Received 21 May 2016; accepted 9 July 2016; published 12 July 2016
1. Introduction
Trypanosomosis is a major disease complex that has caused great set back to profitable farming in sub-Saharan African. The disease complex is caused majorly by T. brucei brucei, T. congolense and T. vivax in cattle [1] [2] . Cattle are differentially susceptible to T. brucei, T. congolense and T. vivax infections [3] - [5] but the mechanism of susceptibility or resistance is not fully understood.
It is generally believed that response to African animal trypanosomoses involves Type-I and/or Type-II immune response [6] , or a combination of both against infection(s) of trypanosomes. Interferon gamma (IFN-γ) and interleukin-2 (IL-2) are the main cytokines produced in Type-I immune response while IL-4, IL-5, IL-6, IL-9 and IL-10 are mainly produced in Type-II immune response. Though, different reports are available on the roles of Type-I and Type-II immune responses in different animal trypanosomosis model, the reports are contradicting [7] - [9] . While proliferation of trypanosomes is said to decreased in mice treated with anti-IFN-γ [10] [11] and IFN-γ gene deficiency [10] , other reports indicate that IFN-γ stimulates trypanosome proliferation [12] . Furthermore, most reports on the immune responses in animals to trypanosomoses are experimental murine models [13] - [15] , few are available on experimental bovine T. congolense infections [16] - [20] and no report is available on immune responses of animals to T. vivax infection.
Cardiac troponin I (cTnI) is a very sensitive biomarker for assessing the cardiac status [21] . It is considered “gold standard” for the non-invasive diagnosis of myocardial injury in human and animal diseses. But no work has been done to assess the effect of trypanosomosis on the serum level of cTnI in infected cattle.
In Nigeria, single and mixed infections of Trypanosomoses have been reported [2] , but reports on the immune responses of cattle to trypanosomoses caused by T. brucei brucei, T. congolense and T. vivax are minimal, where it exists, it is experimental report [22] . To the best of our knowledge no data is available on cytokines and cTnI responses to natural bovine trypanosomosis. This study therefore, quantified the serum levels of interferon gamma (IFN-γ), interleukin-10 (IL-10) and cardiac troponin (cTnI) in cattle that were naturally infected with T. brucei, T. congolense and T. vivax, and also associated the effect of parasitaemia and PCV on the serum levels of the assayed cytokine and cTnI.
2. Materials and Methods
2.1. Study Population
A total of 411 cattle consisting of 308 cattle kept under traditional management system of free grazing (nomadic) and 103 cattle from various abattoirs and slaughter slabs were randomly selected for sampling between October and December, 2010. Those animals that tested positive for trypanosomes by microscopy and confirmed by polymerase chain reaction (PCR) were selected for this study. The study was approved by the Ethical Committee of the College of Veterinary Medicine, Federal University of Agriculture, Abeokuta, Nigeria.
2.2. Sample Collection
Blood samples were collected from the jugular vein of each cattle into 5ml tubes containing ethylene diamine tetraacetic acid (EDTA) as anticoagulant and 5ml tubes without the EDTA for serum analysis. The samples were transported in mobile refrigerator to the laboratory within 3 hours of collection. The blood samples without the anticoagulant were set on tray slanted and allowed to stay for 24 hours in the laboratory for serum harvest. Sera were collected in clean and sterile eppendorf tubes and stored in −20˚C freezer until use while the blood, in the EDTA bottles were stored at 4˚C prior to parasitological examination and DNA extraction.
2.3. Parasitological Diagnosis and PCV Determination
Parasitemia was determined using rapid matching method [23] . From each tubes containing anticoagulant, blood was transfer into three capillary tubes which were sealed at one end with plasticin. The capillary tubes were spun in microhaematocrit centrifuge, Haematospin 1400 (Hawksley and Son Ltd. UK) at 220 g for 5 minutes at room temperature. After centrifugation, the packed cell volume (PCV) was determined using hematocrit reader. The buffy coat and upper most layer of red blood cells of one capillary tube was extruded onto a microscope slide and examined with a phase-contrast microscope (Olympus CX21, Pennsylvania, USA) at ×400 magnification for the presence motile trypanosomes. Not less than 50 fields were examined before positive or negative was declared for each sample. While the haematocrit centrifugation technique (HCT) positive samples were further processed as thin smear stained with Giemsa (EMD Chemicals, USA) for trypanosome species identification, thick blood smear was also prepared, stained with Giemsa and all examined under ×100 oil immersion objective lens (×1000 magnification).
