Abstract
In a controlled experiment, we compared renal-hepatal, haematological and organ histopathology responses in chickens treated with Crude extracts of Capsicum annuum L. and Carica papaya L. to those treated with synthetic anthelmintics. Twenty-one indigenous seven-week-old chickens were fed on Nuvita® feeds Uganda limited and adlib tap municipal water. They were divided into seven groups of three chickens per group. The treatments were; CPLe (Carica papaya L. ethanol extract), CPLa (Carica papaya L. acetone extract), CAFe (Capsicum annuum L. ethanol extract), CAFa (Capsicum annuum L. acetone extract), levamisole, piperazine and phosphate buffered saline (PBS). CPLe, CPLa, CAFe, CAFa were given at a dose of 0.48 g per bird (1.37 g/kg body weight) as determined from a previous in-vitro experiment. Levamisole hydrochloride was given at 25 mg/kg body weight and piperazine citrate at 100 mg/kg body weight. The control group received 0.2% dimethyl sulfoxide (DMSO) in PBS. The treatments were individually administered orally and repeated on the second day. The birds were bled and sacrificed one week after treatment. Blood was submitted for haematology and harvesting of serum for renal-hepatal function tests. The heart, kidneys and liver were also harvested for histological examinations. CPLe caused significantly raised albumin compared to CAFe (p = 0.02), levamisole caused significantly raised AST compared to CAFe (p = 0.04). All extracts and synthetic anthelmentics increased the number of eosinophils, indicating an increased inflammatory response. CPLa, CAFe, CAFa and levamisole were toxic to the kidneys. All extracts were toxic to the liver except CPLe. Piperazine and levamisole were as well toxic to the liver.
Contribution: Plant extracts are not necessarily safer than synthetic anthelmintics and should be used with caution.
Keywords: extract; herbal; synthetic; toxicity; eosinophilia; sodium; lesions; kidney; liver.
Introduction
Synthetic and natural therapeutic products can both induce toxicities, especially when administered at very high doses (Abdel-Daim et al. 2018). Over the years, natural products, including those from plants, have been used to manage several conditions in animals. Most (81%) of the plant extracts were reported to have toxic effects in various animal tissues, unless dosed prudently (Viegi & Vangelisti 2011). Although many plants have been evaluated for their ethnoveterinary potential, few of them have been assessed for toxicity (Habeeb 2010; McGaw & Eloff 2008; Sunder 2016).
Previous studies determined the cytotoxic effects of several plant extracts on parasitic agents but have been silent on their effects on the hosts. Grades of toxicity may vary for the same plant from different geographical locations because of the possible differences in levels of soil minerals and contaminants (González-Miqueo et al., 2010). Plants accumulate toxic materials, especially in areas of high environmental toxicants (Ssempijja et al. 2020). Many ethnomedicinal plants are known to contain unacceptable levels of heavy metals (Kasozi et al. 2021). High levels of lead and zinc in plants predispose to renal diseases (Wismer 2015). Some plants even containing negligible levels of heavy metals have been associated with nephrotoxicity for unknown reasons (Baudoux et al. 2017).
Hypovitaminosis A in animals has been associated with renal diseases (Cojean, Larrat & Vergneau-Grosset 2020), while hypervitaminosis D3 also causes renal diseases. High vitamin C concentration increases iron uptake, which predisposes animals to renal diseases. Damage to the kidney of any cause manifests with changes in the concentrations of blood urea nitrogen (BUN), creatinine, uric acid and changes in serum osmolality (Gounden, Bhatt & Jialal 2021; Kluwe 1981). Changes in kidney weight and histopathological changes are also plausible indicators of nephrotoxicity in laboratory animals. Although piperazine citrate was said to have wide safety margin, its toxicity manifests as swollen kidney, enlarged ureters and visceral gout (Shumard 1956). Levamisole is said to increase plasma creatinine levels in other species, but it is not known whether this happens even in chicken. The renal function effects of anthelmintics in chicken are not well known.
