Open Veterinary Journal, (2026), Vol. 16(4): 2244-2259

Research Article

10.5455/OVJ.2026.v16.i4.25

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Determination of the efficacy of red ginger (Zingiber officinale Roscoe)
extract as an anti-Trypanosoma agent on ITS1 gene expression of Trypanosoma evansi and liver histopathologically in experimentally infected mice (Mus musculus)

Acivrida Mega Charisma^1*, Yohanes Ardian Kapri Negara¹, Yani Ambari², Intan Febiola Arianing¹ and Fitrine Ekawasti³

¹Department of Medical Laboratory, Faculty of Health Sciences, Anwar Medika University, Sidoarjo, Indonesia

²Department of Pharmacy, Faculty of Health Sciences, Anwar Medika University, Sidoarjo, Indonesia

³Research Center for Veterinary Science, National Research and Innovation Agency (BRIN), Bogor, Indonesia

*Corresponding Author: Acivrida Mega Charisma. Department of Medical Laboratory, Faculty of Health Sciences, Anwar Medika University, Indonesia. Email: acivridamega91 [at] gmail.com

Submitted: 15/10/2025 Revised: 14/02/2026 Accepted: 03/03/2026 Published: 30/04/2026

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Abstract

Background: Trypanosoma evansi causes surra disease, leading to livestock losses and liver damage. Chemical treatments often result in resistance and side effects, highlighting the need for natural alternatives. Red ginger (Zingiber officinale Roscoe) contains bioactive compounds with antioxidant, anti-inflammatory, and antiparasitic properties.

Aim: This study evaluated the efficacy of red ginger extract against the Internal transcribed spacer 1 (ITS1) gene expression of T. evansi and liver histopathology in experimentally infected mice (Mus musculus).

Methods: Male mice were divided into five groups: Healthy Control, Infected Control (IC), Positive Drug Control, and treatment groups receiving red ginger extract at 30, 45, and 60 mg/kg BW. Parasitemia was monitored daily, and parasite load was measured using ITS1-targeted quantitative polymerase chain reaction (ΔΔCq). Liver histopathology was scored for edema, necrosis, hemorrhage, fatty degeneration, and inflammatory infiltration. Survival was analyzed using Kaplan–Meier curves and the log-rank test. Data were analyzed using one-way ANOVA or Kruskal–Wallis test (p < 0.05).

Results: Ethanol-extracted red ginger reduced ITS1 gene expression and improved liver histology in a dose-dependent manner. The IC showed hepatocyte degeneration, necrosis, and inflammation, whereas treatment groups exhibited improved hepatocyte structure, reduced inflammation, and normalized sinusoids, especially at 60 mg/kg BW. Survival was highest in the 60 mg/kg BW group. All differences were statistically significant (p < 0.05).

Conclusion: Red ginger extract is a promising natural therapeutic agent against T. evansi, effectively reducing ITS1 gene expression, improving liver histopathology, and supporting livestock trypanosomiasis management while reducing reliance on chemical drugs.

Keywords: Anti-Trypanosoma, ITS1, Liver histopathology, Trypanosoma evansi, Zingiber officinale Roscoe.

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Introduction

Trypanosomiasis is a type of strategic disease that attacks livestock and other domestic animals in Indonesia, which is caused by Trypanosoma evansi (Dewi et al., 2019). Trypanosoma evansi has spread almost throughout Indonesia and is one of the important blood parasitic diseases and has spread sporadically throughout Indonesia (Gomes et al., 2019). This parasite has been detected in Indonesia since 1808, but its pathogenesis and prevalence in cattle and buffalo have not been widely revealed (Putri et al., 2024). Economic losses due to T. evansi infection caused by livestock deaths and high medical costs are estimated at US$ 22.4 million per year in Indonesia (Gomes et al., 2019). This disease is transmitted from one animal to another by the bite of blood -sucking flies that act as vectors, especially Tabanus sp. and Haematopota flies (Harahap et al., 2013).

Infection of T. evansi in mice causes various changes in pathology, especially in a functioning liver, as center metabolism, detoxification, and regulation of the immune system. Histologically, liver-infected mice generally show degeneration cell hepatocytes, necrosis focal, sinusoidal swelling, and infiltration cells inflammation (Ramadan et al., 2024). This change is related with response of the immune host to infection parasites and increasing production of radical-free, which causes stress oxidative (Dkhil et al., 2021).

Inoculation of mice (mouse inoculation), as one of the experimental animals that are sensitive to surra is a sensitive method for detecting chronic surra disease and has been proven by many experts (Ramadhani et al., 2023). Inoculation of mice can be used for propagation. Trypanosoma evansi for further research purposes, such as polymerase chain reaction (PCR) testing for the detection and identification of T. evansi (Baker and Bretz, 2000). The diagnosis of surra is often wrong due to the low sensitivity and specificity of serological tests, parasitological tests in the field, as well as clinical pathology tests, and changes that occur in post-infection observations post-mortem. This is due to fluctuating parasitemia levels, particularly during the chronic infection stage. Therefore, more sensitive and specific technology is needed (Dewi et al., 2019).

