CC BY
62023, Scienceline Publication
Worlds Veterinary Journal
World Vet J, 13(2): 285-292, June 25, 2023
DOI: https://dx.doi.org/10.54203/scil.2023.wvj30
Treatment Effects of Chitosan Nanoencapsulated Bromelain against Gastrointestinal Nematodes and Coccidia in Goats of Kenya
Ahmota Romain Daiba ' © , John Maina Kagira2* , Maina Ngotho © , James Kimotho © , and Naomi Maina ' ©
'Department of Molecular Biology and Biotechnology, Pan-African University of Institute of Basic Science, Technology and Innovation, Nairobi, Kenya 2Department of Animal Sciences, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya 3Department of Clinical Studies, Faculty of Veterinary Medicine, University of Nairobi, Nairobi, Kenya 4Innovation and Technology Transfer Division, Kenya Medical Research Institute, Nairobi, Kenya 5Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya 6Department of Livestock Science and Technology, National Higher Institute of Science and Techniques of Abeche, Chad *Corresponding author's Email: [email protected]
ABSTRACT
The management of gastrointestinal nematodes (GIN) and coccidiosis of livestock relies on the use of commercial anthelmintic; however, the excessive and frequent usage of these drugs has led to the substantial and dramatic development of anthelmintic and anticoccidial resistance. The present study aimed to evaluate the anthelmintic and anticoccidial efficacy of chitosan nanoencapsulated bromelain (CNB) against a wide spectrum of GIN and coccidia in goats. Additionally, the study assessed the safety of CNB in the goats. Bromelain was extracted from the pineapple peels and then encapsulated using chitosan. A total of 20 healthy male goats naturally infected with GIN and coccidia were used. The goats were separated into four treatment groups, with five goats per each. The CNB was orally administered at dosages of 270 and 90 mg/Kg, once daily for 60 days. Fecal egg counts (FEC), fecal oocyst counts (FOC), packed cell volume (PCV), aspartate aminotransferases (AST), alanine aminotransferases (ALT), urea, and creatinine were determined weekly. The goats were monitored for clinical signs daily, and their body weight was recorded weekly. The findings revealed that FEC reduction rates in the group that received 270 mg/Kg CNB and the group that received Albendazole were 73.41% and 79.54% at day 7 post-treatment. Also, the reduction of FOC in the group receiving 270 mg/Kg CNB at day 7 (84.12%) did not show a significant difference with Diclazuril (82.12%). The FEC and FOC were zero (reduction of FEC and FOC was 100%) at 28 days of treatment in goats treated with 270 mg/Kg CNB. During the monitoring period, no mortalities and no clinical signs were observed in the treated goats. The PCV, AST, ALT, creatinine, and urea levels for the goats in all groups were within normal limits. No pathological lesions were observed in the goat's organs. In conclusion, the results demonstrated that repeated (60 days) dosages of 270 mg/Kg had anthelmintic and anticoccidial effects and were safe for goats. The study recommends further investigation in a field setting involving more animals. This would allow the development of a novel product for managing helminthiasis and coccidiosis in ruminants.
Keywords: Anthelmintic, Anticoccidial efficacy, Bromelain, Chitosan, Encapsulation, Goat INTRODUCTION
Endoparasites such as gastrointestinal nematodes (GIN) and coccidia are a serious health concern in small ruminants across the world (Jansen et al., 2020; Wasso et al., 2020; Wondimu and Bayu, 2022). In many tropical countries, the main GIN affecting small ruminants is Hemonchus contortus, Oesophagostomum spp., Trichostrongylus spp., Ostertagia spp., Nematodirus spp., and Cooperia spp. (Maichomo et al., 2004; Waruru et al., 2005). These parasites cause a decline in milk and meat production resulting in severe economic losses in the livestock industry (Waruru et al., 2005; Ibrahim et al., 2014; Mat Yusof and Md Isa, 2016). On the other hand, coccidiosis is caused by protozoan parasites of the genus Eimeria with the main one being Eimeria ninakohlyakimovae, E. hirci, E. caprina, E. christenseni, E. jolchijevi, E. apsheronica, and E. arloingi (Etsay et al., 2020). These parasites contribute to enteric disease, particularly in young or stressed livestock leading to low production and high mortality (Faizal and Rajapakse, 2001; Mohamaden et al., 2018; Etsay et al., 2020). In most of the ruminants' production systems, GIN and Coccidia manifest as mixed or singles infections which compound the morbidity and mortality cases (Faizal and Rajapakse, 2001; Jansen et al., 2020; Bawm and Htun, 2021). In Kenya, the prevalence of gastrointestinal tract parasites in small ruminants is high, with the prevalence of up to 82% and 85% for GIN and coccidiosis, respectively (Maingi and Munyua, 1994; Waruru et al., 2005; Kanyari et al., 2009).
