Coccidiosis is an economically menacing disease affecting livestock and poultry (Mat Yusof and Md Isa, 2016). In small ruminants, coccidiosis is mainly caused by Eimeria species, which develop in both small and the large intestines and mainly affects young animals (Dakpogan et al., 2019). The disease is responsible for considerable losses in husbandry, due to reduced productivity, mortality, associated clinical and sub-clinical diseases and the cost of treatment and control measures (Etsay et al., 2020; Bawm and Htun, 2021). In Africa, coccidiosis is considered as a leading cause of mortality of young small ruminants and is compounded by the occurrence of other infectious and parasitic diseases such as helminthosis and pneumonia (Kagucia et al., 2020; Etsay et al., 2020). In Africa, prevalence of coccidiosis in goats was reported at  60% in Egypt (Mohamaden et al., 2018), 73% in Nigeria (Ikpeze et al., 2010), 85% in Ethiopia (Etsay et al., 2020) and 45% in Kenya (Maichomo et al., 2004).

 

Despite the aggressive use of anticoccidial drugs, coccidiosis continues to limit the productivity of livestock, especially that of small ruminants (Waller, 2006). This is due unfortunately to the rapid development of anticoccidials resistance (Hema et al., 2015), the higher cost of the latter and consumer concerns of drug residues in animal products. All these factors have led to a growing interest in alternative products to control coccidiosis. Phytochemicals from different types of botanical elements have been explored as sustainable alternatives to management of coccidiosis (Hady and Zaki, 2012). A wide variety of herbal extracts have been shown to have anti-parasitic activity, while others can enhance the immune system and growth performance, thereby helping the host to overcome coccidiosis infection (Zaman et al., 2015; Debbou-Iouknane et al., 2019). Among the plant extracts, bromelain obtained from pineapples (Ananas comosus) has been shown in a previous In vitro study, to have high activity against Eimeria spp. isolated from goats (Daiba et al., 2022). In furtherance of the latter study, we assessed the in vivo efficacy of chitosan nano-encapsulated bromelain (CNB) against coccidiosis in goats and investigated the possible toxic effects of the extract.

Ethical approval

 

The approval for goats’ experiments was obtained from the Animal Ethics Committee of University of Nairobi (REF: FVM BAUEC/2020/339). The study followed the design, animal husbandry practices and protocols approved by the committee.

 

Experimental site

 

The study was carried out from January to March 2022, at the animal facility located in Jomo Kenyatta University of Agriculture and Technology (JKUAT), Juja, in Kiambu County, Kenya. 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  to  1,300 mm while average temperature is 19.5°C (Menge et al, 2014).

 

Extraction and encapsulation of bromelain in chitosan

 

Bromelain was extracted from the peels of pineapples (Ananas comosus) sold at local market in Juja Sub-county, Kenya. The enzyme was extracted using the procedure described previously (Daiba et al., 2022). Briefly, 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 sulphate. Then, after 24 h of incubation at +4°C, extracted bromelain was purified using dialysis membrane (12 kDa). Encapsulation of bromelain in chitosan (Sigma Aldrich, USA) was done by ionic gelation method. The pellet obtained after encapsulation was frozen at -60°C and dried by placing in the freeze-dryer (MRC, Model FDL-10N-50-BA, Israel). The success of encapsulation of bromelain in chitosan nanoparticles was confirmed by Fourier transform infrared spectrophotometer analysis (FTIR).

 

Oral solution of chitosan nanoencapsulated bromelain (CNB) formulation

 

Oral solution of EB was made according the procedure described previously (Niazi, 2009a; Niazi, 2009b). The solvent contained  in the excipients included: Tween 20 (surfactant), sucrose (to enhance solubility and stabilize against denaturation of enzymes), Potassium sorbate (antimicrobial preservative), xanthan gum (suspending and viscosity agent) and propylene glycol (humectant and stabilizing agent) (Rowe et al., 2009). Casein enzymatic assays were performed to evaluate the proteolytic activity and specific activity at diverse storage conditions (temperature, exposition to the light and pH) (Barnes, 2014).