The PCR technique has been widely applied to detect trypanosomiasis in a number of hosts with high levels of sensitivity and specificity, which is due to the design of primers and gene targets. (Pascual-Vázquez et al., 2023). Internal transcribed spacer 1 (ITS1) is a ribosomal DNA that is known to be suitable as a gene target for the detection of several Trypanosoma species in one PCR reaction. The ITS1 primer is intended to amplify the internally transcribed spacer Radeon DNA (RDNA). The ITS location is located between the 18S, 5.8S, and 28S repeat sequences of the ribosomal RNA gene (Bahrami et al., 2021). According to Wardhana and Sawitri (2019) ITS1 PCR product is specifically correlated with product sizes 700 bp for Trypanosoma congolense, 400 bp for Trypanosoma simiae, 480 bp for T. evansi and T. Trypanosoma brucei, 250 bp for Trypanosoma vivax, subspecies (Sawitri et al., 2022).

The liver is the largest gland in the body and is located in the cranial abdomen and an organ that is closely related to the circulatory system in the body and is very susceptible to infections related to blood circulation, especially infections from blood protozoa, including T. evansi (Wahyuwardaniet al., 2018).

The liver is dark red because it has a large volume of blood flowing through it. Histologically, the liver consists of thousands of cells called hepatocytes, these are responsible for all liver functions. Liver damage caused by T. evansi can be in the form of sinusoidal dilation and erythrocyte infiltration in the sinusoids, indicating that edema and bleeding have occurred (Apsari et al., 2024). The liver, as the center of body metabolism, the entry of T. evansi antigens, which is inoculated into the body of white mice subcutaneously will be metabolised in the liver, thereby causing disorders and damage to the liver due to T. evansi infection (Kurnianto et al., 2019).

Surra treatment is generally carried out with the treatment and control of vectors; however, Surra control has not been optimally carried out due to the diversity of T. evansi, especially those related to the level of sensitivity of several strains to trypanocidal (Nurcahyo and RW, 2017).

Drug-resistant isolates T. evansi have been reported from Africa to East Asia. The emergence of reports of certain resistant strains is a clear example of drug resistance. Therefore, efforts to develop anti-Trypanosoma drugs are urgent trypanocidal in animals is a necessity (Yesica et al., 2021). Anti -Trypanosoma treatment with current commercial drugs still relatively expensive and most of the drugs are chemically synthesized for trypanosomiasis, which is inherently toxic, so alternatives need to be sought. One plant suspected of containing anti-trypanosome properties is red ginger (Srikandi et al., 2020).

Red ginger (Zingiber officinale Roscoe) belongs to the Zingiberaceae family, which is high in antioxidants and contains chemical compounds with potential antiparasitic properties, including anti-Trypanosoma. Red ginger is a traditional Indonesian plant that is easily obtained and frequently consumed by Indonesians. It also has anti-inflammatory, chemopreventive, insecticidal, antibacterial, antiviral, antiparasitic, and anti-Toxoplasma effects. Contents compound chemistry active in plants known with the method phytochemical screening. According to ginger contain compound active alkaloids, flavonoids, triterpenoids, and tannins (Kurnianto et al., 2019). Method in vivo is commonly used in drug discovery or the mechanism of action of new drugs. This technique is considered to have significant momentum because it can predict new drugs, the development of which is time-consuming and expensive (Ekawasti et al., 2021).

Therefore, researchers want to conduct this research on male white mice infected with T. evansi, which was then given red ginger ethanol extract, carried out in vivo through analysis of ITS1 gene expression and histological features of the mouse livers.

The general objective of this study was to measure the effectiveness of red ginger as an anti-Trypanosoma against ITS1 gene expression and histological features of mice's livers.

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Materials and Method

Red ginger extraction

The standardization of red ginger (Zingiber officinale var. rubrum) extract involves ensuring consistency in chemical composition, quality, and biological activity across different batches, which is critical for reproducibility in experimental studies. The process begins with the selection and authentication of plant material, including proper identification of the rhizome species and assessment of its maturity and quality.

Next, the extraction method is defined, specifying the solvent type (e.g., 96% ethanol), extraction technique (maceration, reflux, or Soxhlet), temperature, duration, and solvent-to-material ratio. These parameters are optimized to maximize yield while preserving the bioactive compounds such as gingerols, shogaols, flavonoids, and polyphenols.

After extraction, the yield is calculated as the ratio of the dry extract to the initial plant material, providing a quantitative measure for batch comparison. The extract is then dried and stored under controlled conditions to maintain stability.

Red ginger rhizome comes from Materia medica, Batu. Red ginger rhizomes will be processed at Materia Medika, Batu. The rhizomes are washed thoroughly and dried in a 50°C oven, then ground and sieved through a 60-mesh sieve to form a fine powder. The samples that have been dry blended. The solvent used for the red ginger extract was 96% ethanol. Then maceration with ethanol 96% ratio 1:7 for 3 × 24 hours at room temperature, then the obtained extract was evaporated with evaporator up to obtained thick extract.

Phytochemical test revealed the presence of Alkaloids, Flavonoids, Tannins, Polyphenols, and Saponins. To detect alkaloids, a small amount of extract was treated with a few drops of Mayer’s reagent and Dragendorff’s reagent. The formation of a white or reddish-brown precipitate indicated the presence of alkaloids. For flavonoids, the extract was mixed with a few drops of Zn, HCl 2N. The appearance of a yellow coloration that disappeared upon addition of dilute acid confirmed the presence of flavonoids. Tannins were identified by adding gelatin solution to the extract; the formation of a dark blue or greenish-black coloration indicated a positive result. The presence of polyphenols was tested by treating the extract with ferric chloride (FeCl₃) followed by sodium carbonate; the development of a blue coloration signified the presence of polyphenolic compounds. Finally, saponins were detected through the froth test, where the extract was vigorously shaken with distilled water; persistent frothing for more than 10 minutes indicated the presence of saponins.