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To cite this paper: Daiba AR, Kagira JM, Ngotho M, Kimotho J, and Maina N (2023). Treatment Effects of Chitosan Nanoencapsulated Bromelain against Gastrointestinal
The management of GIN and coccidiosis of livestock relies heavily on the use of commercial anthelmintic and anticoccidial drugs (Kaplan, 2004; Noack et al., 2019). However, the excessive and frequent usage of these drugs has led to the substantial and dramatic development of anthelmintic and anticoccidial resistance to GIN and coccidia, principally in cattle, sheep, and goats (Hema et al., 2015; Mickiewicz et al., 2021; Potarniche et al., 2021). Regarding the growing threat to animal health and production posed by drug resistance, innovative medications, including those based on plant extracts need to be considered and developed as potentially sustainable alternatives (Sadr et al., 2022).
One of the potential anthelmintic and anticoccidial plants is pineapple (Ananas comosus), containing cysteine proteinases enzyme (Misran et al., 2019). The pineapple enzyme, bromelain, has been demonstrated to have anthelmintic and anticoccidial activities in vitro and in vivo levels (Githiori, 2004; Hunduza et al., 2020; Wasso et al., 2020; Daiba et al., 2022). The recent in vitro and in vivo studies by Wasso et al. (2020) and Daiba et al. (2022) called for the investigation of the efficacy of nanoencapsulated bromelain at enhanced and repeated doses. The study by Wasso et al. (2020) reported that nano-encapsulated bromelain caused an efficacy of up to 68.8% at 30 mg/Kg for 14 days of treatment. The aim of the current study was to use higher doses of nano-encapsulated bromelain to investigate the in vivo efficacy and toxicity of encapsulated bromelain in chitosan against GIN and coccidia in goats.
MATERIALS AND METHODS
Ethical approval
The approval for the goats' experiments was obtained from the Animal Ethics Committee of the University of Nairobi, Kenya (REF: FVM BAUEC/2020/339). The study followed the design, animal husbandry practices, and protocols approved by the Committee.
Experimental site
This controlled laboratory study was carried out between July 2021 and January 2022, at the animal facility located at Jomo Kenyatta University of Agriculture and Technology (JKUAT), Juja Campus, located just outside Kenya's capital city of Nairobi. The University is located at latitude 1°05 S and longitude 37°00 E, and it lies at an altitude of 1525 m above sea level with rainfall bimodal and ranges from 500 mm - 1300 mm while the average temperature is 19.5°C (Menge et al., 2014).
Extraction and encapsulation of bromelain in chitosan
The University Campus falls within the agricultural zone, where commercial pineapple cultivation forms a substantial part of agricultural activities. Pineapples were readily available at the local market. Bromelain was extracted from the peels of pineapples (Ananas comosus) sold at a local market in Juja sub-county, Kiambu County, Kenya. The bromelain was extracted using the procedure described previously by Hunduza et al. (2020). Fresh ripe pineapples were ground in a blender in sodium acetate buffer (pH = 7.4). The resultant crude extract was precipitated by adding 40% ammonium sulfate. After 24h of incubation at 4°C, extracted bromelain was purified using a dialysis membrane (size of 12 kDa). Bromelain in chitosan (Sigma Aldrich, USA) was encapsulated by the ionic gelation method (Hunduza et al., 2020). The pellet obtained after encapsulation was dried by placing it in the freeze-dryer (MRC, Model FDL-10N-50-BA, Israel) after frozen at -60°C. The Fourier transform infrared spectrophotometer analysis (FTIR) was used to confirm the success of the encapsulation of bromelain in chitosan nanoparticles (Hunduza et al., (2020).
