Trypanosomiasis is an extremely zoonotic, infectious protozoan disease of humans and animals, including cats (Panigrahi et al., 2015). It’s a debilitating and fatal disease caused by multiple species of Trypanosoma. Feline trypanosomiasis is reportedly pre-dominantly caused by Trypanosoma brucei and Trypanosoma evansi (Mohammed et al., 2022), both of which elicit similar clinical signs and symptoms in felids. Natural infections of Trypanosomiasis in felines are very rare as they are more resistant compared to other vertebrates (Misra et al., 2016). The disease is primarily transmitted through biting flies such as Stomoxys, Tabanus and Triatomids bugs, however, certain studies have shown secondary transmission can occur through ingestion of carcasses of the recently infected with cats (Nwoha, 2013), leading to reported transborder migrations (Devi et al., 2025). Clinical signs appear within 2-3 weeks post-bite, initially as a local skin inflammation (chancre) at the bite site, gradually manifests into pyrexia, anorexia, anaemia, enlargement of superficial lymph nodes, conjunctivitis, corneal opacity, peri-ocular unilateral or bilateral edema, due to subsequent spread of the infection into the blood stream and parasitaemia. In protracted cases, progressive paroxysm and emaciation eventually causes death of the animal (Tarello, 2005). Thin blood smear examination using a Romanowsky stain (such as Giemsa) for staining and examination under a microscope at 100× magnification, following the method of Umeakuana et al., (2019), is considered to be the most effective on-field method of diagnosing trypanosomiasis. Species specific confirmatory diagnosis is done using polymerase chain reaction (PCR) (Gaithuma et al., 2019).
       
Therapeutic management of trypanosomiasis chiefly involves usage of anti-protozoal drugs, like diminazene aceturate, but traditionally, the medicine practitioners have been using various ethnoveterinary practices to control and prevent animal trypanosomiasis using medicinal plants as both trypanocidals and fly repellents, respectively. Ethno-veterinary practices prevailed in India since the ancient times of the Atharva Veda, recognised as a reservoir of many traditional medicine practices, including animal treatment prescriptions. As per WHO, more than eighty per cent use traditional knowledge to treat animal and human diseases are used in most underdeveloped and developing countries (Jabbar et al., 2005). Ethno-veterinary can be a major game-changing alternative to antibiotics in treatment of various ailments in animals, hence preventing the multi-drug resistance phenomenon, which is a rising concern in medicine. Despite the vast popularity of traditional ways for treating animal illness, there is little to no documentation about ethno-veterinary practice that are effective in the therapeutic or preventive management. The aim of the current study is to report the prognosis and progression of the disease recovery in the affected cats, subjected to two different therapeutic approaches using modern medicine and ethnoveterinary practices, respectively.

The present work consists of 120 clinical cases presented at the veterinary clinical complex and Department of Veterinary Medicine, Rajendranagar. Cats ranging from 6 months to 2 years of age, were studied over a period from 2022 to 2024 and screened for the presence of Trypanosomia spp, on the basis of patient’s symptoms, duration of the discomfort, history of recurrence and were subjected to various detailed physical and clinical examinations. Initial screening for positive cases was done through wet blood film examination, conducted using blood samples collected from the superficial ear vein. Blood smear examination with Giemsa staining was performed to rule out the presence of hemoprotozoans. Blood samples were collected into sterile vials and subjected to further analysis. Heamtological parameters were estimated using EDAN haematology analyzer and biochemical parameters were estimated using EXIGO Veterinary biochemistry analyzer. Based on the results of the hematological test parameters, the wet blood film examination and the Giemsa blood smear examination, 42 out of 120 cats were screened positive for the presence of Trypanosoma. The results were recorded accordingly.
       
For the confirmatory diagnosis, PCR assay was carried out by conventional PCR method. The sample mixture consisted of 2.5  μl of 10X PCR buffer, 0.5  μl of 10 mMdNTP mix, 1.5 μl of 25 mM MgCl₂, 1.0 U of recombinant Taq DNA polymerase, 1 μl each (20 pmol) of the primers and 5 μl of template DNA was isolated from 2 mL of the collected blood sample. The volume was made up to 25 μl with nuclease-free water. The PCR cycling conditions were initial denaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec and extension at 72°C for 1:30 min and the final extension was performed at 72°C for 5 min. The PCR products obtained were checked for amplification by electrophoresis on a 1.5% agarose gel and visualized using gel documentation system, using ITS-1 gene as a marker for amplification.
       