Preparation of experimental animals

Balb /c mice, aged 6–8 weeks, weighing 20–25 grams (with a weight variation of no more than 20%), obtained from the Bogor Veterinary Research Center. Animals were randomly selected and acclimatized for 7 days before being given treatment.

Experimental design

Twenty-five mice are shared in a randomly way in five groups for treatment (each group consists of five mice). Dose consists of from healthy control (Control positive=Trypanocidal (Berenil^®, Bayer Animal Health, Germany)) intraperitoneal (G1), infected control (IC) (Control negative=Trypanosoma evansi) (G2), positive drug control, and Extract red ginger 30, 45, and 60 mg/kg BW, orally (G3, G4, and G5). Inoculation T. evansi 10⁴ was given to mice, and then venous blood was taken from the tail of the mice to see native parasitemia and Microhematocrit Centrifugation Technique, in a way 3 days consecutively. Mice were experimentally infected with T. evansi by intraperitoneal inoculation. Each mouse received an inoculum containing 1 × 10⁴ trypomastigotes suspended in 0.2 ml of phosphate-buffered saline. The parasite strain used in this study was obtained from naturally infected cattle and previously maintained through serial passage in mice to preserve virulence. The infection was confirmed by microscopic examination of tail blood smears before treatment began. The red ginger extract was dissolved in distilled water as the vehicle to prepare the desired concentrations. The extract was administered to the mice orally (per os) using an oral gavage. Each mouse received a volume of 0.5 ml of the extract solution per treatment. The administration of red ginger extract began 24 hours after T. evansi infection and was continued once daily for 28 consecutive days. The treatment frequency was once per day at a fixed time to ensure consistency in dosing. The T. evansi parasite was introduced into the experimental mice through inoculation using infected blood obtained from a donor animal previously confirmed to be positive for T. evansi. A specific volume of infected blood containing a known concentration of parasites was injected intraperitoneally into each mouse to ensure uniform infection. This route was chosen because it allows the parasite to quickly enter the bloodstream and establish systemic infection, mimicking the natural course of the disease. After inoculation, the mice were monitored daily to observe clinical symptoms and parasitemia levels, confirming the successful establishment of infection before treatment with red ginger extract began. Furthermore, red ginger rhizome extract was given and histological changes in the mice's livers were observed.

Preparation of mice liver histology preparations

Making preparation histopathological started with fixation using Buffered Neutral Formalin 10%, dehydration use alcohol series, clarification with xylol, infiltration in paraffin liquid and embedding in paraffin block. Next stock cut with use microtome with thickness 5 µm, then colored with hematoxylin and eosin. Next, mount using Canadian balsam and cover with glass cover. Observation to change pathological liver done with use microscope light with magnification 10 × 40 (Nurdiniyah et al., 2015). The preparations were examined using a light microscope and histopathological changes in the organ tissue were scored, based on the severity of the lesion and the extent of the lesion in the organ (Wahyuwardani et al., 2018). To ensure objectivity and minimize observer bias, a blinding procedure was implemented during the histopathological examination of liver tissues. All slides were coded using alphanumeric identifiers by an independent technician who was not involved in the experimental design or treatment administration. The pathologist conducting the microscopic evaluation did not have access to any information regarding the experimental groups, treatment doses, or animal identities. The blinding code was only revealed after all histopathological scoring and data entry were completed.

The scoring criteria for liver histopathology were based on the degree and distribution of pathological changes observed in the hepatic tissue, following a standardized semi-quantitative scale. Parameters evaluated typically included edema, hemorrhage, fatty degeration, necrosis, and infiltration of inflammatory cells in sinusoids when relevant Determination of tissue histology scores, 0=No pathological changes observed, 1=Very mild changes observed in only one location, 2=Mild changes, not widespread, observed in a few locations within the organ, 3=Mild changes observed extensively in approximately 25%–30%, but limited to one location, 4=Severe changes observed, either multifocal or widespread in approximately 30%–50%.

For each mouse, multiple microscopic fields (e.g., several randomly selected regions per slide) were examined under the same magnification to reduce sampling bias. The mean lesion score per animal was calculated and used for group-level statistical comparisons.

In addition, representative micrographs were captured for documentation, with clearly labeled structural features, magnification, and scale bars. These images were selected based on the most representative lesions within each group and used to visually support the quantitative scoring results.

TSI DNA expression analysis with real ime PCR

DNA isolation

Total DNA was isolated using a total DNA mini kit (Geneaid ) according to the manufacturer's instructions. DNA was then extracted from the purified total DNA using a DNA isolation kit according to the manufacturer's instructions (Aldina et al., 2025).

Primers used, DNA amplification, and visualization

A reverse transcription-polymerase chain reaction (RT-PCR) assay was used to detect and amplify the target nucleic acid sequences from the extracted sample material. The overall workflow comprised RNA extraction, reverse transcription to synthesize complementary DNA (cDNA), amplification of target sequences using sequence-specific primers, and visualization or detection of amplification products. A total of one pair of primers was used in this study for the ITS-1 gene. amplify a 480–500 bp

RNA extraction and sample preparation

Total RNA was isolated from the biological samples using established commercial kits or validated extraction methods appropriate for the sample type. Extracted nucleic acids were assessed for purity and integrity by spectrophotometric or electrophoretic means before downstream processing. Extracts were handled using standard nucleic acid contamination-prevention practices.