Haematological parameters are affected by animal physiological condition, genotype, age, sex, nutrition, climatic conditions and prevailing pathology. The effects of piperazine citrate on haematological parameters of chicken are not well known. The effects of levamisole hydrochloride on haematological parameters depend on the number of given doses: a single dose increases the parameters, while triple doses decrease the parameters (Kuropka et al. 2022).
There is no information on haematology of indigenous chicken of Uganda when exposed to therapeutic dozes of plant extracts compared to that of commercial dewormers for nematodes (levamisole and piperazine citrate). The effect of ethnoveterinary practices (EVPs) on the haematological parameters of chicken has not been widely documented. This is the first study in Uganda reporting the effects of Carica papaya L. and Capsicum annuum L. on chicken haematological parameters.
Various plant extracts and commercial drugs have been shown to have toxic effects on the cardiovascular system of animals. Some of the pathologic effects of chemotherapeutic agents and plant extracts include, but are not limited to foci of haemorrhages on various organs, myocardial changes, vascular damage and necrosis of myocardium. Other effects may include: acute inflammatory response, fibrinous or oedematous exudates in various tissues including cardiac muscle fibres. Toxic substances may cause congestion of coronary vessels (Lateif et al. 2024).
Hepatoxicity may manifest as hepatomegaly, presence of haemorrhages, focal necrosis, discolorations, accumulation of lipids in hepatocytes, jaundice, biliary tissue proliferation, focal fibrosis and hyperplasia (Sathiyanarayanan & Arulmozhi 2007).
Piperazine and levamisole are known to remain as drug residues in the tissues of chickens, but the organ-toxic effects in chickens have not been progressively assessed (El-Kholy & Kemppainen 2005; Ke, Chen & Lin 2024). Levamisole acute toxicity has been reported in mice (Almawla & Al Baggou 2023), but extensive toxicity studies have not been appraised in chickens.
The use of EVPs has been studied in other species and cause various complications, but such studies have not been conducted in chickens. The effect of EVP on the immune system, nutritional status and metabolism of chicken is not known. The rising campaign for EVP alternatives in chickens requires a concomitant appraisal of their effects on body systems. This study intended to compare the toxicological indices of ethno-anthelmintics (C. annuum L. and C. papaya L.) to the commonly used synthetic anthelmintics (piperazine citrate and levamisole hydrochloride). The indices considered here are haematology, renal functions, liver functions and selected organ histopathology.
Research methods and design
Experimental design
The design was adopted with modifications from the National Research Council (NRC 2006). An experimental design to study the safety of C. papaya L. leaves extract, C. annuum L. fruits extract, piperazine citrate and levamisole hydrochloride chicken anthelmintics was conducted by comparing selected organ histo-toxicology, hepato-renal functional tests and haematological parameters. A cohort of indigenous chicks (Gallus gallus domesticus) in the Soroti district (Uganda) were left to scavenge with flocks for 6 weeks before being selected for caging in the 7th week. In the 7th week, which was also the acclimatisation week, the chickens were fed on standard broiler finisher from Nuvita feeds (Uganda Millers Limited®), and ad libitum tap water was provided. In the 8th week, the chickens were subjected to treatments on 6th and 7th May 2024. The chickens from the different groups were then transported to the College of Veterinary Medicine post-mortem laboratory, where whole blood samples were collected. Chickens were then euthanised, and organs (liver, heart and kidneys) were harvested.
Climatic conditions of Soroti district
Soroti district is located at latitude 1o 42’ 47.4516’’N and longitude 33o36’ 22.986’’ E. In May 2024, the temperature ranged from 18.9 °C to 27.1 °C, with an average humidity of 84%. The rainfall was 192 mm (7.56’’), and the ultraviolet (UV) index ranged from 3 mW/m2 to 5 mW/m2.