Experimental animals
Twenty small East African male goats, which were naturally infected with GIN and coccidia, were purchased from farmers (before purchase, fecal samples were collected to diagnose positive animals for both GIN and coccidia infection) in Makima Ward in Embu County in Northeastern Kenya. Molecular identification by PCR showed that animals were infected by a wide number of nematodes (Haemonchus contortus, Trichostrongylus vitrinus, Trichostrongylus axei, Trichostrongylus colubriformis, Nematodirus filicollis, Oesophagostomum spp., and Ostertagia ostertagi). The average age of the goats was 15 months old, and they weighed between 22-31 Kg. Acclimatization to the diet was done for 2 weeks, and animals were tagged with ear tags for easy identification before the start of the experiment. The goats were group-housed at the JKUAT animal facility. The animals were kept in pens of 3 m x 3 m size and were fed with 1.5 Kg of wheat hay thrice per day, 1 Kg/animal of feed concentrate made up of beet liquid molasses, maize germ, and soybean meal (Aroma Feed Suppliers®, Kenya). The animals had access to essential minerals lick blocks (Aroma Feed Suppliers®, Kenya) and fresh drinking water ad libitum.
Treatment groups and sampling
Fresh fecal samples (10 grams) were collected using sterile gloves from the rectum of goats. These were analyzed to determine the number of eggs per gram (EPG) and coccidia oocysts per gram (OPG) of feces using the McMaster method (Cringoli et al., 2004). The animals formed self-control samples taken at the start of the study. All the animals
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used in the experiments had an EPG and OPG of more than 2,000 and 10,000, respectively. The goats were divided into four treatment groups of five goats. Groups 1 and 2 received 90 mg/Kg and 270 mg/Kg of CNB, respectively, while group 3 was considered as the positive control where goats were administered orally with 7.5 mg/Kg of Albendazole (Sigma Aldrich, USA) and 5 mg/kg of Diclazuril (Sigma Aldrich, USA), and group 4 was served as negative control (untreated goats). The above dosages were chosen based on the results of the bromelain toxicity and efficacy tests obtained in the previous studies (Wasso et al., 2020; Daiba et al., 2022). The treatment was done orally every morning for 60 days. The animals were monitored for 90 days after the last day of drug administration.
In vivo assessment of anticoccidial efficacy
Fecal samples were collected from the rectum of goats every 7 days after the start of drug administration up to the end of the monitoring period (150 days). The fecal samples were analyzed using a modified McMaster technique to determine the EPG and OPG (Coles et al., 1992; Odden et al., 2018). Fecal egg count reduction (FECR) and fecal oocyst count reduction (FOCR) percentages were evaluated as previously described by Coles et al. (1992) and Odden et al. (2018).
Assessment of toxicity effect
Following dosing, the animals were observed for acute toxicity during the first 30 minutes and then every 6 hours during the first 24 hours. Thereafter, the animals were monitored for any changes in general behavior, clinical and other physiological activities (OECD, 2002; Parasuraman, 2011). Rectal temperature of goats was measured daily, each morning (8 a.m. to 9:30 a.m.) coinciding with the time of drug administration) using a digital thermometer (Kruuse Digital Thermometer; Jorgen Kruuse). The body weight of animals was recorded weekly using a 100 Kg spring balance scale (Salter Model, Capital Scales - Pretoria, South Africa). Two mL of blood was sampled weekly from each animal from the jugular vein into a blood collection tube with 4 mL EDTA. The PCV was determined using the micro-hematocrit method (Shamaki et al., 2017). The plasma samples obtained were used to determine the levels of Aspartate aminotransferases (AST), Alanine aminotransferases (ALT), urea, and creatinine using standard diagnostic test kits on an Automated Clinical Biochemistry analyzer (Reflotron Plus System®, model: Cobas 4800 Detection Analyzer; India, Emma et al., 2020; Wasso et al., 2020).