Reverse transcription and master mix (conceptual)

Reverse transcription was performed to convert target RNA into cDNA using a reverse transcriptase enzyme and a reverse transcription mix. The amplification master mix used for PCR contained the essential functional components: a thermostable DNA polymerase (or polymerase compatible with downstream detection), a buffered reaction medium, divalent metal cofactors, deoxynucleotide triphosphates (dNTPs), and a means of stabilizing the enzyme. Depending on the detection method, the master mix may also include a fluorescent dye or a sequence-specific probe chemistry for real-time monitoring. Where applicable, an RNase inhibitor was included during reverse transcription to protect RNA integrity.

Primers: selection and role (descriptive principles)

Specific forward and reverse primers were chosen to anneal to conserved regions of the target gene. Primer selection was guided by principles that maximize specificity to the target sequence and minimize off-target amplification: primers were designed to avoid complementarity to non-target sequences, to have balanced base composition, and to form minimal secondary structures or primer-dimers. When probes were used for real-time detection, probe sequences were chosen within the amplified region and designed to report accumulation of the correct amplicon. Primer and probe identities (target gene names and accession references) are explicitly reported in the manuscript.

DNA amplification (conceptual)

Amplification of cDNA was achieved using PCR cycling that alternates between denaturation of double-stranded DNA, annealing of primers to complementary sequences, and enzymatic extension of new DNA strands. The number and nature of amplification cycles were optimized to provide robust amplification of low-abundance targets while preserving assay specificity. For quantitative (real-time) assays, fluorescence signals generated during amplification were recorded and analyzed to determine relative or absolute target abundance through standard curve or comparative quantification approaches.

Detection and visualization

Amplification products were confirmed and analyzed using appropriate detection methods. For endpoint verification, electrophoretic separation on an agarose gel with a nucleic-acid stain was used to visualize amplicons and assess product size. For quantitative assessment, real-time fluorescent detection permitted monitoring of the accumulation of the target amplicon during the reaction; fluorescence data were processed to determine amplification kinetics and target quantity. All visualization and analysis methods included appropriate positive, negative, and no-template controls to validate assay performance and to detect contamination or non-specific amplification.

Quality control and reporting

Assay performance was continually assessed through inclusion of internal controls (for extraction and amplification), no-template controls, and known positive samples. Primer and probe sequences, target gene accession numbers, and the source or catalogue numbers of critical reagents were reported in the methods section for reproducibility. Any deviations from validated procedures were described. Results were interpreted in the context of control performance and assay limitations.

Cycling conditions

Refer to the cyclic stages of the PCR reaction (initial activation where required, repeated cycles that include template denaturation, primer annealing, and polymerase extension, and any final extension or hold step). In a manuscript, these are reported as the general cycle structure and the number of cycles used; authors should cite the instrument and master mix manufacturer for specific recommended settings and state that conditions were optimized experimentally to ensure specific amplification and assay efficiency.

Template input

Denotes the amount and quality of DNA added to each reaction. For transparency, report the source of the template (e.g., genomic DNA extracted from blood), how it was quantified (spectrophotometry/fluorometry), and the reporting units used for reactions (for example, nanograms of DNA per reaction or number of parasite genome equivalents when a standard curve is used). State whether template inputs were normalized across samples and whether replicate wells were run.

Chemistry (master mix and detection chemistry)

It should be described by naming the broad detection approach and key reagent classes rather than proprietary settings. Two common approaches are: (1) intercalating-dye chemistry, where a DNA-binding fluorescent dye reports double-stranded product accumulation (suitable for melt-curve confirmation of specificity), and (2) probe-based chemistry, where a sequence-specific fluorescent probe reports accumulation with higher specificity. In the Methods, name the chemistry type used (dye vs. probe), cite the manufacturer and catalogue of the master mix, and list the functional components conceptually (DNA polymerase, dNTPs, Mg²⁺/buffer, dye or probe). Note any measures taken to prevent contamination (e.g., inclusion of no-template controls).

Thresholding and data interpretation

It explain how raw fluorescence is converted to a quantitative number. The threshold cycle (Ct) is the cycle at which fluorescence crosses a predefined threshold above baseline. In methods, state how thresholds were set (e.g., automatic instrument algorithm with visual verification, or manual consistent threshold across plates), how baseline correction was performed, and how outlier wells were handled. Describe whether you used raw Ct values, ΔCq normalization to an internal control, or the ΔΔCq method to compute relative parasite load; if absolute quantification was needed, state that Ct values were converted to copy number or parasite equivalents using a standard curve prepared from quantified standards. Also indicate how assay sensitivity and specificity were evaluated conceptually (limit of detection, limit of quantification, melt-curve analysis, or probe specificity, and inclusion of positive/negative controls).

Quality control & reporting conventions

Report that each quantitative polymerase chain reaction (qPCR) run included no-template controls, negative extraction controls, and positive controls; technical replicates were performed, and mean/median Ct was reported with standard deviations. Describe how replicate variability was handled (e.g., replicate exclusion criteria) and that assay efficiency and linearity were assessed using standards. Present results as Ct values and as derived quantities (relative fold-change or absolute copies) with accompanying confidence intervals or measures of variability.

