Livestock Research for Rural Development 28 (5) 2016 Guide for preparation of papers LRRD Newsletter

Citation of this paper

In vivo anthelmintic activity of pineapple (Ananas comosus Merr.) fruit peeling juice in semi-scavenging Philippine native chicken naturally co-infected with Ascaridia galli and Heterakis gallinarum

Joan Marie Y Cormanes, Harvie P Portugaliza and Ana Marquiza M Quilicot

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Visayas State University, Visca, Baybay City, Leyte, 6521-A, Philippines
hportugaliza@vsu.edu.ph

Abstract

Gastrointestinal helminths are important disease-causing agents in free-range and backyards chickens threatening poultry health and welfare, and impeding livelihood of smallholder farmers. The present study aimed to evaluate the efficacy of Pineapple (Ananas comosus) Fruit Peeling Juice (PFPJ) against Ascaridia galli and Heterakis gallinarum in naturally infected semi-scavenging Philippine native chickens. Specifically, the study investigated the anthelmintic property of PFPJ by using the following parameters: percent Fecal Egg Count Reduction Test (%FECRT), total worm counts, Packed Cell Volume (PCV), weight gain and selected organ weight. A total of 30 naturally infected native chickens of 1-2 month-old were distributed into five treatment groups (Placebo, 20 mg/kg BW levamisole, 1008 mg/kg BW PFPJ, 504 mg/kg BW PFPJ and 255 mg/kg BW PFPJ). Fecal egg per gram (EPG) counts were derived 3 times with one day interval using the modified McMaster technique and utilizing all four chambers of Whitlock Universal slide (analytical sensitivity of 30 EPG/chamber) during the pre-treatment, 7th and 14th days post-treatment periods. At the end of post-treatment, all chickens were weighed, blood collected for PCV determination and necropsied to obtain data on total worm counts and organ weight (i.e. liver, spleen and bursa of Fabricius). The anthelmintic efficacy of PFPJ was categorized into highly effective (>98% FECR), effective (90-98% FECR), moderately effective (80-89% FECR) and ineffective (<80% FECR).

Results showed significant EPG reduction after treatment among treated groups (p<0.05). Arithmetic mean %FECR at the dosage rate of 1008 mg/kg BW PFPJ was effective on the 7th day and highly effective on the 14th day post-treatment, whereas 504 mg/kg and 255 mg/kg BW PFPJ were both effective on the 14th day post-treatment. Significantly better geometric mean %FECR was also observed in 1008 mg/kg BW PFPJ compared to the placebo treated chickens (p<0.05). Worms recovered included A. galli, H. gallinarum and Raillitina spp. Chickens treated with PFPJ has numerically lowest A. galli infection and a significantly better weight gain compared to placebo (p<0.05). There were no significant changes observed in PCV values and selected organ weight. The present results suggest the potential of pineapple fruit peeling juice as an alternative anthelmintic in semi-scavenging native chickens.

Keywords: avian nematode, herbal anthelmintic, organic farming


Introduction

Gastrointestinal helminthiasis in chicken causes intestinal damage leading to blood loss and inefficient feed conversion resulting in emaciation. Ascaridia galli and Heterakis gallinarum are the two most common helminths infecting backyard chickens in tropical countries (Pattison 2008). A. galli is a large nematode that resides in the lumen of the small intestine with the larval stages responsible for mucosal invasion. The parasite causes enteritis and intestinal impaction with severity correlated to worm burden. H. gallinarum, on the other hand, resides in the ceca causing mild infection in chicken and serves as a mechanical vector for Histomonas meleagridis, a protozoan known to induce entero-hepatitis in turkey and other susceptible avian (Schwarz et al 2011).

The likelihood of obtaining high parasitic infections among native chickens in backyard setting is attributed to its scavenging behaviour where various insects, earthworms, human leftovers, and any other feed materials containing the infective stage of the parasite can be eaten. Hence the major concern in backyard poultry raisers, especially those that practice pasture type of farming system, is parasitism (Cabarles 2013; Permin et al 1997). A report of Rabbi et al (2006) demonstrated the highest case of helminthiasis among backyard chickens, wherein at least 80% of them harboured A. galli, H.gallinarum and Raillitina tetragona. Adding all the economic importance, vast distribution and high occurrence of these helminths, it is imperative to come up with a parasite control measure whenever farmer capacitation through chicken dispersal program is implemented in rural areas (Pattison 2008; Permin and Hansen 1998).

