Evaluation of T. evansi Using Different Diagnostic Techniques with Experimentally Infected (Dromedary) Camels

Falmata Kyari¹, Albert Wulari Mbaya¹, Abdullahi Abubakar Biu¹, Lawan Adamu^2* and Ali Abbagana Benisheik^3
*Correspondence: Lawan Adamu, Department of Veterinary Medicine, University of Maiduguri, Borno, Nigeria, Tel: +2348185089090, Email:

Keywords

Camels • Diagnostic techniques • Prevalence • T. evansi

Introduction

Trypanosoma evansi is a protozoan organism of that causes surra disease all through the tropical and subtropical regions of the world, as well as for Asia, Africa and Latin America [1,2]. Livestock trypanosomes triggered by T. evansi, a member of the Trypanozoon subgenus, it has a major socioeconomic effect and limits the performance of animal proteins worldwide [3-5]. It has been the most widely distributed pathogenic animal T. evansi affecting domesticable animals in Africa, Asia, Central and South America [5,6]. In Africa, it is primarily a protozoan disease of camels, prevalent in the Sahel zone, above the Tsetse belt, where it is disseminated mechanically by haematophagous insects such as stomoxys and tabanids [7]. The role of transmission can even be highlighted by the fact that there are serious forms of the disease in horses and camels, while cattle and buffalo are deemed to be impactful sources of the disease [8]. Its economic impact is often underestimated and losses may be due to mortality, reduced animal production, lower productivity resistance, poor carcass quality, weight loss, decreased milk yield, low reproductive output and therapy costs [9]. Classical clinical symptoms of surra entail recurrent fever, anaemia, mostly owing to haemolysis, and erythrophagocytosis [10].

Specific canonical parasitological approaches for the evaluation of trypanosomosis such as microscopic analysis of blood or lymph node aspirates were used. However, these are not very sensitive, but a range of techniques are used in order to enhance the sensitivity by enrichment of sample, inoculation of mouse and DNA methods. In addition to T. evansi, specific detection by microscopy, molecular tools are very useful for species specific diagnosis [11]. Numerous sorts of genetic and molecular approaches have been constantly revised to increase the accuracy of the diagnosis and classification of Trypanozoon species. In particular, the evaluation and comprehensive review of genomes have been strongly linked to T. evansi recognition and can give rise to a verifiable gene hypothesis that may be accountable for variations in dysfunctions amongst parasitic variants or subspecies [12]. Several methods have been developed for the detection of T. evansi infection, but among these, Polymerase Chain Reaction (PCR) is identified to be the most sensitive and specific, and it has a wider range of application [13,14]. Reliable epidemiological research (such as PCR-based methods) is a precondition for designing effective control programs against trypanosomosis. PCR allows detections of specific trypanosomal DNA through the use of species-specific primers [15]. PCR is likely attributed to the parasitic and tests of antigen detection owing to its responsiveness in assessing the pre-patent and protracted process of infection [16].

Early detection may play a role for the control of T. evansi, and amidst the adoption of a number of screening procedures, the diagnosis of trypanosomosis remains difficult and the widely used tests have significant drawbacks, and diagnosis basically relies on traditional Giemsa staining of thin and thick blood smears. Tests of Serology that are used for antibodies detection against pathogens, faces reproducibility drawbacks owing to antigenic heterogeneity and large proportions of false negative and false positive outcomes [17-19]. To resolve the sensitivity and specificity limitations enforced by other diagnostic approaches, the molecular identification of T. evansi Deoxyribonucleic Acid (DNA) is favored, and some molecular markers have been widely constructed to detect, differentiate, and study trypanosome species such as Internal Transcribed Spacer-1 (ITS-1) and Rhode Trypanozoon Antigen Type 1.2 VSG gene (RoTat-1.2 VSG) [20,21].

Therefore, the present study was designed to determine the diagnostic sensitivity of the current parasitological techniques for the detection of T. evansi in experimentally infected camels using (Thin Blood Smear (TBS), Wet Blood Film (WBF), Buffy-coat Technique (BCT), Mouse Inoculation (MI), antibody detection test (CATT/T. evansi) and PCR. Furthermore, to compare all these techniques, ROC-curve was employed to identify the most sensitive technique amongst the different methods of identifying T. evansi.