 

Experimental animals

 

Fifteen (15) Small East African goats, which were naturally infected with coccidia were purchased from farmers in Makima Ward in Embu counties in Kenya. The average age of the goats was 15 months old and weighed between 22 and 31 Kg. Acclimatization was done for two weeks and animals were tagged with ear tags for easy identification before the start of the experiment. The goats were kept in JKUAT’ goat house, where they were fed with 1.5 kg of wheat hay thrice per day, 1 Kg of concentrate made up of beet liquid molasses, maize germ, and soybean meal (Aroma Feed Suppliers, Kenya), and supplemented with feed blocks minerals (Aroma Feed Suppliers, Kenya).

 

Study groups and sampling of animals

 

The fecal samples were collected using sterile gloves from the rectum of goats and analyzed to determine the number of coccidia oocysts per gram (OPG) of feces using the McMaster method (MAFF, 1986). All the animals used in the experiments had an OPG of more than 10,000. The goats were randomly allocated into three (3) treatment groups. Groups 1 and 2 received 90 and 270 mg/Kg of CNB, respectively, while group 3 acted as the positive control where goats were administered Diclazuril at 5 mg/Kg (Diclazuril®, Sigma-Aldrich). The  above  dosages  were  chosen  based  on  th 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 3 days and the goats were monitored for 28 days after last day of drug administration.

 

In vivo assessment of anticoccidial efficacy

 

Fecal samples were collected from the rectum of goats, once per week, during the 4 weeks of monitoring. The fecal samples were analyzed using a modified McMaster technique to determine the coccidia oocyst counts (FOC) under light microscopy (Optical Element Corporation, Melville, USA) at 100× magnification (Joachim et al 2018). First, the burden of oocyst was evaluated before the treatment (pre-treatment samples at day 0), followed by evaluation of treatment efficacy, by assessing post-treatment fecal samples (days 7, 14, 21 and 28). Fecal Oocyst Count Reduction (FOCR) percentage was evaluated as previously described (Odden et al., 2018).

 

Assessment of acute toxicity effect

 

Following dosing, the animals were observed during the first 30 min and then periodically during the first 24 h. Special attention was given during the first 4 h, and daily thereafter, for a total of 28 days to observe any changes in general behavior and other physiological activities (OECD Guidelines, 2002; Parasuraman, 2011). Rectal temperature of goats was measured daily, each morning (8 h 30-9 h 30 am) using a digital thermometer (Kruuse Digital Thermometer; Jorgen Kruuse). The body weight of animals was recorded weekly. Two (2 mL) of blood was sampled weekly from each animal, from the jugular vein into blood collection tube 4 mL EDTA. The PCV was determined using the micro-hematocrit method (Shamaki et al., 2017). Afterward, blood samples were centrifuged for 14,000 rpm for 10 min to obtain the plasma. 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 Automated Clinical Biochemistry analyzer (Reflotron Plus System®, model: Cobas 4800 Detection Analyzer; India) (Emma et al., 2020; Wasso et al., 2020).

 

At 28^th day, post last day of drug administration, the goats were euthanized and gross pathology conducted according the procedure described previously (King et al., 2013). Sections of liver, kidney, spleen and heart were collected and preserved in 10% buffered formalin for 24 h before being processed for histology as described previously (Rousselle et al., 2019).

 

Statistical analysis

 

The collected data were entered into and analyzed using Graph Pad Prism 8.4.3. for data analysis. Descriptive statistics (means and standard deviations) were determined before conducting other statistical tests. The FOCR, PCV, weight, temperature and biochemical parameters of different groups were compared using Students t-test, with p < 0.05 indicating statistical significance.

Necropsy findings

 

There were no gross changes at necropsy. All the organs were normal and similar in both treatment groups to control   groups.  There   were   no   histological    lesions observed in the organs in both the treated and control groups (Figure 1).

