CSIRO PUBLISHING

Animal Production Science

http://dx.doi.org/10.1071/AN16540

Impact of dietary Moringa oleifera leaves on intestinal

pathogenic load and histological structure of growing
rabbits raised under heat-stress conditions

A. Y. El-Badawi A, I. El-Wardany B, S. I. Abd El-Moez C, F. I. S. Helal A,

Nematallah G. M. Ali B, M. I. Shourrap B and O. M. Aboelazab A,D
A
Animal Production Department, National Research Centre, Dokki, 12622 Giza, Egypt.
B
Poultry Production Department, Faculty of Agriculture, Ain Shams University,
Shoubra El-Kheima, 11241 Cairo, Egypt.
C
Microbiology and Immunology Department, National Research Centre, Dokki,
12622 Giza, Egypt.
D
Corresponding author. Email: [email protected]

Abstract. Forty-five growing New Zealand White (NZW) male rabbits weighed 750.0  5.8 g aged 6 weeks were
used in a feeding experiment lasting 56 days. Rabbits were housed in a semi-climatic chamber at a mean ambient
temperature of 33.1C and relative humidity of 43%. The rabbits were placed in three equal groups of five replicates.
The first group was fed on free moringa diet (control, R1), while R2 and R3 groups were fed diets supplemented with
0.5% and 1.0% moringa dry leaves respectively. Daily feeds and water were offered ad libitum. At the end of the
feeding period, three random animals of each group were slaughtered to investigate intestinal and caecal contents of
pathogens and changes of intestinal and caecal tissues. The results indicated that there were no statistical differences
among groups concerning daily feed intake, average daily gain and feed conversion ratio; however, slight improvement
was noticed with rabbits fed R3. Intestinal microbial load, namely total plate count of Salmonella, Escherichia coli,
Staphylococcus aureus, Clostridium perfringens and mould, was reduced (P < 0.01) with R2 and R3 diets compared
with the control (R1). A similar trend was also noticed on caecal microbial load. Histological examination of jejunum
tissues showed clear increases in villus height, villus : crypt ratio and obvious depth of caecal tunica mucosa, which
might reveal better absorption of nutrients and microbial metabolites. It could be concluded that rabbits fed moringa-
supplemented diets showed better feed conversion and intestinal and caecal histological structure, with clear reduction
of gastro-intestinal pathogenic bacterial load under the hot climatic conditions.

Additional keywords: growth performance, intestinal histology, pathogenic microbial load.

Received 28 August 2016, accepted 5 April 2017, published online 24 May 2017

Introduction Recently, several feed additives derived from plants have

High ambient temperature is the major environmental factor been established to contain aromatic properties that affect gut
adversely affecting livestock productivity. Rabbits, in particular, microflora, nutrient digestibility and intestinal morphology of
are sensitive to heat-stress conditions, where their productive and poultry (Cross et al. 2007). Moringa oleifera has the ability
reproductive aspects are dramatically reduced (Marai et al. 2001; to prevent effectively morphological changes and oxidative
Pei et al. 2012). damage by enhancing the activities of antioxidant enzymes,
Heat stress has a negative effect on intestinal mucosa and reducing the intensity of lipid peroxidation and reducing
microbiota (Quinteiro-Filho et al. 2010). It is well known that generation of free radicals (Sreelatha and Padma 2009).
the commensal intestinal and caecal bacterial populations have M. oleifera leaves have multiple antioxidants such as phenolic
an important physiological and pathological effects on the host. acids (gallic, chlorogenic, ellagic and ferulic acid), glucosinolates
Stable intestinal microflora can protect the host from pathogen and flavonoids (kaempferol, quercetin and rutin; Mbikay 2012).
colonisation by competing for epithelial binding sites and Furthermore, moringa dry leaves have been reported to be a
nutrients, strengthening the intestinal immune response, and valuable source of alpha-carotene (precursor of vitamin A) and
producing bacteriocins that act against some feedborne bacteria vitamin (B-complex, C, D and K; Doora et al. 1975). Moreover,
(Burkholder et al. 2008). Abd El-Moez et al. (2014) reported that rabbits fed diets

Journal compilation  CSIRO 2017 www.publish.csiro.au/journals/an

B Animal Production Science A. Y. El-Badawi et al.