Preparation and storage of plant extracts
Leaves of C. papaya L. and fruits of C. annuum L. were obtained from Soroti district, Eastern Uganda, during the rainy season (May 2023). The plants were identified and authenticated by senior botanists from the College of Natural Sciences, Makerere University. A conventional Soxhlet extraction method was used to obtain plant extracts (Hirondart et al. 2020). Freshly collected plant material was washed with tap water to remove observable debris. Plant material was air dried in the shade for 2 weeks before grinding to a fine powder using a coffee grinder. Ten grams of ground plant material and 5 g of pumice stones were placed in a cellulose thimble plugged with cotton wool. The setup was placed in the conventional Soxhlet extraction apparatus containing 300 mL of solvent. Extraction was performed using a solid to liquid ratio of 1 to 12 (g/mL) for 8 h. The extraction was done in duplicate using analytical-grade ethanol and acetone as solvents. The C. papaya L. ethanol extract was labelled CPLe and the acetone extract CPLa. The C. annuum L. ethanol extract was labelled CAFe and the acetone extract CAFa. The extract was concentrated under vacuum and conserved at 4 °C.
Management of study chickens
Traditionally hatched chicks of indigenous breed were left to scavenge free-range with the hens to expose them to nematodal parasites in the environment. Faecal samples were collected from the chickens and examined for nematode infection in the 7th week. The extent of infection was graded, and chickens with a worm burden of at least 200 eggs per gram of faecal sample and weighing between 300 g and 350 g were selected for the experiments. The chickens were then put into cages in groups of three chickens per cage, with each cage having an area of 0.16 m2. They were fed on grower mash from Nuvita feeds (Uganda Millers Limited®) and given ad libitum tap water. The detail of the composition of the feeds is shown in Table 1. Birds were left to acclimatise in the cages for 1 week before treatments were given. During the free-ranging period, no supplementation was provided; however, the animals received vaccination against Newcastle disease.
| TABLE 1: Composition of grower finisher feed used in the study. |
Administration of treatments
Seven separate treatment groups were set up in triplicate: CAFa, CAFe, CPLa, CPLe, piperazine citrate, levamisole hydrochloride and 0.2% DMSO. The lowest concentration of compound that paralysed the highest number of Ascaridia galli worms in the in vitro experiments (0.08 g/mL) was doubled to determine the concentration of the extracts that were used in chickens (Zirintunda et al. 2025). This was to simulate the discriminating concentration concept. The extracts were administered in 3 mL volumes (0.48 g) per chicken per day. Piperazine citrate was given at 100 mg/kg body weight as recommended by the manufacturer and levamisole hydrochloride at 25 mg/kg body weight also as recommended by the manufacturer. DMSO (0.2%) in phosphate-buffered saline (PBS) was administered as the negative control. The treatment was repeated on the following day. Birds were sacrificed a week after treatments in the post-mortem room of the College of Veterinary Medicine, Makerere University.
Organ collection and histological slide preparation
The chickens were euthanised by mechanical cervical dislocation (Boyal et al. 2020). Chickens were sacrificed by severing the cervical region using a surgical blade. Post-mortems were conducted, and the target organs (heart, kidneys and liver) were collected in 10% neutral buffered formalin solution for 48 h. The fixed specimens were trimmed and placed in labelled cassettes, then placed in formalin again and finally transferred into the tissue processor. The tissues were processed in a semi-automated processor, based on an established procedure (Dey 2018).
Measurement of hepato-renal function
Blood urea nitrogen, sodium (Na+), chloride (Cl-) and creatinine (Cre) were determined using a semi-automatic clinical analyser, DIRUI-7000 (Jilin Jingquan Medical Equipment Limited®, Changchun, China). Uric acid and the liver function parameters – aspartate aminotransferase (AST), alanine transaminase (ALT), glutamyl transferase (GT), total bilirubin (TB), direct bilirubin (DB), total proteins (TPs), albumin (ALBU) and globulins (Globu) – were determined using Cobas® C311 analyser (Roche, Germany).
Measurement of haematological and blood parameters
Blood was collected by jugular puncture (Kelly & Alworth 2013) into pink top tubes containing ethylenediaminetetraacetic acid (EDTA). Gentle inversion was done to mix the blood with the anticoagulant, and processing for haematological analysis was conducted immediately.
Manual blood smear review (MBSR) and manual differential leukocyte counts (MDLCs) were done as described by Comar, Malvezzi and Pasquini (2017). Meticulous examination of well-prepared stained blood smears with keen assessment of any morphological changes was conducted by a team having 10 years to 40 years’ experience in MBSR, in the central diagnostic laboratory of the College of Veterinary Medicine and Biosecurity, Makerere University.