Materials and Methods

Source of trypanosomes

The Trypanosoma evansi used in the present study was isolated from natural infections of surra in camels at the University of Maiduguri, Faculty of Veterinary Medicine, Department of Veterinary Parasitology and Entomology. It was defined in the negative Blood Inhibition and the Infectivity Test (BIIT) and for the reason of phenotype, stabilized by six passages in Wistar rats, and sent to the Nigeria Institute for Trypanosomiasis and Onchocerciasis Research (NITOR) Vom, Nigeria where it’s been validated as T. evansi (Code: T.e.mguri 2018) by a series of parasitological and molecular characterization of the isolate. The dose of the inoculum, 1.0 x 10³ tryps/ ml was inoculated intraperitoneally into Wistar rats to enlarge the inoculums, when parasitaemia has produced nearly 5.0 x 10³ tryps/ ml of blood, the infected blood was collected from the rats by cardiac puncture and successively diluted with phosphate buffered glucose saline (PBSG, pH 7.2) until 1.0 x 10³ trypanosomes/0.5 ml was attained. Each camel was subsequently inoculated by means of the abdominal vein laterally with 0.5 ml of the infected blood.

Experimental animals

Thirty-five apparently healthy adult camels of both sexes were being used for this investigation and all the camels were evaluated for intestinal, blood, and external arthropod parasites and permitted to accustom to the environment for 60 days before the research began. They were housed in concrete floors and fly-proof stalls and fed wheat bran, groundnut husks, 5 kg of dry fodder daily per camel, watermelons, and sliced cucumber, whereas drinking water was given ad-libitum during the research.

Experimental infection

Peripheral blood samples were collected from the dromedary camels on days 0, 4, 8, 12, 16, 20, 24 and 28 post-infection. Two blood samples were collected from each animal, one with an anticoagulant (EDTA) for conventional parasitological diagnosis (thin blood smear, wet blood film, Buffy coat technique and mouse inoculation) and PCR. The second sample was placed in a plain vacutainer tube for serum collection.

Procedures for diagnosis of Trypanosoma evansi

Conventional parasitological methods: Wet Blood Film: Ten microliters of peripheral blood was placed on a clean glass slide, mixed gently with 1 ml of phosphate buffered saline, covered with a coverslip and examined at a magnification of x 100 for 15-20 mins [22].

Thin blood smear: At one end of a clean microscope slide a small drop of blood was placed and a thin film was drawn. It was fixed in methyl alcohol for 1 minute air dried. The timing used was 25 min for the smears to be stained with Giemsa (one drop of Giemsa+1 ml PBS, pH 7.2), and this was followed by rinsing the slides in tap water and drying. The thin smears of Giemsa stain were examined at a magnification of x 100 with immersion oil [22].

Mouse inoculation: Two Wistar rats were intra-peritoneally inoculated with 0.2-0.3 ml of camel blood. Three days post infection, blood samples were collected from the tail vein and blood films were prepared and examined by light microscopy for detection of trypanosomes.

Buffy-coat technique: Microhaematocrit centrifuge tubes were filled with blood containing anticoagulant and sealed with plasticine and centrifuged in microhaematocrit centrifuge at 1500 g for 5 minutes. A smear was prepared by scratching and breaking the capillary tube 1 mm below the surface of the Buffy-coat and one drop of the Buffy-coat was expelled onto a microscope slide, smeared and covered with a coverslip and examined at a magnification of x 100 for 15-20 mins [23].

Serological technique

Sera have been screened for antibodies to T. evansi

using the CATT/T.evansi test (Institute of Tropical Medicine, Antwerp, Belgium). An antigen roughly measuring 45 μl was conveyed to the test card and combined with 25 μl of the test sera diluted at 1⁄4 with PBS pH 7.2 as instructed by the manufacturer. The card was stirred for 5 min and the reaction was tested in a clear light with a positive reaction verified by the monitoring of agglutinations (blue agglutinates) [24].

Molecular technique (polymerase chain reaction)

Genomic DNA from whole blood specimens was collected using an obtainable commercially DNeasy blood and tissue kit (Qiagen, Inc. Germany) as instructed by the manufacturer. The specimens were evaluated using the primer set RoTat 1.2 forward and reverse targeting 205bp fragments of the predominant surface glycoprotein of T. evansi [25]. The PCR amplification reaction was accomplished in a total reaction volume of 25 µl enclosing 50 ng of template DNA and 12.5 µl of commercially obtainable PCR Mastermix (Qiagen, Germany). The primer was utilised at a concentration of 10 pmol/ µl, and the PCR amplifications were performed in a Biometrathermocycler.