supplemented with particular levels of moringa leaves showed ad libtum and water was freely available during the
significantly lower intestinal Escherichia coli, Staphylococcus experimental period. Ambient temperature and relative
aureus and Salmonella. humidity were measured twice daily. Experimental rabbits
In that way, the present study was conducted to investigate were weekly weighed and recorded. Oarts were weekly collected
the impact of supplementing diets of growing rabbits with and weighed to calculate the actual feed intake.
moringa dry leaves on growth performance, antimicrobial
potentiality and intestine histological changes.
Bacteriological analysis
Materials and methods A quantity of 10 g of the intestinal or caecal sample was weighed
The present study was conducted at The Rabbits Breeding Farm in a sterile stomacher bag, and then 90 mL from maximum
and the Poultry Physiology Laboratory, Poultry Production recovery diluents was added to the sample, the sample was
Department, Faculty of Agriculture, Ain Shams University, then well mixed using stomacher machine. After incubation,
Cairo, Egypt, and Animal Production Department Laboratory, the counting of the colonies was undertaken using the Stuart
National Research Centre, Giza, Egypt. Digital colony counter.
Enumeration of total plate count was performed according
The housing system and climatic control to ISO (2003). The plates were incubated for a maximum of
24–72 h at 30C (including the control plates).
A semi-climatic concrete double-wall chamber of 3 by 3 by 2.5 m Detection of Salmonella was performed according to
dimentions fitted with controlled-speed suction fan, electrical modified ISO (2002). The typical colonies of Salmonella in
heaters, plastic wash basins, thermometer and hygrometer were XLD were red with a black centre. Biochemical reaction
used to keep ambient tempreature at 33.1  0.5C and relative (triple-sugar iron agar, lysine iron agar, citrate agar and urea
humidity at 43  3% for the whole experimental period (see agar) was used for confirmation of typical Salmonella colonies.
Table 1). The chamber was equiped with individual galvanised Enumeration of Escherichia coli was performed according to
metal-wire cages with separate feeding and water troughs. ISO (2001). A volume of 5 mL of violet bile lactose agar medium
was added and a 10 mL second layer of the same media was
Experimental diets and feeding added after the solidification of the agar and then incubated
Three batches of rabbit diets, each of 100 kg, were formulated at 44C for 1 day. Suspected colonies were confirmed to be
to contain 32% alfalfa hay, 21% soybean meal (44%), 16% E. coli by IMViC tests.
ground yellow corn, 16% barley, 9.2% wheat bran, 3% cane- Enumeration of coagulase-positive Staphylococcus aureus
molasses, 1% lime stone, 0.6% di-calcium phosphate, 0.5% was performed according to ISO (1999). A volume of 1 mL of
sodium chloride, 0.5% vitamin and mineral premix and 0.2% each dilution was plated onto Baird–Parker agar (Oxoid, UK).
methionine. Moringa dry leaves in a powder form were added Then, the plates were incubated aerobically at 37C for 24 h.
and thoroughly hand-mixed with other feed ingredients at 0%, Black colonies surrounded by a hallow zone were confirmed by
0.5% and 1.0% moringa for R1 (control), R2 and R3 respectively. biochemical tests including catalase, oxidase, indole, methyl red,
Experimental rations were pelleted at 0.3-cm diameter and Voges Proskauer, Simmon’s citrate, urease test, hydrogen sulfide
packed in polyethylene bags until feeding. production in triple-sugar iron agar medium, sugar fermentation
A feeding trial lasted 56 days and was conducted in the semi- test using different sugars, arginine hydrolysis test, hippurate
climatic chamber, where 45 male growing New Zealand White hydrolysis test, nitrate reduction test and coagulase test, which
rabbits (NZW) aged 6 weeks, with an average bodyweight of were performed according to Quinn et al. (2002).
750.0  5.8 g, were randomly distributed by weight in three Enumeration of yeast and moulds was performed according
equal groups, each with five replictes. The first group of animals to ISO (2008); 15 mL of Dicholoran DG18 medium (Oxoid)
was fed R1 (free moringa ration, control) while second and third was added to the Petri plates and incubated at 30C for 3 days.
groups were fed R2 and R3 respectively. Feed was offered Enumeration of Clostridium perfringens was performed according
to ISO (2004); 5 mL of TSC agar medium (Sigma-Aldrich, Inc.,
USA) was added and a 10 mL second layer of the same media
Table 1. Average of temperature (8C), relative humidity (%) and
was added after the solidification of the agar and then incubated
temperature–humidity index (THI, calculated according to Marai
et al. 2001) during the experimental period anaerobically at 37C for 1 day.