Statistical analysis
Data were entered in Microsoft (MS) Excel for cleaning and transferred to SPSS® version 26 for analysis. Descriptive statistics were done to obtain the mean, median, standard deviation and confidence intervals. One-way analyses of variance (ANOVA) was performed, followed by Tukey’s honest significant difference (HSD) multiple comparison. Statistical significance was considered at p ≤ 0.05.
Toxicological assessment
The protocols were adopted with modifications from the Organisation of Economic Cooperation Development (OECD 1996). The renal function tests, liver function tests and the haematology parameters of the treatments were compared to their control; a treatment having one or more aspects significantly different was considered toxic. The significance levels and the number of significant aspects showed the toxicity levels. For organ histopathology, the heart, kidney and liver of the three birds per treatment were assessed. The number of organs with observable lesions over the total number of organs per treatment was the scoring method. Having no observable lesions for a treatment (n = 0/9) was considered safe, having one organ with observable lesions (n = 1/9) was considered moderate and having more than one organ with observable lesions was considered toxic (safe < n = 1/9 > toxic).
Ethical considerations
Ethical clearance to conduct this study was obtained from the Uganda National Council for Science and Technology (UNCST Ref A220ES). The guidelines for research during the coronavirus disease 2019 (COVID-19) pandemic concerning safety of respondents (UNCST 2020) were followed. An ethical review certificate was acquired from the School of Veterinary Medicine and Animal Resources Institutional Review Board (IRB) (SVAR_IACUC/93/2021). Permission to conduct research was also granted by the Soroti District Veterinary Office.
Results
Renal function parameters
The results of BUN, sodium, chloride, creatinine and uric acid measurements are shown in Table 2.
| TABLE 2: Renal function parameters in chickens treated with herbal and synthetic anthelmintics. |
Blood urea nitrogen
There was no significant difference between the different plant extracts and the control treatments. There was no significant difference in the BUN levels for the extracts and piperazine citrate treatments (p > 0.05). There was no significant difference in the BUN levels for the extracts and levamisole hydrochloride (p > 0.05).
Sodium
There was no significant difference between the different plant extracts and the control treatments; however, CAFa extract demonstrated significantly different (lower) levels of sodium compared to CPLe extract (p = 0.046). There was no significant difference in the sodium levels for the extracts and piperazine citrate treatments (p > 0.05). There was no significant difference in the sodium levels for the extracts and levamisole hydrochloride (p > 0.05).
Chloride
There was no significant difference between the different plant extracts and the control treatments. There was no significant difference in the chloride levels for the extracts and piperazine citrate treatments (p > 0.05). There was no significant difference in the chloride levels for the extracts and levamisole hydrochloride (p > 0.05).
Creatinine
There was no significant difference between the different plant extracts and the control treatments (Table 3). There was no significant difference in the creatinine levels for the extracts and piperazine citrate treatments (p > 0.05). There was no significant difference in the creatinine levels for the extracts and levamisole hydrochloride (p > 0.05).
| TABLE 3: Multiple comparisons of renal functions in chickens treated with herbal and synthetic anthelmintics. |
Uric acid
There were no significant differences in the uric acid levels among the different extracts (p > 0.05). There was no significant difference in the uric acid levels for the extracts and piperazine citrate treatments (p > 0.05). There was no significant difference in the uric acid levels for the extracts and levamisole hydrochloride (p > 0.05).
There was no difference in the effects on renal parameters between the plant extracts (CPLa, CPLe, CAFa and CAFe) and the synthetic anthelmintics (piperazine citrate and levamisole hydrochloride) Table 3 (p > 0.05).
There was no significant difference in BUN, sodium, chloride, creatinine and uric acid when comparing the treatments and control birds. There was a significant difference in Na+ levels when comparing chickens treated with CAFa and CPLe (p = 0.046), CAFe (p = 0.005) and piperazine citrate (p = 0.04).