Cycling circumstances were as follows: initial denaturation at 94/3 mins (1 cycle), accompanied by 94/min (denaturation), 57˚C/1 min (annealing), and polymerization at 72˚C/1 min (40 cycles), final extension at 72˚C/5 min. Amplified products were electrophoresed through 1.5% agarose containing ethidium bromide and the voltage was set at 60 V for the electrophoretic mobility to visualize the amplified DNA and contrasted to a standard DNA100 bp (Qiagen, USA).

Statistical analysis

Receiver Operating Characteristic Curves (ROCC) is widely used to represent the relationship/trade-off between clinical sensitivity and specificity for each potential cut-off for a test or a series of tests in a graphical format. On the y-axis, sensitivity was plotted, while 1-specificity was plotted on the x-axis. It's determined by comparing the proportion of true positives (sensitivity) to the proportion of false positives (specificity or 1-specificity) based on the total number of real negatives. On both axes, they have values between 0 and 1. The capacity of the ROC curve as a diagnostic test to depict correctly certain diagnostic techniques based on the sensitivity and specificity is provided by the region to the left and above the diagonal line known as the line of no discrimination. On the other hand, the region under a ROC curve is used to assess the effectiveness of tests since it is a measure of the effectiveness of a test in general, with a larger area indicating a more sensitive test (Figure 1). A diagnostic test's sensitivity is defined as its ability to correctly depict true positives, while specificity is defined as its ability to correctly depict false positives. The data obtained were analyzed using receiver operating characteristic curve (ROC-Curve) for sensitivity and Chi-Square test at 95% confidence interval with JMP Version 11 (SAS, Inc. NC, USA).

Results

Table 1 showed the detection rate of T. evansi infection in camels using the different techniques. On day 4 and 8 of the experimental period, WBF, BCT, TBS and MI tests did not detect T. evansi. However, CATT/T. evansi showed a detection rate of 70% on day four and PCR detection rate was 100 %, while on day eight CATT/ T. evansi and PCR tests detection rate were 100%. On day 12 of the study, the detection rate of WBF, BCT, TBS and MI was 80%, conversely, the detection rate of CATT/T. evansi and PCR techniques were 100%. On day 16 of the study, the detection rate of WBF and TBS were 80% while CATT/T. evansi, BCT, MI, and PCR were 100%. On day 20 of the experimental study, the detection rate of WBF, TBS and MI was 80% while that of the CATT/T. evansi and PCR were 100%. On day 20 the detection rate of BCT, CATT/T. evansi and PCR were 100% while WBF, TBS and MI were 80%. On day 24 of the experimental study, the detection rate of WBF, TBS, BCT, MI, CATT/T. evansi and PCR produce similar results for the entire tests with 100%. On day 28 of the experimental study, the detection rate of WBF, TBS, BCT, TBS, and MI were 80% respectively whereas CATT/T. evansi and PCR have a detection rate of 100%.

Table 2 showed the prevalence of T. evansi using different diagnostic techniques with experimentally infected camels. The number of positive tests for WBF was 21 (60%), BCT and MI were 23 (66%), TBS was 22 (63%), CATT/T. evansi was 29 (83%) and PCR was 32 (91%).

Table 3 showed the association between T. evansi infection and the different diagnostic techniques in experimentally infected (dromedary) Camels, the association was strong and significant (Chi-squared=13.720,degree of freedom (df)=5, p=0.0175).

Figure 1 showed the sensitivity of different diagnostic techniques in the detection of T. evansi in experimentally infected Camels using the ROCcurve. The sensitivities of the following techniques were; BCT=50.0%, CATT/T. evansi=71.84%, MI=61.63%, PCR=81.43%, TBS=68.37% and WBF=68.37% respectively.

Discussion

The present study aimed at comparing the detection rate of PCR using RoTat 1.2 primers set specific for Trypanosoma evansi with traditional parasitological methods (WBF, BCF, TBS and MI) and serological method (CATT/T. evansi) in camels experimentally infected with T. evansi. The detection rate of WBF, BCF, TBS and MI were much lower than that of CATT/T. evansi and PCR techniques in the early days, especially on days 4 and 8 of the diagnosis, which isn't unforeseen in view of the high detection rate of molecular and serological tests.