The use of chemical anthelmintic has long been considered in treating parasitic infections in poultry, however, widespread reports of anthelmintic resistance motivated many researchers to explore an alternative source (Behnke et al 2008; Islam et al 2008). Moreover, many commercial anthelmintic were often unaffordable and unavailable in remote areas so that majority of the farmers utilized indigenous plants as dewormer (Islam et al 2008). Pineapple (Ananas comosus) is one of the pioneer plants used and studied for its anthelmintic activity (Hossain et al 2015; Begum et al 2010; Hoque et al 2006).

The current study screened the anthelmintic potential of pineapple fruit peelings which are waste products in the pineapple market. Specifically, the study (i) evaluated the efficacy of Pineapple Fruit Peeling Juice (PFPJ) based on the percent fecal egg count reduction (% FECR) and total worm counts in Philippine native chickens co-infected with A. galli and H. gallinarum, and (ii) assess the packed cell volume (PCV), weight gain and weight of liver, spleen and bursa of Fabricius of PFPJ treated chickens.

The results of the study would serve as a reference tool for prospective anthelmintic alternatives intended to help local farmers in treating nematode infections.


Materials and methods

Study area

The study was conducted at the Animal House and Diagnostic Laboratory of the College of Veterinary Medicine, Visayas State University, Baybay City, Leyte, the Philippines (coordinate 10.7444 N, 124.7921 E). Experimental chickens were purchased from backyard raisers in the neighbouring villages. In general, the climate of the study cite is characterized by evenly distributed rainfall with no pronounced dry season. Humidity and ambient temperature may range from 80 to 90% and 27.2 to 28.2C, respectively (Portugaliza and Bagot 2015; LGU-Baybay 2013).

Preparation and management of the native chicken

A total of 30 semi-scavenging Philippine native chickens aged 1 to 2 months were derived from different backyard farms in selected barangays of Baybay City, Leyte. All chickens underwent fecalysis for parasite detection. Eggs of A. galli and H. gallinarum were counted together since they both resembled morphologically (Wongrak et al 2015). Chickens with fecal egg per gram (EPG) count above 150 were included in the study (Coles et al 1992). They were initially weighed, marked for identification and housed individually for one week acclimatization. The design of the cage was an adaptation from Ernst et al (1988) but involved the utilization of bamboo and wire mesh to construct a cage dimension of 9 inches x 12 inches x 14 inches. Fecal samples were collected through catching pan set below each chicken’s cage from 5:00 AM to 8:00 AM. During the entire study period, experimental chickens were fed twice a day with cracked corn grit and commercial feeds containing 19% crude protein. Clean water was always available and changed daily. Antibiotics, a combination of amoxicillin, bromhexine, and tiamulin with a dose of 250 mg/kg BW and electrolytes were given to individual chickens exhibiting signs of respiratory disease.

Preparation of pineapple juice from fruit peelings

Pineapple fruit peelings were obtained from the fruit stalls of Ormoc City Market. The peelings were weighed, minced finely, blended and sieved separating the juice from the pulp. The total volume recovered was recorded and weighed. This resulted in a weight per volume ratio of 84 g/mL. The physical characteristics which included odor, color, opacity, and consistency were noted.

Pineapple fruit peeling juice dosage and administration

Three dosage levels representing high, medium, and low dosages were prepared out of the stock solution. Experimental chickens under the positive control group were treated with 20 mg/kg BW levamisole, while the negative control were treated with water (placebo). Chickens in respective PFPJ treatment groups were treated per os after calculating the dose based on the formula below (Wanamaker and Massey 2009).

The stock solution used for PFPJ has a concentration of 84mg/mL and the dosage rates involved 1008 mg/kg body weight (BW), 504 mg/kg BW and 255 mg/kg BW.

Percent fecal egg count reduction (% FECR) and parasite recovery

Two grams of fecal samples per bird was processed following the modified McMaster technique of Zajac and Conboy (2012). Fecalysis was conducted three times with one day interval. All four chambers of Whitlock Universal Slide with an analytical sensitivity of 30 EPG per chamber were examined for eggs in every fecal sample. Eggs of A. galli and H. gallinarum were counted as one because differentiating the two parasites based on egg morphology is unreliable and difficult (Wongrak et al 2015). Fecal egg per gram (EPG) counts were derived during the pre-treatment, 7 th and 14th days post-treatment periods. The indicators for the anthelmintic efficacy of the varying dosage levels of PFPJ were the arithmetic (AM) and geometric (GM) means of the % FEC calculated based on the formula of Coles et al (1992)

Parasites located within the gastrointestinal tract were collected during necropsy. Collected parasites were placed in a petri dish plate containing Alcohol-Formalin-Acetic acid solution (AFA) and was counted. The number and localization of the parasites were recorded. Identification of the parasites were based on their host, localization and gross morphology as described by Taira et al (2003), Ramadan and Abouznada (1992) and Soulsby (1982).