Period Temperature Relative humidity THI Histological examination

1st week 33.2 41 29.76 At 14 weeks, specimens (six from treated groups) from the
2nd week 32.8 44 29.61 jejunum, as the second upper three-fifths of small intestine,
3rd week 33.5 42 30.07 and the caecum, as the middle region, were carefully dissected
4th week 32.8 46 29.72 at the slaughter time. These samples were first rinsed with
5th week 32.8 44 29.61
saline (0.85% Nacl) and then cross-sectional lengths of 1 cm
6th week 33.4 42 29.98
7th week 33.5 42 30.07
were placed into 10% buffered neutral formaldehyde solution.
8th week 32.7 44 29.52 A routine staining procedure was performed using hematoxylin
and eosin (H and E). The slides were examined under ·10
Overall 33.1 43 29.80
magnification.
Heat stress remains a major challenge for rabbits Animal Production Science C

Histometric measurements of the intestine (El-Badawi et al. 2015). The conflict with the present results
The images of the intestine were analysed using stereological concerning moringa intake per kilogram liveweight indicated
image software, Image J 1.50b (Wayne Rasband, National that heat-stressed rabbits might need a higher dose of moringa
Institute of Health, Bethesda, MD, USA), with its scale than those raised under ambient climatic conditions.
calibrated to the micrometre unit (mm) using a micrometric
ruler (PZO: Warszawa, Poland). Jejunum villus height was Intestinal and caecal microbial load
measured from the tip of the villus to the villus–crypt junction. Moringa leaves as a feed additive had a highly significant
The mucosal depths of caecum were recorded by measuring the (P < 0.001) effect on bacterial and mycotic counts of intestinal
length from the top of submucosa to the tip of the mucosa. and caecal contents of rabbits (Tables 3, 4). All tested samples
showed negative results for the isolation of C. perfringens,
Statistical analyses whether in intestinal or caecal samples.
Collected data were subjected to one-way ANOVA by using the Total plate count of intestinal contents was the highest in
general linear model procedure (GLM) of the Statistical Analysis the control group (R1), followed by moringa groups, and the
System (SAS 1998). Differences among treatment means were same trend was observed in caecal contents. E. coli count of
detected by using Duncan’s multiple-range test. intestinal contents was high in the control group (R1), while
E. coli was non-detectable in the other experimental groups. It
is worth mentioning that all caecal samples did not record any
Results and discussion count of E. coli. A high count of Salmonella was detected in
Growth performance the intestinal content of the control group (R1), while the
Bodyweight gain, average daily gain, feed intake and feed moringa groups did not record any count of E. coli. Further, no
conversion ratio of rabbits fed experimental rations are given Salmonella was recorded in caecal samples of any of the
in Table 2. There were no significant differences among experimental groups.
experimental groups, but the data showed numerical increases
in the final bodyweight and bodyweight gain of R3 group, Table 3. Intestinal microbial load of heat-stressed New Zealand White
(NZW) rabbits fed with Moringa oleifera leaves
associated with a slight decrease in feed conversion ratio of
R1, control; R2, 0.5% Moringa leaves; R3, 1% Moringa leaves. Means
the same group. within a row followed by the same letter are not significantly different. cfu,
The effects of moringa leaves on growth performance were colony-forming unit. s.e.m., standard error of the mean. **, P  0.01; n.d.,
noticed in some previous studies. Nuhu (2010) reported that not detectable
the daily weight gain of weaner rabbits was clearly improved
(P < 0.05) with an increasing supplementation level of M. oleifera Item Experimental group s.e.m. Significance
leaf meal in rabbit diet. Moreover, Onu and Aniebo (2011) R1 R2 R3
reported that inclusion of M. oleifera leaf meal in broiler
diets improved the final bodyweight and average daily gain Total plate count 289.0a 6.7b 5.9b 1.59 **
significantly. Moreover, El-Badawi et al. (2014) reported that (·104 cfu/mL)
Salmonella (·101 cfu/mL) 29a n.d.b n.d.b 0.58 **
bodyweight gain, average daily gain and feed conversion ratio
Eschrichia coli (·103 cfu/mL) 249a n.d.b n.d.b 10.97 **
of rabbits fed 0.15% and 0.30% moringa-supplemented rations Staphylococcus aureus 31.7a n.d.b n.d.b 2.03 **
improved significantly (P < 0.05) more than did those of (·102 cfu/mL)
rabbits fed 0% or 0.45% moringa rations. The same authors Clostridium perfringens n.d. n.d. n.d. – –
found that utilisation of digestible energy and protein were Mold (·103 cfu/mL) 15a 3.9b 3.8b 2.18 **
significantly deteriorated with the highest level (0.45%) of
moringa supplementation equivalent to 485 mg/kg bodyweight
Table 4. Caecal microbial load of heat-stressed New Zealand White
Table 2. Growth performance and feed intake of heat-stressed New (NZW) rabbits fed with Moringa oleifera leaves
Zealand White (NZW) rabbits fed with Moringa oleifera leaves R1, control; R2, 0.5% Moringa leaves; R3, 1% Moringa leaves. Means within
R1, control, R2, 0.5% Moringa leaves, R3, 1% Moringa leaves. s.e.m., a row followed by the same letter are not significantly different. cfu, colony-
standard error of means, n.s., non-significant forming unit. s.e.m., standard error of the mean. **, P  0.01; n.d., not
detectable
Item Experimental group s.e.m. Significance
R1 R2 R3 Item Experimental group s.e.m. Significance
R1 R2 R3
Initial weight (g) 755 757 752 5.2 n.s.
Final weight (g) 1915 1908 1954 97.2 n.s. Total plate count 33.00a 2.37b 5.27b 1.50 **
Bodyweight gain (g) 1160 1151 1202 97.5 n.s. (·104 cfu/mL)
Average daily gain (g) 20.7 20.6 21.5 1.72 n.s. Salmonella n.d. n.d. n.d. – –
Daily feed intake (DM) 72.3 70.7 73.8 2.7 n.s. Escherichia coli n.d. n.d. n.d. – –
Feed conversion 3.50 3.47 3.44 0.27 n.s. Staphylococcus aureus 89.00a 0.77b 0.50b 5.20 **
(kg DM/kg gain) (· 102 cfu/mL)
Calculated intake of moringa – 265 545 – – Clostridium perfringens n.d. n.d. n.d. – –
(mg/kg live bodyweight) Mold (·103 cfu/mL) 230.0a 21.0b 15.0b 17.42 **
D Animal Production Science A. Y. El-Badawi et al.