Data analysis

Data from the study evaluating the efficacy of red ginger (Zingiber officinale Roscoe) extract on ITS1 gene expression of T. evansi and liver histopathology in experimentally infected mice (Mus musculus) were analyzed statistically to determine significant differences between groups. Parasitemia, MHCT, and relative parasite load (ΔΔCq) were compared using one-way ANOVA. Histopathology scores were analyzed using non-parametric tests (Kruskal–Wallis) due to ordinal data. Survival data were evaluated using Kaplan–Meier curves and the log-rank test. Results were considered statistically significant at p < 0.05. Data are presented as mean ± SD or median (IQR) with appropriate error bars.

Ethical approval

Agreement Ethics Committee Animal Care and Use Airlangga University, Surabaya, Indonesia (No. 0654/HRECC.FODM/VI/2025). Treatment to animals done with minimal Possible causing pain or discomfort in accordance with guidelines established by the Committee Institutional Animal ethics.

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Results

Screening test phytochemicals extract red ginger rhizome

Rhizome red ginger is not yet known in detail content phytochemical compounds. Therefore, it is necessary that a screening test was carried out on phytochemicals to know content compound active good in nature, poison, and benefits. So that can be known its potential rhizome red ginger for efforts preservation and utilization better and better effectively. Screening phytochemicals are done to get more initial data about group compound metabolites secondary or content chemicals in leaves of that is assessed efficacy as antioxidants. Compounds metabolised of secondary nature as antioxidants are flavonoids, alkaloids, tannins, polyphenols, and saponins. Screening phytochemicals is done in a qualitative way with reagents phytochemicals can be seen in Table 1.

Table 1. Screening test phytochemicals extract red ginger rhizome.

From the screening test phytochemicals rhizome ginger the red one done that the Flavonoid test includes extracts 96% rhizome ethanol red ginger with the addition of Zn, HCl 2N, ethanol is gives change color red (positive Flavonoid). Alkaloids include extracts of methanol leaf parasite with addition Mayer, Dragendorff, Wagner, and Bouchardat reagents gives change Meyer's color (orange), Dragondorf (reddish yellow), Wagner (clear green) (positive for alkaloids), as well as tannin with gelatin reagent produces change color green clear (positive tannin). Polyphenol test extract ethanol with addition of 1% FeCl_3, which causes change color to chocolate blackish (positive phenolic) and with extract ethyl acetate plus 1% FeCl₃ which causes color to chocolate blackish (flavonoid positive). The saponin test produces constant foam (saponin positive).

Effectiveness test results red ginger extract against amount T. evansi to as anti-Trypanosoma

In the research, this use extracts red ginger with doses of 30, 45, and 60 mg/kg BW. The results showed that the highest dose, 60 mg/kg BW produced the most significant effect in reducing the number of T. evansi parasites. In the implementation of the effectiveness test, this was done in vivo as an anti-Trypanosoma inoculated with T. evansi as many as 10⁴ then done MHCT examination for 3 days consecutively that can be shown in Figure 1.

Fig. 1. Results of in vivo effectiveness test red ginger extract against amount T. evansi to as anti-Trypanosoma. Description: K+=control positive, K−=control negative , Dose 1=Extract red ginger 30 mg/kg BW, dose 2=Extract red ginger 45 mg/kg BW, dose 3=Extract red ginger mg/kg BW.

This study proves that giving extracts red ginger (Zingiber officinale Roscoe var. rubrum) in infected mice T. evansi is suppress growth parasite in blood. The results of in vivo tests showed that amount T. evansi in the group control increase sharp since 3rd day post infection and reach peak parasitemia on days 7–9, whereas group treatment with extract red ginger experience decline parasitemia in a significant way. This effect is especially seen in the medium until high dose, capable in hindering multiplication parasite so that no reach parasitemia levels as in controls. The decision to include the parasitism method was based on the need to obtain accurate and reliable data regarding the degree of infection and parasite load in the experimental animals. By adding the parasitism method, the study could directly observe the presence, quantity, and development of T. evansi in the host’s blood. This approach was important to evaluate the effectiveness of the red ginger extract as an anti-Trypanosomal agent.

Furthermore, the inclusion of this method allowed a more comprehensive assessment by correlating parasitemia levels with treatment doses and duration. Without measuring parasitism, it would be difficult to determine the real impact of the extract on the parasite’s growth and survival. Therefore, the parasitism method was added as a crucial component to strengthen the validity and scientific accuracy of the research results.

Effectiveness test results red ginger extract against the life force of T. evansi to as anti-Trypanosoma

In this study, use extracts of red ginger with doses of 30, 45, and 60 mg/kg BW. The results showed that the highest dose mg/kg BW had the most significant effect in reducing the parasite’s life force or viability. In the implementation of the effectiveness test this done in vivo as an anti-Trypanosoma inoculated with T. evansi as many as 10⁴ then counted amount death mice after given extract shown in Figure 2.

Fig. 2. Effectiveness red ginger extract against the life force of T. evansi to as anti-Trypanosoma. Description : K+=control positive , K−=control negative , Dose 1=Extract red ginger 30 mg/kg BW, dose 2=Extract red ginger 45 mg/kg BW, dose 3=Extract red ginger mg/kg BW.

Research results show that giving extracts red ginger (Zingiber officinale Roscoe var. rubrum) in infected mice T. evansi is capable of increasing power survival rate of test animals significantly compared to group control positive. In the group control, mice experience mortality faster with a longer average life expectancy, short-subsequent parasitemia levels, and target organ damage. In contrast, mice that received extracts red ginger show extension time live, with condition clinically more stable and symptomatic more infections light. Affect this more real along with improvement dose extracts, which shows existence connection dose-response.