Liver function parameters
The results of the liver function parameters are shown in Table 4. There was no significant difference in the effects on liver function parameters for plant extracts compared to the control (p > 0.05). There was no significant difference in the effects on liver function parameters for piperazine citrate compared to the control (p > 0.05). There was no significant difference in the effects on liver function parameters for levamisole hydrochloride compared to the control (p > 0.05). There was no significant difference between the effects of the various plant extracts on the liver function parameters (p > 0.05) except for serum albumin, where CPLe caused significantly higher albumin levels compared to CAFe (p = 0.02), see Table 5.
| TABLE 4: Liver function parameters in chickens treated with herbal and synthetic anthelmintics. |
| TABLE 5: Multiple comparisons of liver function parameters. |
There was no significant difference between the effects of the plant extracts and piperazine citrate on liver function parameters (p > 0.05). There was no significant difference between the effects of the plant extracts and levamisole hydrochloride on liver function parameters except for serum AST, where levamisole hydrochloride caused significantly higher levels of AST compared to CAFe (p = 0.04), see Table 5.
Haematology
The results of the haematological parameters are shown in Table 6.
| TABLE 6: Haematological parameters from chickens on herbal and synthetic anthelmintics. |
There were no significant differences in the haematological parameters when comparing chickens treated with CPLa, CAFa, CPLe, CAFe, piperazine citrate, levamisole hydrochloride and those in the control group, except for eosinophils. There was a significant difference in the percentage of eosinophil between chickens exposed to CPLa and the control group (p = 0.017). There was a significant difference in the percentage of eosinophils between chickens exposed to CAFa and the control group (p = 0.000). There was a significant difference in the percentage of eosinophils between chickens exposed to CPLe and the control group (p = 0.017). There was a significant difference in the percentage of eosinophils between chickens exposed to CAFe and the control group (p = 0.001). There was a significant difference in the percentage of eosinophils between chickens exposed to levamisole hydrochloride and the control group (p = 0.001). There was a significant difference in the percentage of eosinophils between chickens exposed to piperazine citrate and the control group (p = 0.000) (Table 7).
| TABLE 7: Multiple comparisons of haematological parameters in chickens treated with herbal and synthetic anthelmintics. |
| TABLE 7 (Continues…): Multiple comparisons of haematological parameters in chickens treated with herbal and synthetic anthelmintics. |
There was no significant difference in haematological parameters when comparing chickens that were treated with plant extracts (CPLa, CPLe, CAFa and CAFe) and those that were treated with synthetic anthelmintics (piperazine citrate and levamisole hydrochloride) (p > 0.05).
Organ histopathology
Heart
For all treatments, there was no necrosis of myocardium, no foci of haemorrhages or form of vascular damage observed, Figure 1.
 |
FIGURE 1: Histopathology micrographs of heart sections from chickens treated with plant extracts and synthetic anthelmintics (H&E stain): (a) heart of chicken treated with CAFa, (b) heart of chicken treated with CAFe, (c) heart of chicken treated with CPLa, (d) heart of chicken treated with CPLe, (e) heart of chicken treated with piperazine, (f) heart of chicken treated with levamisole and (g) heart of chicken treated with PBS (control). In all treatments, there were no lesions in the heart. |
|
Kidneys
CAFa caused observable kidney lesions in 1 out of 3 of the chickens, CAFe caused observable kidney lesions in all the three chickens (n = 3/3), CPLa caused observable kidney lesions (n = 2/3) chickens, CPLe did not cause any observable lesions in any of the three chickens (n = 0/3), piperazine citrate did not cause any observable lesions in any of the three chickens (n = 0/3), levamisole hydrochloride caused observable kidney lesions in 1 out of 3 of the chickens, PBS did not cause any observable lesions in any of the three chickens (n = 0/3), Figure 2.