Following the last day of drug administration, the goats (2 goats per group on day 60 and the rest on day 150) were sacrificed using 0.25 mL/Kg of Pentobarbital sodium (Leary et al., 2020) and gross pathology conducted according to the procedure described previously by King et al. (2013). Sections of liver, kidney, spleen, and heart were collected and preserved in 10% buffered formalin for 24 hours before being processed for histopathology (Rousselle et al., 2019).
Statistical analysis
Data were entered into Microsoft office excel (Microsoft® Office 365™, Microsoft, USA) and analyzed using Graph Pad Prism 8.4.3 (San Diego, CA, USA) for data analysis. Descriptive statistics (means and standard deviations) were determined. The FECR, FOCR, weight, temperature, PCV, and biochemical parameters of different groups were compared with those of non-treated goats using Students t-test (p < 0.05).
RESULTS
Efficacy assessment of chitosan nanoencapsulated bromelain
The results showed a relatively high reduction in EPG count mean in all treated goats (Figure 1). On day 7 of treatment, 73.41% (580 EPG), 62.61% (780 EPG), and 79.45% (530 EPG) of reduction of FEC were recorded for goats treated with 270 mg/Kg, 90 mg/Kg of CNB and Albendazole, respectively. Contrariwise, the FEC increased by 107.42% in the untreated group (negative control). The reduction in FEC count reached 100% between day 28 to 77 post-first day of treatment for goats treated with 270 mg/Kg of CNB. During the first month of treatment, there was no significant difference (p > 0.05) in fecal egg counts in goats treated with 270 mg/Kg of CNB and the group treated with 7.5 mg/Kg of Albendazole. In the negative control group, the FEC increased by 127.18% on day 28.
Efficacy assessment of chitosan nanoencapsulated bromelain on fecal oocyst count reduction
At the start of the study, the mean OPG in goats in all the groups was similar. Following treatment, the oocyst count reduced gradually between days 7 and 35 of treatment (Figure 2). The reduction percentage in oocyst counts on day 7 post-treatment was higher (p > 0.05) for the goats treated with 270 mg/Kg CNB (84.12%, 2,660 OPG) and Diclazuril (82.12%, 3,180 OPG) than those treated with 90 mg/Kg CNB (44.83%, 7,220 OPG). On the other hand, there was an increase in FOC in the animals in the negative control group. This represents a statistically significant reduction in FOCR, which was highest (p < 0.05) for goats treated with 270 mg/Kg CNB in comparison to those treated with 90 mg/Kg CNB. In addition, after 28 days of treatment, the FOCR was 100% for the goats treated with 270 mg/Kg.
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Toxicity assessment of chitosan nanoencapsulated bromelain
Clinical observations
During the entire period of observation, the administration of 270 mg/Kg of CNB for 60 days did not show any mortalities or clinical signs in the group. The rectal temperature of the animal body varied between 37 to 38.5oC and was within a normal range (Radostitis et al., 2000; Al-Bulushi et al., 2017) for the Small East African goat breed. Following the treatment, there were no significant differences in body temperatures (p > 0.05) in goats from different groups. The body weights of the goats before treatment were 25.00 ± 2.58 Kg (positive control Group), 16.50 ± 1.00 Kg (negative control), 16.25 ± 1.26 Kg (270 mg/Kg CNB), and 16.50 ± 1.00 Kg (90 mg/Kg CNB). After three weeks of treatment, the weight had increased to 18.88 ±1.93 Kg and 18.75 ±1.70 Kg of goats treated with 270 mg/Kg and 90 mg/Kg CNB, respectively, while the body weight decreased to 24.88±1.84 Kg for positive control (goats treated with Albendazole and Diclazuril) and to 16.13±1.08 Kg for negative control. The mean of body weights for the following treatment groups showed a significant (p < 0.05) increase (2.25 Kg) at the end of the study. This increase (compared to day 0) was statistically significant (p < 0.05). However, there was a decrease in weight by 0.62 to 1.62 Kg in goats of the positive control group (p < 0.05).