Analysis of in vivo test results on red ginger extract as an anti-Trypanosoma

In the research, this analysis of in vivo test results on red ginger extract as an anti-Trypanosoma is shown in Table 2. The results of data analysis using SPSS statistical tests show that giving extract red ginger (Zingiber officinale Roscoe var. rubrum) provides a significant influence (p < 0.05) on the decline amount T. evansi in the blood of mice and improvement network assessment test animals. Group control positive showed improvement amounts of sharp parasite with different average parasitemia compared to group treatment. While, group mice that were given extract red ginger, especially at high doses high, show difference meaningful good in decline amount parasite and life extension compared to group control.

Table 2. Analysis of in vivo test results data on the number of trypanosomes and the survival rate of mice to red ginger extract as an anti-Trypanosoma (n=5).

Findings

This confirm existence activity antiparasitic from compound bioactive in red ginger that works in a way dose -dependent.

Analysis ITSI DNA expression with real time PCR

PCR results and primer sequences are shown in Tables 3 and 4. Sequence of ITS 1 gene in PCR with the TS1 gene can be seen in Figures 3 and 4. Analysis results of the RT-PCR to ITS1 gene expression of T. evansi shown in Table 5. This data shows change level gene expression in groups control and group treatment given extract red ginger (Zingiber officinale Roscoe). The presentation results in this aim to evaluate effectiveness extracting red ginger as anti-Trypanosoma agent through decline ITS1 gene expression. Positive results under mark control, more and more small mark ct his so increasingly tall positive and vice versa.

Table 3. Determination the efficacity of red ginger (Zingiber officinale Roscoe) extract as an anti-Trypanosoma agent on PCR test.

Fig. 3. Determination the efficacity of red ginger (Zingiber officinale Roscoe) extract as an anti-Trypanosoma agent on primer sequence PCR test.

Fig. 4. Determination the efficacity of red ginger (Zingiber officinale Roscoe) extract as an anti-Trypanosoma agent on RT-PCR test graph.

Table 4. Determination the efficacity of red ginger (Zingiber officinale Roscoe) extract as an anti-Trypanosoma agent on primer sequence PCR test.

Histological image results of the liver of mice infected with T. evansi

Histological picture of the liver infected mice T. evansi after giving extract red ginger (Zingiber officinale Roscoe) is presented in Table 6 and Figures 5 and 6a–e. These data show difference level damage network liver in each group treatment, therefore it can be used for evaluating potential extract red ginger in repair condition the existence to pathology liver consequence infection T. evansi. Infection T. evansi in mice cause various change pathology, especially in a functioning liver as center metabolism, detoxification, and regulation immune system in histological, liver infected mice generally show degeneration cell hepatocytes, necrosis focal, sinusoidal swelling, and infiltration cells inflammation.

Table 5. Determination the efficacity of red ginger (Zingiber officinale Roscoe) extract as an anti- Trypanosoma agent on RT-PCR test.

Table 6. Histological scores of the liver in mice infected with T. evansi.

Fig. 5. Average score results histopathology liver mice infected with Trypanosoma in each treatment. Description Average Score: K+=control positive, K−=control negative , Dose 1=Extract red ginger 30 mg/kg BW, dose 2=Extract red ginger 45 mg/kg BW, dose 3=Extract red ginger 60 mg/kg BW.

Fig. 6. (a) Control positive (Tryponicidal), Description: Arrow Red: Bleeding , Black: Necrosis, Yellow: Inflammation, Blue: Fat degradation, White: Edema, Magnification 400×. (b) Control negative (T. evansi), Description: Arrow Red: Bleeding , Black: Necrosis , Yellow: Inflammation, Blue: Fat degradation, White: Edema, Magnification 400×. (c) Extract red ginger 30 kg/mg BW, Description: Arrow Red: Bleeding, Black: Necrosis, Yellow: Inflammation , Blue: Fat degradation, White: Edema, Magnification 400×. (d) Extract red ginger 40 kg/mg BW, Description: Arrow Red: Bleeding, Black: Necrosis, Yellow: Inflammation, Blue: Fat degradation, White: Edema, Magnification 400×. (d) Extract red ginger 40 kg/mg BW, Description: Arrow Red: Bleeding, Black: Necrosis, Yellow: Inflammation, Blue: Fat degradation , White: Edema, Magnification 400×.

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Discussion

Screening test phytochemicals extract red ginger rhizome

The phytochemical screening of red ginger (Zingiber officinale var. rubrum) rhizome extract revealed the presence of several important bioactive compounds, including alkaloids, flavonoids, tannins, polyphenols, and saponins. These compounds are known to contribute significantly to the pharmacological and therapeutic activities of red ginger.

The detection of alkaloids indicates the potential of the extract to possess biological activities such as antimicrobial, antiparasitic, and analgesic effects. Alkaloids are nitrogen-containing compounds that can interfere with the metabolism of pathogens and disrupt their cellular functions.

Flavonoids were also found abundantly in the extract. These compounds are well known for their strong antioxidant properties, which help neutralize free radicals and protect biological tissues from oxidative stress. Moreover, flavonoids may enhance immune responses and inhibit the growth of various microorganisms, including protozoan parasites such as T. evansi.

The presence of tannins and polyphenols further supports the potential health benefits of red ginger extract. These compounds can precipitate proteins, inhibit microbial enzymes, and strengthen cell membranes, thereby preventing infection and inflammation. Their antioxidant capacity also plays a vital role in maintaining the stability and function of host cells during parasitic infection.