2.4. DNA Extraction and PCR Diagnosis
DNA was extracted from the blood in EDTA bottle using Quick-gDNA™ MiniPrep (Zymo Research Corporation, Irvine, CA 92614, U.S.A ) as described by the manufacturer. The primers sets (Bioneer Inc, USA); TBR1 & 2, TCS1 & 2, TCF1 & 2, TCK1 & 2 and ILO1264 & 1265, as shown in Table 1, were selected for amplification of T. brucei, T. congolense-savannah, T. congolense-forest, T. congolense-kilifi and T. vivax DNA, respectively. Polymerase chain reaction amplification was performed in 20 μl final reaction volume containing equivalent of 20 ng of genomic DNA, 10mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 µM KCl, 200 µM each of dNTPs, 40 ng of each of the primers and 1unit of Thermus aquaticus DNA polymerase (Bioneer USA) as described in the previous work [2] . Ten microliter of the PCR products were electrophoresed through 1% agarose gel in 1 × TAE (40 mM TRIS-acetate and 1 mM EDTA) at 90 V for 80 min. along with 10 µl of biological marker, GENEMate Quanti-Marker 100 bp DNA ladder (BioExpress, UT, USA). Gels were stained with GelRedR Nucleic Acid Stain (PHENIX Research Product, Candler, NC, USA) at 5 µl/100 ml of the agarose gel suspension. After electrophoresis, the PCR products were visualized using ultra violet transilluminator (SpectrolineR TC 312 E). The PCR products of T. brucei, T. vivax and T. congolense (savannah and forest) strains were sequenced using Big dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the sequences were aligned with available published sequences of Trypanosoma species from GenBank.
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Table 1. Names and sequences of primers set optimized for Trypanosoma species detection.
Note: T. congolense (s); T. congolense savannah-type, T. congolense (f); T. congolense forest-type and T. congolense (k); T. congolense kilifi-type.
2.5. Quantification of Cytokines (IFN-γ and IL-10) and Cardiac Troponin-I (cTnI)
Cytokines and the cardiac troponin I were quantified in serum. Bovine Interferon-gamma (IFN-γ) ELISA was carried out using AbD serotec Bovine Interferon-γ ELISA kit (Catalog number MCA5638KZZ), interleukin-10 (IL-10) ELISA was carried out using Cusabio Biotech Co., Ltd. IL-10 ELISA kit (Catalog number CSB- E12917B) and Cardiac troponin I ELISA was carried out using Life Diagnostic, Incorporation diagnostic ELISA kit (Catalog number 2010-8-HS) following manufacturer’s suggested protocols as contained in the kit manuals. The absorbance at 450 nm was measured with micro-titer plate ELISA reader ( BioTek ELx 800, Highland Park, USA) and the concentration of the cytokines and the cardiac troponin in each well was calculated by comparison with a standard curve plotted using Microsoft excel program.
2.6. Statistical Analysis
Data were presented as the mean ± SEM. Significance of differences was determined by ANOVA and association within the measured parameters was done using Pearson correlation analysis in SPSS version 19 software. Differences were considered significant at p < 0.05.
3. Result
3.1. Parasitological and PCR Results
Parasite detection using microscopy observation showed 62 samples infected by one or more species of trypanosomes. Out of this, 14, 19 and 25 samples were single infection of T. brucei, T. congolense-savannah type and T. vivax, respectively. Using PCR, 7, 22 and 21 of those samples detected as single infections by parasitology were confirmed as T. brucei, T. congolense(s) and T. vivax, respectively.
3.2. Parasitaemia and PCV
The mean ± SEM parasitaemia of infected and non-infected cattle were not significantly different. The mean ± SEM of PCV of T. vivax-infected cattle was relatively lower than T. brucei and T. congolense-infected cattle, but non-infected cattle was had highest PCV that significantly different from others (Table 2).
3.3. Interferon Gamma (IFN-γ)
The serum levels of interferon gamma (IFN-γ) of T. brucei, T. congolense, T. vivax and non-infected cattle were 0.022 ± 0.002 pg/ml, 0.219 ± 0.183 pg/ml, 0.065 ± 0.023 pg/ml and 0.018 ± 0.005 pg/ml, respectively. Though the serum IFN-γ values of infected cattle were generally higher than non-infected cattle, the changes were not significantly different (p = 0.412). There was no correlation between the levels of interferon gamma expressed in the serum and, the PCV and the parasitaemia (r = −0.134 and −0.15)
3.4. Interleukin-10 (IL-10)
The mean sera levels of Interleukin-10 (IL-10) of T. brucei, T. congolense, T. vivax and non-infected cattle were 13.75 ± 11.95 ng/ml, 11.44 ± 18.05 ng/ml, 16.28 ± 20.27 ng/ml and 41.56 ± 34.64 ng/ml, respectively. While there was no significant (p > 0.05) different between the mean values of interleukin-10 of the trypanosome infected cattle, the mean values of infected cattle were significantly lower (p = 0.049) than non-infected cattle.