The present study showed that the diagnostic detection rate of WBF to T. evansi in experimentally infected (dromedary) camels was 100% on 24 day while, 80% detection rate was recorded at day 8, 12. 16, and 28 with an overall prevalence rate of 60% (21/35). The result of the current finding was higher than the report of Singh et al., who detected T. evansi in 4.14% of the camels examined with concurrent detection rate of 24.32% [26]. However, the detection rate of WBF was high during day 24 of the diagnosis. Moreover, Pathak et al., detected T. evansi in 45.37% (49/108) in naturally infected camels that was much higher than the result obtained in the present study using WBF. The variation in detection rate between the present study and that of Pathak et al., could not be unconnected to the much higher sample size used in their study. Furthermore, Jaiswal et al., reported that microscopic examination is not always sufficient to reveal the epidemiology and magnitude of surra [27,28].

The present result showed variable diagnostic detection rate for BCT with 0% on day 4 and 8. While, on day 12 and 28, 80% of detection rate was recorded, whereas, a 100% detection rate was accounted for on day 16 and 24 with an overall prevalence of 66% (23/35). The result observed in the present study was higher than the finding of Ali et al., who revealed 3.67% of detection rate [29]. The variation in detection rate might be due to the ability of parasitological tests which depends on the number and virulence of the inoculated trypanosomes.

Mouse Inoculation (MI) divulged a diagnostic detection rate of 0%, 0%, 80%, 100%, 80%, 100% and 80% on days 4, 8, 12. 16, 24 and 28 respectively, while the overall prevalence of T. evansi was 66% (23/35) as indicated for BCT, and MI has been reported to have a high detection rate of 20-50% trypanosomes per milliliters, which is considered the most efficient parasitological test for the diagnosis of scanty trypanosomes [30,31].

The diagnostic detection rate of CATT/T. evansi in the present study was 70% on day 4 and 100% on day 8, 12. 16, 24 and 28 respectively, while the overall prevalence of T. evansi was 83% (29/35) which agrees with Davison et al., Pathak et al., and Ngaira et al., who reported a higher detection rate using serological methods compared to parasitological methods [18,27,32]. However, this result was different from the reports of Dia et al., and Verloo et al., who reported a lower detection rate using serology, and this may be attributed to low antibody titers which correlates with the explanation of Verloo et al., who proved that specific antibodies appeared 6-32 days post infection [33-35].

The study also showed the high diagnostic detection rate of PCR in camels experimentally infected with T. evansi, which was as high as 100% on day 4, 8, 12. 16, 24 and 28 respectively, while the overall prevalence of T. evansi was 91% (32/35) and T. evansi specific PCR products were detected 4 days post infection, which was contrary to the results obtained by de Almeida et al., who detected T. brucei by PCR one week after infection [36]. PCR signifies a sustainable contrivance for a broad epidemiological study, and is often used to document on the factual prevalence of trypanosome infections and allows the execution of stratagems for controlling T. evansi. During the current study, there was a significant association between the different diagnostic tests used and T. evansi infection in the experimentally infected Camels. These findings reflect the study of El-Metanawey et al., where they found significant relationships between CATT/T. evansi and MI and also between PCR with MI [37]. After the experiment the camels were treated, and each camel was treated with a single 4 grammes intravenous dose of Naganol and those with positive response were treated after 3 months for T. evansi to disappear from the blood of the dromedary camels.

Using the ROC-curve to interpret the sensitivity of the different diagnostic techniques, the present study divulged variable sensitivities. This could be due to the ability of the molecular, serological and parasitological tests which depends on the number and virulence of the inoculated trypanosomes. The result of the present study showed a sensitivity of 71.8% for CATT/T. evansi which was dissimilar with the sensitivity of the CATT T. evansi evaluated under the TG-ROC analysis, with 97.5% as documented by Camoin et al., [38].

Conclusion

In conclusion, PCR has the highest detection rate and is the most sensitive technique compared to the other contrivances studied in the present study and the association between the different diagnostic techniques and detection of T. evansi infection in experimentally infected Camels was strong and significant.

Authorship

The concept was developed and planned by Falmata Kyari, Albert Wulari Mbaya and Abdullahi Abubakar Biu; the data were gathered by Falmata Kyari. Falmata Kyari wrote the paper, and Lawan Adamu reviewed the manuscript, evaluating and interpreting the data statistically.

Funding

This research received no grant from any funding agency.

Ethical Statement

Collection of blood samples and handling of animals was authorized by the University of Maiduguri, Faculty of Veterinary Medicine.

Conflict of Interest

No conflicting interest to declare.

Acknowledgement

The authors would like to express their deep gratitude to the staff of the Department of Veterinary Parasitology and Entomology, Faculty of Veterinary Medicine, University of Maiduguri, Nigeria and also livestock owners and abattoir personnel in Borno and Yobe States for their cooperation.

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