Meanwhile, saponins were detected through the froth test, indicating their role as natural surfactants. Saponins are known to cause lysis of parasite cell membranes by forming complexes with sterols, which leads to the breakdown of the parasite’s structural integrity. This mechanism contributes to the anti-Trypanosomal activity observed in the red ginger extract.

Overall, the results of the phytochemical screening demonstrate that red ginger rhizome extract contains a rich composition of bioactive compounds that may work synergistically to produce antiparasitic, antioxidant, and immunomodulatory effects. These findings provide scientific evidence supporting the traditional use of red ginger as a natural therapeutic agent and justify further studies on its mechanism of action and potential applications in treating parasitic infections such as T. evansi (Herawati and Saptarini, 2020).

Effectiveness test results red ginger extract against amount T. evansi to as anti-Trypanosoma

In addition, the extract red ginger also works as an immunomodulator with increaseing activity phagocytic macrophages and stimulate production cytokines. So that system defense body mice more optimal in opposing the infection. This repair reflected in the decline amount parasite in blood as well as increased hematological parameters, such as amount erythrocytes and hemoglobin levels. Thus, extract red ginger not only pressing rate development parasites, but also improve condition physiological infected mice (Erlita et al., 2022).

Compared to with antitrypanosomal conventional drug like suramin or diminazene aceturate which is often cause side effect, extract red ginger relatively safer and can used as companion therapy. Although, the effect antiparasitic from red ginger tend nature dose-dependent, meaning the taller dose within safe limits, the more its effectiveness is also great in reduce parasitemia. Because that, research more carry on still required for determine optimal dose and security term long before applied in a way more wide (Chekwube et al., 2014).

Overall, results study show that extract red ginger potential as agent phytotherapy in control infection Trypanosoma in mice, through mechanism antiparasitic direct, role antioxidants, as well as modulation system immune. Findings this support utilization red ginger as alternative herbal therapy that is complementary to treatment conventional trypanosomiasis (Peter et al., 2009).

Effectiveness test results red ginger extract against the life force of T. evansi to as anti-Trypanosoma

Findings this in line with report (Pascual-Vázquez et al., 2023) which states that herbal extracts with content polyphenols and flavonoids play a role important in reduce parasitemia in animal models of trypanosomiasis. Likewise, results study (Idris et al., 2017) who reported that giving extract antioxidant -rich plants capable hinder development T. brucei with pressing stress oxidative and damaging membrane cell parasites. Content bioactive red ginger, such as gingerol, shogaol, and flavonoids, are thought to work through similar mechanism, which is called bother metabolism energy parasites as well as trigger damage Trypanosoma plasma membrane.

Improvement network assessment mice in groups treatment close relation with ability extract red ginger in pressing development parasites inside blood. Content compound bioactive, such as gingerol, shogaol, and flavonoids are known own activity antiparasitic as well as antioxidants, which do not only hinder multiplication Trypanosoma, but also reduces stress oxidative and damage network host. Mechanism this supports life extension mice post infection.

This result in line with study (Feyera et al., 2014) who reported that decrease in parasitemia in test animals correlates direct with repair survival rate. Another study by Fotsing Yannick Stéphane et al. (2021) also showed that use extract antioxidant-rich plants capable extend age animal infected Trypanosoma through protection to damage liver and spleen which are the main target organs infection.

Additionally, a study by Idris et al. (2017) reports that giving extract plant with content phenolic tall can improve physiological status and endurance stand life mice infected T. brucei , with mechanism main in the form of modulation system immunity and suppression replication parasites. Research results this also supports findings (Egbe-Nwiyi et al., 2020) which states that therapy combination herbal extracts with drug trypanocidal conventional generate life span more animals long compared to use drug single.

Analysis of in vivo test results on red ginger extract as an anti-Trypanosoma

Research result this consistent with findings (Feyera et al., 2014) who reported existence connection significant between reduction of parasitemia with increased survival rate of test animals after giving herbal therapy. Research (Ramadhani et al., 2023) also shows results similar, where the statistical test show existence difference significant (p < 0.05) between group treatment with control in infected animals T. brucei, proves effectiveness extract antioxidant -rich plants in hinder development parasites.

In addition, (Mergia et al., 2016) find that using extract polyphenol-rich plants extend life span test animals significant, with analysis statistics which shows difference meaningful compared to control. These results support findings study now, where red ginger, which is rich in gingerol, shogaol, and flavonoids, can improve physiological status mice through inhibiting stress oxidative and damage network consequence infection T. evansi.

Analysis TSI DNA expression with real time PCR

In a molecular way, ITS1 in Trypanosoma rDNA is often used as a PCR/qPCR target because drinking coffee plural and give good specificity for detection and quantification burden parasites. With targeting ITS1, qPCR is capable of detect parasites at very low levels and monitoring parasitemia dynamics in general, more sensitive compared to parasitological conventional techniques. In African Animal Trypanosomosis, ITS1-based qPCR has been shown to be sensitive for T. brucei, T. congolense, and T. vivax, as well has adapted for T. evansi through the design of TaqMan/ITS1 specific primers.

In context therapy phytopharmaceuticals, extracts red ginger (rich in 6-gingerol, 6-shogaol, zingerone, and phenolics) indicates activity antitrypanosomal in vivo: reduces parasitemia and improves continuity life mice infected Trypanosoma (Priyowidodo et al., 2023 ). Proposed mechanism covers stress oxidative directed at parasites, disorders membrane, as well as modulation inflammation more hosts controlled. Studies on infected mice T. brucei report decline parasitemia and safe levels in a way toxicology of extracts methanol ginger; findings this give base biological that component active ginger can pressing burden parasites (Suprihati et al., 2022).