Performance parameters

Weight gain of chickens, packed cell volume (PCV) and weights of the liver, spleen, and bursa of Fabricius were the selected performance parameters used in the study. Live weights were collected before and after the treatment period. Blood was also collected during the post-treatment period either from the jugular or wing vein using a 3 mL syringe with 23G needle. Three replicates of the microhematocrit were processed to get the mean PCV per bird. Chickens were euthanized and necropsied to obtain the weights (g) of selected organs, i.e. bursa of Fabricius, spleen and liver.

Experimental design

The experiment was laid out following a completely randomized design. There were five treatment groups with five replicates (1 chicken = 1 replicate). Each chicken has at least 1,157.50 EPG. There were 2 control groups; placebo served as the negative control treated with water and 20 mg/kg BW levamisole which served as the positive control. The PFPJ treated groups of the study represented the high (1008 mg/kg BW), medium (504 mg/kg BW), and low (255 mg/kg BW) dosages.

Data management and analysis

All data were managed in MS Excel 2013 and initially tested for Levene’s homogeneity test. Heterogeneous data (p<0.05) were normalized using the formula log (x+1). A Repeated Measures Analysis of Variance (ANOVA) was conducted to determine the statistical difference of mean fecal EPG count across time (pre-treatment versus post-treatment) and between subjects effects. Mauchly’s Test was run to check if sphericity was met (p>0.05), otherwise a Greenhouse-Giesser or HuynhFeldt correction was needed. Bonferroni test was used for the post hoc comparison. The % FECRT, weight gain, parasite recovery and performance parameters (PCV and organ weight) were analyzed using One-Way ANOVA with Tukey HSD as post hoc test. All statistical analyses were run in IBM SPSS Statistics 21. Evaluation of anthelmintic efficacy based on % FECR was made using the characterization of Kassai (1999), wherein: <80% FECR means the anthelmintic drug is ineffective; 80-89% FECR, anthelmintic drug is moderately effective (still acceptable); 90-98%, anthelmintic drug is effective; >98% efficacy, anthelmintic drug is highly effective.


Results

Amount and physical characteristics of pineapple fruit peeling juice

A total of 370 g of peelings from the queen variety of pineapple were extracted to produce a final volume of 100 mL PFPJ after the extraction process which involved mincing through knife and blender, and sieving twice to extract the juice. The pure PFPJ had a concentration of 84 mg/mL. The organoleptic characteristic of the juice included sour odor, dark-yellow coloration, turbid and watery in consistency.

Percent fecal FECRT

Fecal egg counts among replicates during the pre-treatment period ranged from 1,157.50 to 8,837.50 EPG, an indication that all chickens met the required minimum EPG count of 150 (Coles et al 1992) to conduct the %FECRT. Statistically, all pre-treatment EPG data were homogenous and had insignificant mean variations (p>0.05) prior to treatment.

Results of the repeated measures ANOVA with Sphericity Assumed showed that mean EPG count differed significantly between pre-treatment and two post-treatment readings (F(2,34) = 47.02; p<0.01). Mean fecal EPG count during pre-treatment was significantly highest compared with either 7 th or 14th day post-treatment EPG (p <0.01). This suggested a significant reduction of parasite’s egg count over time from pre-treatment to post-treatment period as shown in Figure 1. However, mean differences between treated and control groups were not significant (p>0.05).