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Table 2. Parasitaemia and the PCV of trypanosomal infected cattle.
Note: Trypanosoma congolense(s); Trypanosoma congolense savannah-type.
There was no correlation between the levels of interleukin-10 expressed in the serum and the PCV and the parasitaemia (r = 0.048 and 0.122).
3.5. Cardiac Troponin (cTrI)
The mean values of serum cTrI of T. brucei, T. congolense, T. vivax and non?infected cattle were 0.11 ± 0.02 ng/ml, 0.14 ± 0.07 ng/ml, 0.16 ± 0.42 ng/ml and 0.10 ± 0.03 ng/ml, respectively. Though the serum cTnI values of infected cattle were generally higher than non-infected cattle, the changes were not significantly different (p = 0.44). There was no correlation between the levels of cardiac troponin expressed in the serum and, the PCV and the parasitaemia (r = −0.187 and 0.008).
4. Discussion
The immune response to African animal trypanosomoses has been studied extensively in experimental mice and bovine model, but no attention has been paid to natural infections in cattle. In this study, we assessed the level of interferon-gamma (IFN-γ), interleukin-10 (IL-10) and cardiac troponin-I (cTnI) in the sera of naturally infected cattle in Nigeria.
The circulating levels of IFN-γ and IL-10 assayed in this study, exhibited relatively higher levels in the infected cattle than non-infected cattle. Though, report on the plasma/serum level of IFN-γ and IL-10 in naturally infected cattle is not available. [6] [14] Reported elevated and decreased levels of IFN-γ in highly susceptible BALB/c and resistant C57BL/6 mice, respectively, infected with T. congolense, [5] reported significantly higher plasma IL-10 and IFN-γ levels in BALB/c mice infected with STIB247 strain of T. brucei. Though these reports are all from murine trypanosomosis model, they are in agreement with our findings except the report of [14] . However, in experimental bovine trypanosomoses, emphasis had been more on T. congolense infections [1] [18] [20] than T. vivax infection [24] . IFN-γ increased significantly in experimental T. congolense-infected cattle [25] [26] , but the increase in T. vivax-infected cattle was small and transitory [24] whereas IL-10 is elevated in susceptible Boran and trypanotolerant N’Dama cattle infected with T. congolense [27] . These reports are in partial agreement with our findings as we recorded significant changes in the levels of IL-10 when compared with the non-infected cattle.
The serum levels of cTnI of infected cattle were slightly elevated than the levels in non-infected cattle. This finding could not be compared due to paucity of data. Though, cTnI level in the serum of cattle naturally or experimentally infected with trypanosomes, has not been documented up to date in Nigeria and elsewhere, it’s increase has been reported in bovine theileriosis [28] , canine babesiosis, ehrlichiosis and canine heart worm diseases [29] - [31] . We reported higher levels of cTnI in infected cattle than non-infected cattle. Contrary to our expectation, the increase was highest in T. vivax infection, a strict intravascular parasite, than infection of T. congolense and T. brucei, both which are capable individually, of attaching to red cells and extravascular foci, respectively. Neither PCV nor parasitaemia had positive correlation with the levels of cytokines and cTnI measured in this study, a finding that is in variant with the report of [32] who indicated that increased levels of cytokines are associated with anemia.
We report for the first time in Nigeria the sera levels of cardiac troponin-I (cTnI) and cytokines (IFN-γ and IL-10) in natural bovine trypanosomoses. Elevation of cardiac troponin may be an indication of myocardiac injury due to trypanosomes. Both cytokines measured were higher than non-infected animals. This is suggestive of both cytokine being critical in immune-modulation in bovine trypanosomoses. The significantly higher serum levels of IL-10 than IFN-γ in the infected animals support the recent report of [13] , who indicated that murine trypanosomosis elicit both Type I and Type II immune responses. The process involves sequential switch from Type I cytokine (IFN-γ and TNF-α) production during the early stage of infection to Type II cytokine (IL-10 and/or IL-4) production during the late stage of infection.
5. Conclusion
In this study, we provide information on serum levels of IL-10, IFN-γ and cTnI in natural bovine trypansomosis. However, the low number of animals considered and absence of follow up makes further investigation imperative to ascertain the specific effect of trypanosomes on myocardiac integrity and interaction between the two cytokine in natural bovine trypanosomosis.
Acknowledgements
Financial support provided by the Educational Trust Fund of the Federal Republic of Nigeria and College of Agriculture and Life Sciences at Cornell University, Ithaca, NY, USA is gratefully acknowledged. We also thank the entire staff of the Department of Parasitology and Entomology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria for permission granted to use their laboratory facilities for part of this study, with special gratitude to Prof. I. A. Lawal and Dr. O. O. Okubanjo.
NOTES
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*Corresponding author.