Fig. 1. Cross-section through small intestine (jejunum) from heat-stressed

New Zealand White (NZW) rabbits treated with different doses of Moringa Fig. 2. Cross-section through caecum from heat-stressed New Zealand
oleifera leaves at 14 weeks. M, muscularis mucosa; SM, submucosa; Cr, White (NZW) rabbits treated with different doses of Moringa oleifera
crypts of Lieberkühn; V, villi; and L, lumen. Original magnification, ·100 leaves at 14 weeks. M, muscularis mucosa; SM, submucosa; MU, mucosa;
(haemotoxylin and eosin). R1, control; R2, 0.5% Moringa oleifera leaf meal; Cr, crypts of Lieberkühn; and L, lumen. Original magnification, ·100
and R3, 1.0% MOLM. (haemotoxylin and eosin). R1, control; R2, 0.5% Moringa oleifera leaf
meal (MOLM); and R3, 1.0% MOLM.
Heat stress remains a major challenge for rabbits Animal Production Science E

Table 5. Histomorphological parameters of the Jejunum and the The means of jejunum villus height, crypt depth, villus
caecum in heat-stressed New Zealand White (NZW) rabbits fed with height : crypt depth ratio and caecum mucosa depth for the
Moringa oleifera leaves different dietary treatments are illustrated in Table 5. It is clear
R1, control; R2, 0.5% Moringa leaves; R3, 1% Moringa leaves. Means within
that moringa supplementation at both levels (R1 and R2)
a row followed by different letters are significantly different. cfu, colony-
forming unit. s.e.m., standard error of the mean. **, P  0.01; n.s., not
significantly increased the villus height and crypt depth (P <
significant 0.05) compared with the control group; however, villus
height : crypt depth ratio was not significantly (P > 0.05)
affected. There was no significant effect on the caecum
Item Experimental group s.e.m. Significance
R1 R2 R3
mucosa depth by moringa supplementation in either R2 or R3
when compared with control, in spite of the improvement of the
Jejunum mucosa depth of the caecum of rabbit from R2. The visual
Villus height (mm) 661.64b 930.32ab 798.31a 47.66 ** investigations of the caecum indicated that the number and
Crypt depth (mm) 148.88b 277.87a 261.38a 16.26 ** size of the crypts are greater in R3 sections than in the other
Villus height : crypt 4.27 4.10 3.74 0.44 n.s.
treatments.
depth
Caecum
As a result of increasing the villus height that was observed in
Mucosa depth (mm) 113.84 123.17 116.44 4.766 n.s. R2 and R3 groups, more nutrients could be absorbed due to the
greater villus surface area (Caspary 1992). It is well known that
the villus crypt is considered as the villus factory. So, deeper
The count of S. aureus in the intestinal content was the highest crypts indicate fast tissue turnover to permit the renewability of
in the control group (R1), while the moringa groups had non- villus as needed in response to normal collapse or inflammation
detectable S. aureus. Also, the same trend (as of the intestinal from pathogens or their toxins and high demands for tissue
contents) was established for caecal contents. Mold count of (Yason et al. 1987). Moreover, increases in the villus height
intestinal contents was the highest in the control group (R1), and villus height : crypt depth ratio are directly correlated with an
while the other groups were similarly equal in their mould count. increased epithelial cell turnover (Fan et al. 1997), and longer villi
The same trend was observed in the mould count of the caecal are associated with activated cell mitosis (Samanya and
contents. Yamauchi 2002).
It is well known that heat stress increases the free-radical Also, the depth of caecum tunica mucosa is important for
production (Belhadj Slimen et al. 2016), and, as a result, causes increasing the absorption of microbial products in the caecum
an increase in the number of pathogenic bacteria and a reduction (Abdel-Khalek et al. 2011). The tunica mucosa plays an
in the number of beneficial bacteria. Furthermore, natural important role in the interaction (competition) between the