The % FECRT (Coles et al 1992) of the EPG arithmetic mean (AM) and geometric mean (GM) on the 7th and 14th day post-treatment are presented in Figure 2. The calculated % FECR of the arithmetic mean showed no significant difference on the 7th and 14th day post-treatment between the treated and control groups (p>0.05). Similar pattern as well was observed on the 7th day post-treatment of the geometric mean % FECRT. However, higher fecal egg count reduction (76% FECR) compared to the placebo treated group (26.68% FECR) was observed on the 14 th day post-treatment of GM % FECRT at the dosage rate of 1080 mg/kg BW PFPJ (p<0.05)

Evaluation of the anthelmintic activity of AM %FECRT of PFPJ following the categorized efficacy of Kassai (1999) showed that the per os dosage of 1008 mg/kg BW PFPJ was effective (96.26%) on the 7th day and highly effective (99.3%) on the 14th day post-treatment (Figure 2). Meanwhile, the dosage of 504 mg/kg BW PFPJ and 255 mg/kg BW PFPJ were only considered effective on the 14th day with 91.1% and 93.6% fecal egg count reduction, respectively. The placebo treatment was ineffective throughout, while the commercial anthelmintic (20mg/kg levamisole) was highly effective (98.48%) on the 14th day post-treatment.

Total worm count

Total worm count based on the parasite recovered during necropsy was also determined (Table 1). Parasites recovered during necropsy revealed 3 types of helminths in the gastrointestinal tract namely, A. galli, H. gallinarum, and Raillitina spp. A. galli was the only helminth and found highest in the placebo group with a total of 28 worms (mean: 9.33 [0.40]) recovered. Traces of A. galli was found in groups treated with 1008 mg/kg BW PFPJ (2 worms) and 504 mg/kg BW PFPJ (1 worm), and no A. galli was recovered in chickens treated with 255 mg/kg BW PFPJ. H. gallinarum was also found in low numbers in all treatment groups and zero in the placebo group. Raillitina sp. was found infecting chickens except in the placebo treated group which may explain the insignificant weight gain in the levamisole and the two PFPJ treated groups.

Performance parameters

The selected performance parameters obtained three weeks post-treatment is shown in Table 1. Results showed that chickens treated with 255 mg/kg BW PFPJ had a significantly higher weight gain compared to the placebo group. Moreover, PCV and weights of the liver, spleen, and bursa of Fabricius revealed no significant differences among treatment groups (p>0.05). PCV values of all treatment groups were within the normal range at 25-37% (Reece 2013).

Figure 1. Log transformed mean fecal egg per gram (EPG) counts across time points of A. galli infected native
chickens treated with varying concentrations of Pineapple Fruit Peeling Juice (PFPJ) indicating a
significant reduction of pretreatment (a) EPG over the post-treatment (b) EPG counts (p<0.05)

Figure 2. Percent fecal egg count reduction test (%FECRT) (Coles., 1992) of the EPG arithmetic mean (AM) and geometric mean (GM) on the 7th and 14th day post-treatment. Treatment
groups having different letters are statistically significant (p<0.05). n= 5



Table 1. Parasite recovery and selected performance parameters obtained 3 weeks post-treatment in native chickens naturally co-infected with A. galli and H. gallinarum and treated with Pineapple Fruit Peeling Juice (PFPJ)

Placebo

Lev

PFPJ

SEM

P-value

High

Medium

Low

Weight gain, g

108a

215ab

179ab

173ab

235b

13.7

0.03

PCV, %

37.4

35.3

37.5

37.5

32.8

1.20

0.72

BF, g

0.27

0.2

0.18

0.34

0.13

0.06

0.80

Spleen, g

0.57

0.80

1.08

0.84

0.80

0.09

0.63

Liver, g

11.3

12.1

11.3

13.6

13

0.60

0.71

A.galli

9

0

0.50

0.20

0

1.14

0.08

H. gallinarum

0

0.20

0.75

2

1.25

0.29

0.32

*Raillitina spp.

0

1

1

1

1

0.10

0.21

Lev= levamisole (20 mg/kg BW); High = 1008 mg/kg BW; Medium = 504 mg/kg BW; Low = 255 mg/kg BW
PCV= Packed Cell Volume; BF = Bursa of Fabricius
* Raillitina spp. was recorded as present (1) and not observed (0) per replicate.
abc Means in the same row without common letter are different at P<0.05


Discussion

Gastrointestinal helminths are important disease-causing agents in free-range and backyards chickens threatening poultry health and welfare, and hampering the livelihood of smallholder farmers (Permin and Hansen 1998). High cost and unavailability of commercial anthelmintics in remote rural areas, and the development of anthelmintic resistance and risks posed by residues in poultry meat produced by commercial raisers have reinforced the need for safe anthelmintics derived from plants along with the concept of organic farming (Fernandez et al 2013).