For therapeutic management, the 42 case studies were divided into two groups Group 1 and Group 2, based on the history, duration and severity of the clinical signs, tentative prognosis and hematological values. Written consent of the owners and Animal Ethic committee approval has been collected and preserved by the author(s) for the study. The cats were subjected to two different therapeutic protocols, namely the allopathic medicine and the ethno-veterinary medicine (EVM), respectively. Group 1, comprising of cats (n=21) with extremely poor prognosis, severe hematological abnormalities and history of recurrent loss of body condition, was treated with Diminazene aceturate at a dosage of 3.5 mg/kg body weight, following the allopathic medicine protocol.
       
The leaf extract of the medicinal plant, Moringa oleifera, belonging to the order Brassicales and family Moringaceae, has been used for the ethno-veterinary treatment of the subjects of Group 2, comprising of cats showing initial onset of symptoms (n=21). The Moringa oleifera leaves were procured from the Shamirpet forest-farm land, in Telangana, India. Preparation of the M. oleifera leaves extract (MOLE) was done following the protocol described by Abubakar et al., (2022) in his experimental studies. These procured moringa leaves were then washed thoroughly in distilled water and allowed to air dry on a clean decontaminated table, in a ventilated room. The dried leaves were then pulverised into powder extract using clean and dry mortar and pestle. Two hundred grams of the powder was soaked in 1 litre of ethanol for two days, with proper care taken to stir periodically to prevent clumping. The extract was filtered and the filtrate was concentrated and collected as the moringa oleifera leaf extract (MOLE), ready to be used as medicine for oral administration route.
       
Control group comprising of healthy cats was used for recording the statistical analysis of the treatment protocols followed.

On physical examination, the common clinical signs observed among the affected cats included anorexia, fever, enlarged superficial lymph nodes, conjunctivitis with corneal opacity (Fig 1), peri-ocular edema, bilateral lacrimation, anterior uveitis (Fig 2), pale mucus membranes, restlessness, dehydration and emaciation in protracted cases.




       
The average of the results obtained are as depicted in (Table 1 and Table 2).




       
The haematological examination revealed decrease in the Haemoglobin concentration, erythrocytic count, neutrophils and PCV values, accompanied by leucocytosis and eosinophilia. Thrombocytopenia, without clumping of platelets were recorded. Dehydration was a consistent finding in all the affected cats.
       
The biochemical evaluation revealed Hypoglycaemia, hypoproteinaemia, hypoalbuminemia, decrease in both total and direct bilirubin values, with significant increase in the blood urea nitrogen (BUN). Serum globulin, ALT, ALP, cholesterol and creatinine values were unremarkable and well within the normal range.
       
The Giemsa-stained thin blood smear examination revealed extra-erythrocytic spindle-shaped trypomastigote forms of Trypanosoma spp (Fig 3) and motility of the same was recorded in the wet blood film examination.


       
As the confirmatory diagnosis, gel electrophoresis done by using (ITS1) gene as the marker for gene amplification between molecular size marker 100-1,200 base pair, recorded the presence of Trypanosoma evansi at 205-300 bp ladder sequence, Lane 1 (Fig 4).


       
Treatment protocols included Group 1 comprising of 21 out of the 42 positive cases, were treated with Diminazene aceturate at a dosage of 3.5 mg/kg body weight deep i/m single dose, along with fluid therapy at 30 ml/kg body weight, supportive and symptomatic therapy. The treatment was continued at the frequency of one dose weekly, for five consecutive weeks, in cats that did not show any noticeable improvement after the single dose of Diminazene aceturate (Ganjwala, 2025). All the 21 cases showed slow and gradual recovery based on their respective prognosis, with 100% recovery within 7-10 weeks. 17 cases showed an improved appetite after 2 sessions of the treatment. Group 2 comprising of the other half, i.e.; 21 positive cases, were treated with Ethnovet therapy using the leaf extract of Moringa oleifera, commonly known as the “Drumstick leaves”, as used by a large percentage of tribal community as an anti-trypanosomal herb even to this day, in many communities where immediate allopathic treatment is not available or affordable. Leaf extract from Moringa oleifera was administered orally at 200 mg/kg daily for 14 days. During the treatment, it was recorded that the recovery of 11 cases was gradual with good prognosis, while 6 cases showed fluctuating recovery, with unpredictable prognosis. 4 out of 21 cases undergoing ethnovet therapeutic management succumbed to their deteriorating health condition and died. All the cats in Group 1 showed positive response to Dimenazene aceturate and complete clinical recovery was observed in all the cases. 
       