 |
FIGURE 2: Histopathology micrographs of kidney sections from chickens treated with plant extracts and synthetic anthelmintics: (a) kidney of chicken treated with CAFa (wide haemorrhages in renal cortex and medulla), (b) kidney of chicken treated with CAFe (renal haemorrhages and interstitial nephritis), (c) kidney of chicken treated with CPLa (renal congestion and haemorrhages), (d) kidney of chicken treated with CPLe (no observable lesions), (e) kidney of chicken treated with piperazine (no observable lesions), (f) kidney of chicken treated with levamisole (renal congestion and haemorrhages), (g) kidney of chicken treated with PBS (no observable lesions). |
|
Liver
CAFa caused observable liver lesions in 1 out of 3 chickens, CAFe caused observable liver lesions in 1 out of 3 chickens, CPLa caused observable liver lesions in 1 out of 3 chickens, CPLe did not cause any observable liver lesions in any of the three chickens (n = 0/3), PBS did not cause any observable liver lesions in any of the three chickens (n = 0/3), piperazine citrate caused observable liver lesions in 1 out of 3 chickens and levamisole hydrochloride caused observable liver lesions in 1 out of 3 chickens, Figure 3.
 |
FIGURE 3: Histopathology micrograph of liver sections from chickens treated with plant extracts and synthetic anthelmintics: (a) liver of chicken treated with CAFa (severe periportal necrotising hepatitis), (b) liver of chicken treated with CAFe (multi-focal hepatitis), (c) liver of chicken treated with CPLa (severe widespread periportal necrotising hepatitis), (d) liver of chicken treated with CPLe (no observable lesions), (e) liver of chicken treated with piperazine (periportal hepatitis), (f) liver of chicken treated with levamisole (moderate periportal hepatitis) and (g) liver of chicken treated with PBS (no observable lesions). |
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Summary of organ toxicity findings
The safety score was assumed as: safe < 1 out of 9 > not safe. The order of toxicity was CPLe and control (n = 0/9), piperazine citrate (n = 1/9), levamisole hydrochloride and CAFa (n = 2/9), CPLa (n = 3/9) and CAFe (n = 4/9).
CAFa caused no observable lesions in chicken hearts. It caused observable lesions in the kidneys of 1 out of 3 chickens and in the liver of 1 out of 3 chickens. It generally caused a toxicity score of two out of nine (n = 2/9) and was considered not safe.
CAFe caused no observable lesions in chicken hearts. It caused observable lesions in the kidneys of 3 out of 3 chickens and in the liver of 1 out of 3 chickens. Its general toxicity score was four out of nine (n = 4/9) and was therefore considered not safe.
CPLa caused no observable lesions in chicken hearts. It caused observable lesions in the kidneys of two chickens out of three (n = 2/3) and in the liver of one chicken out of three chickens (n = 1/3). Its total toxicity score was three out of nine (n = 3/9) and was also considered not safe.
CPLe caused no observable lesions in chicken hearts, kidneys or liver. Its total toxicity score was 0 out of 9 (n = 0/9), the same as PBS and was considered safe.
Piperazine citrate caused no observable lesions in the chicken hearts or kidneys but caused observable lesions in the liver of 1 out of 3 chickens (n = 1/3). Its total toxicity score was 1 out of 9 (n = 1/9) and was considered moderately safe.
Levamisole hydrochloride caused no observable lesions in the chicken hearts but caused observable lesions in the kidneys of 1 out of 3 chickens (n = 1/3) and in the liver of 1 out of 3 chickens (n = 1/3). Its total toxicity score was 2 out of 9 (n = 2/9) and was considered not safe.
Discussion
The results were analysed by linear regressions followed by Tukey’s honestly significant difference test. CAFa, CAFe, CPLa and CPLe, when compared to PBS, did not significantly affect renal function parameters; however, CAFa needs to be used with caution because it led to lower sodium in blood. The extracts did not show significant effects on the electrolyte balance with regard to BUN, sodium, chloride, creatinine and uric acid. Reading renal functions, the extracts are safe for use at the given concentrations. Although the extracts did not affect the sodium electrolyte balance, CAFa caused minor lowering of blood sodium compared to other extracts and piperazine citrate. Lower blood sodium predisposes to decreased blood pressure, increased uric acid, increased haematocrit, shock and heart failure (Julian 1987; Sad 2016). The plant extracts were as safe as the synthetic anthelmintics regarding their effects on BUN, sodium, chloride, creatinine and uric acid balance. The extracts, as well as piperazine citrate and levamisole hydrochloride, had no effects on the metabolic actions that yield uric acid. The treatments did not affect the glucose transporter 9, which regulates serum uric acid in chicken (Ding et al. 2021). The extracts did not significantly affect the liver functions. Chronic toxicity studies are required to confirm the findings. However, CPLe caused higher albumin compared to CAFe, implying that it led to greater dehydration than CAFe. Levamisole hydrochloride increased AST compared to CAFe, implying that levamisole chloride is more likely to cause liver damage compared to CAFe (Khanam et al. 2016).