 

 

The current study is a follow-up of the previous study which had assessed the in vitro activity of nano-encapsulated bromelain (CNB) on oocysts of Eimeria spp. isolated from goats in Kenya (Daiba et al., 2022). The previous study reported high anticoccidial activity of CNB comparable to that of commercial drug (Diclazuril). In the current study, CNB was been administered as an oral solution made by adding other excipients in order to increase its activity in the animal’s body (Rowe et al., 2009). The current study showed that, the proteolytic activity of oral solution of CNB was higher than crude encapsulated bromelain reported in previous studies (Hunduza et al., 2020; Wasso et al., 2020; Daiba et al., 2022). This high activity of the oral solution of CNB could be due to the lower pH (pH = 4.7 to 5) which would contribute to accelerate the release of bromelain from chitosan  nanocarriers during  casein protease assay. As described by Hunduza (2018), acidic conditions chitosan nanocarriers released more bromelain into solution than at neutral pH, and thus had a higher proteolytic activity. Further, the oral solution prepared using Tween 20, sucrose, potassium sorbate, xanthan gum and propylene glycol could have enhanced the stability of CNB in the gut of the goat making it more effective against the coccidial parasites. Previous studies have used water as the solvent (Wasso et al., 2020).

 

The results of the present study showed that the administration of oral solution of CNB for three days, significantly reduced the excretion of coccidia oocysts during the monitoring period. Our findings are in concordance with experiments using cysteine proteinase from others plants, which show that the enzyme having an efficacy against coccidian infections (Abdel-Tawab et al., 2020; Dakpogan et al., 2019; Juasook et al., 2017; Molan et al., 2009). Juasook et al. (2017) treated Eimeria tenella infected broilers with pineapple (Ananas comosus) crude extracts, and observed that the oocyst output decreased significantly and also inhibited sporulation. In their experiment, Dakpogan et al. (2019) reported a reduction of 59% of oocyst per gram (OPG) in chicks treated with Carica papaya crude extracts. On the other hand, Abdel-Tawab et al. (2020) showed that Astragalus membranaceus extracts reduced oocyst output and sporulation of Eimeria papillata infection in mice by 57%. As reported in a recent study in vitro efficacy (Daiba et al., 2022), the activity of CNB was due to the damage it causes to coccidia oocysts. The drug causes degradation of the coccidia shell wall, softening and destruction of the central cytoplasmic mass (Daiba et al., 2022, Juasook et al., 2017).

 

The present study also evaluated the toxicity effect of CNB on goats. As reported in a recent study evaluating the acute toxicity (Wasso et al., 2020), during the present study, there were no mortalities and clinical signs in the experimental goats.  On the other hand, a slight weight gain of the treated animals and this could show that the drug enhanced the growth performance of the goats.

 

During the current study, the levels of the assessed biochemical parameters were normal and were similar to the results reported by Wasso et al. (2020) where the goats were treated with EB at a dose of 30 mg/kg for 14 days, and in a single dose of 90 and 270 mg/kg. Likewise, this was confirmed by the absence of macroscopic and microscopic changes in the examined organs (Moss et al., 1963; Pavan et al., 2012). Moss et al. (1963) in rats have shown that bromelain when administered at 500 and 1500 mg/Kg/day was not toxic. Further, Pavan et al. (2012) did not observe toxic effects after 6 months administrating bromelain at 750 mg/Kg/day to dogs. All these results show that the drug is quite safe for use in different animals.

The current study reported high efficacy of CNB against coccidial infections in goats. Further the oral solution of CNB had no toxic effects at 270 mg/Kg on goats. Accordingly, this formulation of bromelain can contribute to management of coccidiosis in small ruminants. Further studies should be carried out to determine dosing regimens which can increase the efficacy of CNB against coccidiosis to 100%. 

The authors have not declared any conflict of interests.