Effect of treatments on packed cell volume and biochemical parameters
Before treatment, the PCV levels were 32% (positive control group), 27.25% (negative control), 28.50% (270 mg/Kg CNB), and 28.25% (90 mg/Kg CNB). During the treatment, the PCV levels ranged between 25.25 to 30.00% for the goats treated with 270 mg/Kg and 90 mg/Kg CNB, 26.35 to 30.50% for positive control, and 18.25 to 26% for negative control. There were no significant differences (p > 0.05) in the PCV levels during the entire observation period.
During the treatment, AST ranged between 101 and 111 U/L for goats treated with CNB and between 115 and 121 U/L for the positive control group of animals. The ALT level was between 14.89 and 16.74 U/L and 16.51 and 17.93 U/L for goats treated with CNB and positive control, respectively. The creatinine level ranges were between 0.710 and 0.792 mg/L, while that for urea was between 33 and 35.06 mg/L for 270 mg/Kg and 90 mg/Kg CNB during the entire observation period.
Gross pathology and histopathological findings
No gross changes at necropsy were done on days 60 and 150 post first day of treatment. All the organs were normal and similar in both treatment and control Groups. The histopathological assessment showed no changes in the tissues in both groups treated with CNB (90 mg/Kg and 270 mg/Kg).
Figure 1. Fecal egg counts reduction in goats treated with chitosan nanoencapsulated bromelain (270 mg/Kg and 90 mg/Kg), Albendazole (7.5 mg/Kg), and untreated goats (negative control) in Kenya
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To cite this paper: Daiba AR, Kagira JM, Ngotho M, Kimotho J, and Maina N (2023). Treatment Effects of Chitosan Nanoencapsulated Bromelain against Gastrointestinal
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Figure 2. Fecal coccidial oocyst counts reduction in goats treated with chitosan nanoencapsulated bromelain (270 mg/Kg and 90 mg/Kg), diclazuril (5 mg/Kg), and untreated goats (negative control) in Kenya
DISCUSSION
The present study is a continuation of a previous investigation that evaluated the in vivo activity of chitosan nanoencapsulated bromelain (CNB) against GIN in Small East African goats in Kenya (Wasso et al., 2020). In the laboratory study, Wasso et al. (2020) observed an anthelmintic efficacy of 68.8% after administering CNB at 30 mg/Kg for 14 days. Administration of a single oral dosage of 90 mg/Kg and 270 mg/Kg CNB caused an efficacy of 9.5 % and 32.4%, respectively, in goats at 28 days post treatment (Wasso et al., 2020). The current study addressed the efficacy of CNB against natural infection of GIN and coccidia in goats. Present study results showed that administering CNB at higher doses considerably decreased the number of GIN eggs excreted by goats and reached 100% by 30 days post-treatment. Previous research on the anthelmintic action of pineapple (Ananas comosus) in sheep and goats has found that bromelain to have high efficacy against helminths (Hordegen et al., 2003; Domingues et al., 2013). However, when powdered leaves and plain bromelain were administered to sheep, Hordegen et al. (2003) and Domingues et al. (2013) reported a decrease in nematode eggs. The increased activity of CNB described by Wasso et al. (2020) and in the present study might be attributed to the encapsulation of bromelain with chitosan (Bhatnagar et al., 2014; Cheung et al., 2015). Nanoparticles, such as chitosan, boost the efficacy of drugs by avoiding enzymatic breakdown and increasing the drug's ability to access sites that would otherwise not be accessible to other drugs (Cheung et al., 2015). Bromelain, as previously described, induces cuticle deterioration and death of mature nematodes (Stepek et al., 2006; Behnke et al., 2008). Thus, the drop in egg excretion reported in vivo might be related to either adult worm death or a fall in female worm prolificacy. Stepek et al. (2005; 2006) found that papaya latex, which contains cysteine protease, significantly reduced worm load and egg yield in rodent GIN (Trichuris muris and Heligmosomoides polygyrus). Pascal et al. (2017) gave Newbouldia laevis (Bignoniaceae) to sheep and found it to be 55% effective against H. contortus and 19% effective against T. colubriformis. Hounzangbe-Adote et al. (2001) reported an 80% effectiveness rate of papaya (Carica papaya) seed powder on sheep strongyles after ten to fifteen days of therapy.