The plant extracts, as well as piperazine and levamisole, caused increased blood eosinophils. Although helminth infestations can also cause eosinophilia (Fuentebella, Fridge & Bass 2011; Maxwell 1987), the findings point to more than that. The placebo with higher helminth loads had less blood eosinophils compared to the treatments possibly because the inflammatory action of the treatments was greater than that of the helminths, which leads to eosinophilia. Eosinophils are involved in the starting inflammatory responses, can act as antigen-presenting cells, are particularly responsible for defence against parasitic infections and modulate host’s general immune responses (Rothenberg & Hogan 2006). Like piperazine and levamisole, the extracts possibly induced hypersensitivity, autoimmune disorders or neoplasm defence mechanisms, which manifest as eosinophilia (Kanuru & Sapra 2023); however, the mechanisms driving eosinophilia require further investigation. The monocytosis observed in levamisole hydrochloride treatments implies that it caused more stress and autoimmune disorders compared to the control. Monocytosis is observed in stressful conditions and during the onset of autoimmune disorders (Taebipour et al. 2017). The plant extracts were as safe as piperazine citrate but safer than levamisole chloride regarding their effects on the haematological parameters balance.
The different plant extracts, piperazine and levamisole did not show any observable lesions in the heart muscle sections of chickens. The anthelmintic extracts are not toxic to the heart muscles and major vessels. The findings relating to C. annuum L. (acetone red pepper extract and ethanol red pepper extract) are in agreement with Mandal et al. (2023), who reported that C. annuum L. is cardio-protective. Sanati, Razavi and Hosseinzadeh (2017) also reported that C. annuum L. has beneficial effects on the heart. The findings relating to C. papaya L. (acetone pawpaw leaves extract and ethanol pawpaw leaves extract) are in agreement with Haramaki et al. (1995), Hasimun et al. (2020), Kong et al. (2021), who described the cardio-protective action of C. papaya L.
Acetone red pepper extract (CAFa), ethanol red pepper extract (CAFe) and acetone pawpaw leaves extract (CPLa) were toxic to kidneys (Figure 2). The actions of C. annuum L. are in agreement with Jabar and Jassim (2023), who observed kidney histological alterations among animals treated with C. annuum L. Yuca (2022) also reported kidney damages among animals that received very high doses of C. annuum L. The mechanisms of CPLa toxicity are unknown because C. papaya L. is reported in the literature as nephro-protective (Francis et al. 2020). It is possible that acetone yields some compounds with nephro-toxic effects compared to ethanol. However, further investigations are needed to prove the variations in the compounds yielded with the different solvents and the renal effects of such compounds. Levamisole hydrochloride was equally toxic to the kidneys, but piperazine citrate was safe. Nephro-toxic effects of levamisole have been reported in mice (Almawla & Al Baggou 2023); however, there are no such earlier reports in chickens. There is a need to confirm the findings regarding the safety of levamisole in chickens at the efficacious doses.
All extracts were toxic to the liver except CPLe; equally, piperazine and levamisole were toxic. The observed hepatotoxic effects of C. annuum L. in chickens are in disagreement with (Das et al. 2018; Effendi & Sukmanadi 2021; Oloruntola et al. 2024), who reported its hepato-protective actions in other species. The concentrations in this experiment were very high compared to the concentrations in literature where C. annuum L. is reported to have had protective action. Carica papaya L. is also reported to be hepato-protective as for CPLe (Awodele et al. 2016; Shaban et al. 2021). However, CPLa was toxic, possibly because of the difference in compound yields of acetone compared to ethanol. The hepatic findings of the effects of piperazine were in agreement with those of Bakhrebah, Abo-Znada and Ramadan (2011), who observed histological changes in the chicken liver. The findings regarding levamisole were in agreement with Miah et al. (2020), who reports that high doses of levamisole are detrimental to the liver. Overall, all plant extracts were toxic, except CPLe. Levamisole was also toxic, and piperazine was moderately toxic. CPLe was as safe as the placebo and safer than the two synthetic anthelmintics (piperazine and levamisole) that are commonly used by commercial chicken farmers in Uganda.
