesting normocytic, normochromic anaemia. Although diminazene aceturate can clear the parasitemia but unable to check trypanosome induced anaemia. The haematological findings of mice treated with diminazene aceturate in combination with human recombinant erythropoietin were consistent with uninfected control animals of group I. Thus the study establishes the use of combination of therapy of antitrypanosomal drug Diminazene aceturate along with human recombinant erythropoietin to treat trypanosoma induced anaemia. Since anaemia is a critical factor in the progressive pathogenesis and tissue damage caused by the parasite in domestic animals, contributing to the morbidity and mortality the control of anaemia could limit pathogenic effects to a larger extend. Thus beneficial effect of erythropoietin may be utilized to alleviate the Trypanosoma evansi induced haemolytic anaemia and associated tissue damage.
The authors would like to thank Director IVRI, Izatnagar, for providing necessary infrastructural facilities. Financial assistance provided to Aron Jacob by Indian Council for Medical Research, New Delhi is acknowledged.