The physio-clinical findings of lethargy, anorexia, gradual emaciation, muco-purulent ocular discharges, ataxia, pale mucous membranes, edema around eyes observed in the infected cat with Trypanosoma spp. were similar to those reported by Tarello (2005); Priyowidodo et al., (2016); Aregawi (2019) and most recently by Shekhar et al., (2026). Ocular signs might be due to the localization of organisms or formation of immune complexes in the uveal tract, with concentrated granular deposition along the inner angle of the cornea, gradually causing severe damage to the corneal endothelium, alongside causing clouding of the cornea affecting vision (Greene, 2012). Anaemia could be attributed to the damaged erythrocytic membranes caused by lipid peroxidation (Sivajothi and Reddy, 2018), while the virulence of the infecting parasite population, age, nutritional status and breed of the host influence the severity of anaemia, as corroborated by Thirunavukkarasu (2000). Distinct decrease in TRBC, HB concentration and PCV (Al-Eodawee et al., 2024) resulting in distinct anaemia, can be due to the parasitaemia, as explained by the studies of Da silva et al. (2009). The increase in WBCs due to an overall increase in lymphocytes, eosinophils, basophils and monocytes and decrease of neutrophils recorded can be attributed to the secondary bacterial infections, as corroborated by the other studies (Al-Badrani, 2012; Marwa and Aloba, 2022). Trypomastigote stage of the Trypanosoma species were observed in microscopic examination of blood smears stained with Giemsa, while distinct movements of motile intracellular trypanosomes were observed in the wet blood film examination (Solikhah et al., 2019). Trypanosoma organisms compete with host for blood glucose, which can be the cause of noticeable hypoglycaemia observed among the Trypanosoma infected cats, recorded as a consistent biochemical parameter in many cases. A single dose of Diminazene aceturate DIM @ 3.5 mg/kg Bwt was recorded to completely eliminate Trypanosomes from the bloodstream, just few hours after its administration (Giordani et al., 2016). According to reports from previous studies carried out around the nation (Tolossa et al., 2013), medicinal herbs, including the crude leaf extracts of moringa plants, are the most effective when administered orally, compared to other parenteral routes of administration. In rural India, locally available medicinal plants and their extracts, are commonly utilized to manage livestock parasitic diseases, due to prior traditional practices leading to satisfactory results and lack of immediate availability of allopathic medicines. A total of 18 types of medicinal plants were indicated for ethnoveterinary practices against animal trypanosomiasis and as a tsetse fly repellent, through studies conducted around the world.
       
Moringa oleifera supplement reduces tissue and organ damage caused during trypanosomal infection (Shetty and Naryana, 2007), as a result of the potent antioxidant effect of the crude extract (Cosirini et al., 1997). Antitrypanosomal activity of Allium sativum L. (Garlic) were recorded in previous studies (Sharma, 2009; Salem, 2006). Withania somnifera (Sharma, 2009), Aloe vera (Sharma, 2009; Dutta, 2007) and Nicotiana tabacum L. (Rocha, 2005) were also reported for their antileishmanial effect, alongside Moringa leaves.

From the above study and statistical analysis, it can hence be concluded that Moringa oleifera leaf extract, as used as a key extract in EVM against trypanosomiasis, even though very much effective, have not yet been scientifically validated for their claimed antitrypanosomal or trypanocidal activity. Hence further research on the ethnovet protocol and anti-protozoal management is required, to be used at par with the current allopathic therapy. The documentation of use of ethnovet protocols will lay the baseline data for further research on phytochemical and pharmacological constituents of the same, alongside the toxicological studies and their anti-oxidant properties, to confirm Ethnoveterinary uses, in near future.

The present study was supported by PVNRTVU.
 
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The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal Care and Handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.