To provide an alternative and organic approach for the treatment of gastrointestinal nematodes in chicken, the present study explored the in vivo efficacy of pineapple (Ananas comosus) fruit peeling juice (PFPJ) in 1-2 month-old native chickens naturally co-infected with common roundworm ( A. galli) and cecal worms (H. gallinarum). The anthelmintic activity of PFPJ was evaluated based on the following parameters: mean fecal egg per gram (EPG) count, percent fecal egg vount reduction test (%FECRT) (Coles et al 1992), total worm recovery and selected performance parameters, i.e. weight gain, PCV and weight of three reticuloendothelial organs.

Significant reduction of the mean EPG count was observed across time. Also, PFPJ treated chickens showed at least 90% AM FECR on the 14th day, which indicates effective anthelmintic activity (Kassai, 1999). Reduction in EPG count could be attributed to the death and expulsion of the nematodes as evident by the low total worm count among PFPJ treated groups. A. galli numbers were in fact highest in the placebo group and absent to very low in anthelmintic treated groups necropsied on the 21st day post-treatment. This observation is concordant to the findings of Begum et al (2010) who conducted an in vivo study using aqueous pineapple leaves extract and reported reduction of EPG count after the 21st day post-treatment. Hoque et al (2006) also described that commercial anthelmintic, piperazine citrate and levamisole, were able to vacate worms within the intestine by 14th and 7th day post-treatment, whereas pineapple leaves extract took 28 days for the worms to be cleared out. It is good to note that during fecal examination, A. galli and H. gallinarum eggs were counted together since they resemble morphologically (Wongrak et al 2015). This could be the reason why levamisole and 255 mg/kg BW PFPJ treated groups had fecal egg count despite the absence of A. galli during necropsy.

The significant improvement of weight gain in the 255 mg/kg BW PFPJ treated group compared to placebo indicates nematode elimination, although the weight gain result might be confounded by the presence of Raillitina spp. which is a voracious tapeworm that snatches nutrients from chicken (Soulsby 1982). The presence of tapeworms may explain the insignificant weight gain of levamisole and two PFPJ treated groups (1008 mg/kg and 504 mg/kg BW) when compared to placebo. Meanwhile, the PCV values of chickens were within the reference interval (Reece 2013) and showed no difference between placebo and anthelmintic treated groups. This observation is not in agreement with the findings of Patra et al (2010) who observed negative correlation between the level of PCV and A. galli infection. Usually, untreated birds showed low PCV, haemoglobin and erythrocyte count which are attributed to the penetration of larvae into the mucosa causing haemorrhage, and nutrient competition needed for erythropoiesis of the adult worms (Deka and Borah 2008). Similar to PCV value, the weights of liver, spleen and bursa of Fabricius between groups showed no significant difference. The weight of reticuloendothelial organs may serve as indirect measure of chicken health status (Willis et al 2007).

The potential of pineapple fruit peeling juice as alternative anthelmintic in chicken is attributed to the unique phytochemicals that can damage the cuticle of nematode. Rowan et al (1990) described pineapple fruits to contain four cysteine proteinases namely, fruit bromelain, stem bromelain, ananain, and comosain. Fruit bromelain had the highest concentrations in pineapple fruit pulp. Cysteine proteinases attach to the protective cuticle of nematodes which rapidly lead to the dissolution of the parasite, a unique mechanism compared to other actions of various anthelmintic. Studies of Stepek et al (2007, 2006 and 2005) showed that transverse wrinkles were formed on the nematode cuticle followed by the shrinking and blistering of cretes (longitudinal cuticular ridges) disrupting the nematode’s cuticular architecture. At its weakest point, derangement of hydrostatic pressures within the coelomic cavity caused the worms to open up until expulsion of its intestine and internal structures (Lalchhandama et al 2009; Behnke et al 2008). The mechanism has been provenin vivo on a number of nematode species such as Heligmosomoides bakeri, Protospirura muricola andTrichuris muris in mice (Stepek et al 2007, 2006 and 2005) and in cestodes such as Hymenolepis diminuta and H. microstoma (Mansur et al 2014). The slim chance of developing resistance against this compound may be attributed to its mode of action which involved major lyses of the parasite’s cuticle. Cysteine proteinases also showed a sex-biased effectivity in which females were more susceptible than the males. Efficacy against female will reduce nematode egg contamination in the environment hence halting the life cycle. However, the reason behind sex-biased effectivity was not clearly identified (Stepek et al 2007, 2006 and 2005; Behnke et al 2008).


Conclusion


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Received 23 March 2016; Accepted 1 April 2016; Published 1 May 2016

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