antioxidants such as those found in moringa leaves, together normal and pathogenic flora, and, furthermore, it is considered
with antimicrobial activity, could play an effective role in as a major site for digestion and absorption of nutrients
resistance to pathogenic bacterial accretion. The beneficial Gallois et al. (2004). Furthermore, the villus along the
effects of moringa leaves on the reduction of pathogenic small-intestine sites plays an important role in the immunity
bacterial counts were very clear in the present study, and of the animal, since intestinal epithelium tissue acts as a
may have been a result of the presence of short-chain natural barrier to pathogenic bacteria and toxic substances
monosaccharides in M. oleifera leaves, which are the main presented inside the small intestine (Paul et al. 2007). Also,
components that function as competitive attachment sites for Rao and Samak (2012) reported that M. oleifera leaves
pathogenic bacteria, carrying them out of the gut, rather than contain glutathione, a conjugated element of glutamate,
their binding to the intestine, hence enhancing the growth of which is the most abundant amino acid in the blood, and
beneficial gut microflora (Nkukwana et al. 2015). Moreover, has an important function in maintaining mucosal integrity.
the fluid in the upper small intestine plays a protective role, Recent research has indicated that supplementation of
keeping pathogenic bacteria in suspension and washing them broiler diet with M. oleifera leaf meal increases villi length
downstream, while encouraging the proliferation of growth- and villus surface area in different intestinal segments
enhancing bacteria (Hoerr 2001). (Nkukwana et al. 2015). Furthermore, Baurhoo et al. (2009)
The present results are in close agreement with those of reported that M. oleifera leaves as an antimicrobial growth
Rajamanickam and Sudha (2013) and Abd El-Moez et al. promoter (AGP) contain short-chain carbohydrates, glycosides,
(2014) who reported that M. oleifera ethanol extract and which cause major increases in the height of the intestinal
moringa dry leaves showed significant inhibition on S. aureus, villi. M. oleifera extract preserves the micro-anatomical
E. coli, Bacillus sp., Pseudomonas aeruginosa, Cornebacterium, integrity and dimensions of the various layers of duodenum
Klebsiella pneumonia and Acinetobacter sp. in the intestinal of Wistar rats and prevents a decrease in gastrointestinal
content of NZW rabbits. surface area following an ethanol-induced injury, via inhibition
of oxidative damages that accompany ethanol-induced
Histological examination gastrointestinal injuries (Olaibi et al. 2014).
The histological architecture of ileum and caecum, from growing
rabbits of different dietary treatments, are shown in Figs 1 and 2
respectively, showing muscularis mucosa layer, submucosa, Conclusions
mucosa, crypts of Lieberkühn and villi. This description was From the results of the present study, it could be concluded that
also provided by William and Linda (2000). rabbit diet supplemented with 0.5% or 1.0% dry moringa leaves
F Animal Production Science A. Y. El-Badawi et al.

do not influence daily feed intake or feed conversion under hot ISO (2002) Method 6579. Microbiology of food and animal feeding stuffs.
climatic conditions. However, great reductions in intestinal and Horizontal method for detection of Salmonella spp. (ISO: Geneva,
caecal pathogens, with increases in intestinal villus height and Switzerland)
crypt depth, were observed in rabbits fed moringa-supplemented ISO (2003) Method 4833. Microbiology of food and animal feeding stuffs.
Horizontal method for the enumeration of microorganisms. Colony-count
diets.
technique at 30C. (ISO: Geneva, Switzerland)
ISO (2004) Method 7937. Microbiology of food and animal feeding stuffs.
Horizontal method for the enumeration of Clostridium perfringens.
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