When extracting red ginger given in the T. evansi and load models parasite measured with qPCR targeting ITS1, the expected results is increase Ct value in the group treatment compared to control infected, which means decline the number of ITS1 genes (aka decreased burden parasite) (Perrone et al., 2018). Overall analytics, decline relatively can counted with method ΔΔCt; its interpretation not a “blackout” ITS1 transcription” (because ITS1 is an rDNA spacer), but rather decrease copy of ITS1 because the decline amount parasites. Thus, " the decline ITS1 expression” here equivalent with decline cargo parasite.

Validity approach

This reinforced by studies showing superiority qPCR (ITS1/TaqMan) sensitivity for T. evansi compared to method parasitology (Behour et al., 2019).

From the side diagnostic molecules, it is necessary to note that a number of studies comparing markers. Primer TBR-1/2 in conventional PCR is sometimes reported more sensitive than ITS1 for T. evansi local, but ITS1 remains superior for quantification of cross species and surveillance (especially in qPCR or amplicon-sequencing format) so that it is still relevant for therapy monitoring based change burden parasites. Combinations of ITS1 with other markers are also common for increase accuracy taxonomytype (Dongmei and Fan, 2022).

In a way biological, decline post-delivery ITS1 signal extract red ginger can associated with: (1) effect cytotoxic selective on parasites (e.g. disruption membrane/mitochondria, imbalance redox), (2) increase control immune host through characteristic antioxidant-anti-inflammatory ginger pressing damage pro- parasitic networks and environments, as well as (3) inhibition proliferation parasites, so that rate replication (and accumulation) copy of ITS1) is reduced. Evidence review latest also confirms capacity antioxidant/immunomodulator relevant ginger for condition infection chronicle (Gaithuma et al., 2019).

Histological image results of the liver of mice infected with T. evansi

Giving extract red ginger (Zingiber officinale Roscoe) is proven to give effect protective to damage network liver consequence infection T. evansi. it is not a separated from content bioactive main red ginger, those are, gingerol, shogaol, and zingerone which have anti-inflammatory properties antioxidant, anti-inflammatory, and immunomodulator (Leigh et al., 2015). Compound This capable pressing lipid peroxidation, inhibiting production cytokines proinflammatory, as well as increase activity enzyme endogenous antioxidants such as superoxide dismutase and catalase (Cooper 2024). Similarity study (Ramadan et al., 2024) is infection T. evansi in mice cause various change pathology, liver infected mice generally show degeneration cell hepatocytes, necrosis focal, sinusoidal swelling, and infiltration cells inflammation. This Change is related with response immune host to infection parasites and increasing production radical free which causes stress oxidative (Dkhil et al., 2021).

Observation result histology in the group treatment show existence repair structure hepatocytes compared to with group IC without therapy. In the group given extract red ginger, visible decrease degeneration hepatocytes, reduced infiltration cell inflammation, as well as more sinusoidal structures regular. This is indicates that extract red ginger own potential hepatoprotective through mechanism emphasis stress oxidative and inflammatory (Sharma et al., 2023).

Study previously support findings this, for example studies (Dkhil et al., 2021) which shows that giving extract ginger in animals test capable protect liver from damage oxidative consequence material chemistry hepatotoxic. In addition, a report by (Bekkouch et al., 2022) also showed that extract ginger capable repair architecture histology liver and lower signs inflammation. Thus, improvements in histology liver infected mice T. evansi after giving extract red ginger show that plant this potential used as therapy supportive in reducing impact pathological trypanosomiasis.

In a way overall, mechanism protection this possibility big related with ability red ginger in lower stress oxidative, reducing response inflammation, as well as increase power stand body to infection parasites. These strengths the utilization of red ginger as candidate material experience in support treatment of trypanosomiasis.

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Conclusion

Utilization of red ginger extract (Zingiber officinale Roscoe) has been shown to have potential as an anti-Trypanosoma agent. Administration of the extract was able to reduce the expression of the T. evansi ITS1 gene. Significantly compared to IC, indicating an inhibitory effect on parasite development. In addition, histopathological examination of the livers of mice showed improved tissue structure, characterized by reduced necrosis, degeneration, and inflammatory cell infiltration in the treatment group compared to the negative control. These results indicate that red ginger extract not only plays a role in suppressing T. evansi infection molecularly but also provides a protective effect against liver damage. Thus, red ginger extract has the potential to be developed as a natural-based alternative therapy for the control of trypanosomiasis.

Acknowledgments

The researchers would like to thank the Faculty of Health Sciences, Anwar Medika University, National Research and Innovation Agency (BRIN), Bogor, Indonesia, Research Center for Parasitology, Center for Assembly and Modernization Veterinary, Bogor for all the facilities provided during the research.

Conflict of interest

The authors declare no conflict of interest

Funding

The authors acknowledge Ministry of Education, Research, and Technology.

Author's contributions

A.M.C., Y.A.K.N., Y.A., I.F.A., F.E., D.A.K.: conceived the idea and manuscript drafting. A.M.C., I.F.A., F.E., D.A.K.: acquisition, analysis, and interpretation of data. YES, YES: critically read and revised the manuscript for intellectual content. All authors have read and approved the final manuscript. All authors have read, reviewed, and approved the final version of the manuscript.

Data availability

All data are available in the manuscript.

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