The present study showed that CNB significantly decreased the number of oocysts excreted by the goats to a level of efficacy comparable to the commercial drug (Diclazuril). This suggests that the enzyme is effective against coccidian infections as has also been reported elsewhere (Molan et al., 2009; Juasook et al., 2017; Dakpogan et al., 2019; Abdel-Tawab et al., 2020). Juasook et al. (2017) reported that after treating broilers chicken with crude pineapple extracts (Ananas comosus), Eimeria tenella oocyst production fell considerably (p < 0.05). On the other hand, Dakpogan et al. (2019) showed a 59% decrease in OPG in chicks fed with Carica papaya crude extracts. Bromelain has been shown to induce coccidia shell wall disintegration, softening, and loss of the core cytoplasmic mass (Juasook et al., 2017; Daiba et al., 2022).
The current study also evaluated the toxicity of CNB in goats. Similar to a previous study by Wasso et al. (2020), there were no mortalities or adverse clinical signs in the experimental animals. Further, the blood parameters such as PCV were within the species' normal range (Radostitis et al., 2000; Al-Bulushi et al., 2017), which implies that the
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longer administration duration was safe. The levels of AST, ALT, creatinine, and urea levels were also within the species' normal range (Radostitis et al., 2000; Jackson and Cockcroft, 2002). Similarly, there were no pathological lesions observed in the goats at postmortem, which agrees with the study by Wasso et al. (2020).
CONCLUSION
The current study found that CNB in chitosan exhibited high activity against GIN and coccidia infections in goats by lowering eggs and oocysts excretion. Further, CNB demonstrated no harmful effects on goats at repeated dosages of 270 mg/Kg. This sets the background and justification for further studies on strategies to improve efficacy through adjusting the dosage and duration of treatment with CNB. This should eventually lead to on-farm trials on the effectiveness of CNB against nematodes and coccidian. This could serve as validation ahead of a commercial venture to utilize this natural product as an alternative treatment of helminths and coccidian in small ruminant stock, but further study needs to prove this theory.
DECLARATIONS
Availability of data and materials
The data presented in this study are available by reasonable requests from the authors. Funding
This study was fully funded by the Pan-African University of Institute of Basic Science, Technology, and Innovation (PAUSTI),
Ethical consideration
All the authors have carefully examined all ethical issues concerning plagiarism, approval to publish, data fabrication and/or falsification, double publication, and submission.
Acknowledgments
The authors acknowledge Pan-African University of Institute of Basic Science, Technology and Innovation (PAUSTI), and Jomo Kenyatta University of Agriculture and Technology (JKUAT) for financially sponsoring this research and providing technical and infrastructural support used during the project.
Competing interests
No competing interests.
Authors' contributions
All authors contributed to the conception of the experiments. Ahmota Romain Daiba, John Maina Kagira, and Maina Ngotho planned the study design. Ahmota Romain Daiba, James Kimotho, and Maina Ngotho performed sample collection and laboratory work. Naomi Maina conducted the data analyses. Ahmota Romain Daiba and John Maina Kagira wrote the draft of the manuscript. All authors corrected, read, and approved the final manuscript.
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To cite this paper: Daiba AR, Kagira JM, Ngotho M, Kimotho J, and Maina N (2023). Treatment Effects of Chitosan Nanoencapsulated Bromelain against Gastrointestinal