Strength and limitations
The experimental design used the same cohort of chickens to triangulate the toxicity evaluation using the parameters of haematology, renal function tests, liver function tests and organ histotoxicity. This integrated assessment is used to derive a trustable conclusion. The concentrations of the extracts used were proven in previous efficacy tests. The concentration of piperazine citrate and levamisole hydrochloride was that recommended by the manufacturers and other researchers. Phosphate-buffered saline treatment is included as the placebo.
The study focusses on the toxic effects at the effective concentration of the extracts. The effects of extremely high doses were not studied. Immediate effects were not studied because samples were collected 1 week after the last treatment. The study does not consider the effects of chronic use of the treatments because chickens were not kept for a year or 2 years. Isolated or individual parasite effects as possible confounders of toxicity were not assessed.
Implications or recommendations
Capsicum annuum L. and Carica papaya L. are possible treatment alternatives to modern pharmaceuticals for chicken helminth infestation. The organ toxicity study showed that CPLe was the safest extract. Other extracts caused observable organ toxicity lesions in the order of CAFa < CPLa < CAFe. The study demonstrates the relative toxicity of piperazine citrate and levamisole hydrochloride at recommended concentrations compared to plant extracts used as anthelmintics in chickens.
We recommend evaluating the toxicity of pure extracts such that the most effective fractions are scaled up as alternatives to synthetic anthelmintics. Toxicological assessment after prolonged use of the extracts should be evaluated with a larger sample size before recommending for wider use in chickens. The study should be repeated in different breeds of chicken in various geographical zones to identify any possible variations. The safety concerns of piperazine citrate and levamisole hydrochloride are critical requiring more studies for confirmation and to make decisions on concentrations. Safety studies of highly purified extracts are recommended.
Conclusion
The extracts C. papaya L. and C. annuum L. caused minor lowering of blood sodium and eosinophilia, and they were toxic to the kidneys and liver. Generally, there was no observed difference in safety between the plant anthelmintics and the synthetic anthelmintics (piperazine citrate and levamisole hydrochloride); all should be used with caution.
Acknowledgements
The authors are grateful for the technical help, goodwill and counsel provided by Mr Ojambo Vincent, Miss Atoo Juliet, Mrs Nanteza Sylvia Zirintunda, Mr Muteguya John Paul, Dr Muzoora Saphan, Mr Kisekka Mohammed, Mr Isabirye Bryan, Mr Kanyike and Mr Muyombya.
Competing interests
The authors reported that they received funding from the Busitema University staff development fund under the Vice chancellor’s office (2021/2022). The project was also supported by the Makerere University Research fund (MAK-RIF2021/2022), which may be affected by the research reported in the enclosed publication. The author has disclosed those interests fully and has implemented an approved plan for managing any potential conflicts arising from their involvement. The terms of these funding arrangements have been reviewed and approved by the affiliated university in accordance with its policy on objectivity in research. The authors have no other competing interests to declare.
Authors’ contributions
G.Z. and J.O.-A. conceptualised the study. J.K. contributed to the article writing (editing and review). G.Z. and P.V. sourced the funding; G.Z. performed the experiments supervised by S.B. and J.K. G.Z analysed the histological sections; P.V., A.S.N. and G.Z. analysed and interpreted the results. J.O.-A. revised the article and prepared it for submission.
Funding information
The research was partly supported by the Busitema University staff development fund under the Vice chancellor’s office (2021/2022). The project was also supported by the Makerere University Research fund (MAK-RIF2021/2022).
Data availability
Data are available at https://doi.org/10.6084/m9.figshare.28129970 (Zirintunda 2025).
Disclaimer
The views and opinions expressed in this article are those of the authors and are the product of professional research. It does not necessarily reflect the official policy or position of any affiliated institution, funder, agency or that of the publisher. The authors are responsible for this article’s results, findings and content.
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