Coccidiosis is a major disease problem in the poultry industry. Outbreaks usually
result in enormous economic losses as a result of the associated morbidity and
mortality (Oluyemi and Roberts, 2000). The disease is controlled
mainly by hygiene and the use of chemical anticoccidial agents (Soulsby,
1982). However, the development of drug resistance by the causative parasites
and the escalating cost of drug development have greatly reduced the commercial
incentive to develop new chemical anticoccidials (Gordon and
Jordan, 1982; Permin, 1998; Oluyemi
and Roberts, 2000). Consequently, the development of alternative, safer
and environmentally friendly anticoccidial agents have become priority in most
parts of the world (Youn and Noh, 2001).
In Nigeria several plants have been claimed traditionally to have medicinal
value for the treatment of various ailments in both man and animals (Nwude
and Ibrahim, 1980; Akinniyi and Sultanbawa, 1983).
However, their efficacy and safety remain doubtful as only a few of these have
been properly identified and documented (Mbaya et al.,
2007; Nwosu et al., 2004, 2008).
Previous studies have shown that many plants said to be traditionally useful
in the treatment of human and animal ailments have scientifically been shown
to be either very toxic or have no therapeutic effect. In most cases, their
toxic actions have been attributed to the contained active principles or over
dosage due to the absence of standard dosage system in herbal medicine (Onyeyili
et al., 2000; Hashemi et al., 2008).
The semi-arid zone of Northeastern Nigeria is known to account for about 30%
of the domesticated livestock and poultry in the country (Nwosu
et al., 2007). In this zone, several plant extracts including the
stem barks of Butyrospermum paradoxum, Khaya senegalensis and Annona
senegalensis are used traditionally by the natives in the treatment of several
ailments including avian coccidiosis. Although previous experimental studies
have reported the therapeutic efficacy of the stem bark extract of B.
paradoxum against trypanosome species (Mbaya et
al., 2007) and K. senegalensis against nematode species
(Onyeyili et al., 2000) the extracts also produced
toxic effects manifested in various degrees of behavioural changes, morbidity
and sometimes mortalities when administered intraperitoneally to rats. However,
no previous studies have been conducted to assess their toxicity and anticoccidial
efficacy in chickens. This study was therefore designed to evaluate the toxicity
and anticocidial efficacy of these three plants (Anona senegalensis,
Butyrospermum paradoxum and Khaya senegalensis) said to be traditionally
useful in the control of avian coccidiosis in the region.
MATERIALS AND METHODS
Collection and processing of plant materials: The plants used for
this study were Khaya senegalensis, Butyrospermum paradoxum and Annona
senegalensis also, respectively known as Kadanya, Madaci and
Gwandan daji in Hausa, the predominant language in the study area. The
stem barks of the plants were collected in February 2004 from the respective
trees in Maiduguri, the capital of Borno state and the largest urban centre
in semi-arid Northeastern Nigeria. The identity of the plants was confirmed
by a botanist in the Department of Biological Sciences of the University of
Maiduguri, Nigeria, where voucher specimens of the plants were deposited.
The stem barks of the plants were dried under shade for 10 days at 8 h per
day and then ground into powder using a pestle and mortar. The powdered extracts
were sieved to remove excess coarse plant materials and then individually exhaustively
soxhlet extracted with water for 8 h at 60°C (WHO, 1992;
Onyeyili et al., 2001). The soluble extract was
then concentrated in a conical flask placed in a water bath maintained overnight
at 60°C. Thereafter, the concentrated extract now in a gel form was collected,
weighed and stored at 4°C for later use in the study.
Experimental animals: A total of two hundred and twenty-five pullet chicks were used in this study. They were purchased at day-old (ECWA Rural Development Ltd., Jos, Nigeria) housed at the Experimental Animals House in the Parasitology Unit of the Department of Veterinary Microbiology and Parasitology, University of Maiduguri, Nigeria and maintained on standard feed (Vital Feed, Grand Cereals and Oil Mills, Bukuru, Nigeria) containing 14.5% crude protein, 7% fat, 7.2% crude fibre, 1% calcium and 68.1% carbohydrate. Water was provided ad libitum. The birds were four weeks old when they were used for the experimental studies.
Experimental drug: Amprolium (Amprolium 200 NTCOX 20%, Sam Pharmaceuticals Ltd., Nigeria) a commercially available anticoccidial drug for the routine treatment of avian coccidiosis (due to Eimeria species) in Nigeria was used to compare the anticoccidial effects of the plant extracts.
Collection and culture of Eimeria oocysts: The Eimeria
oocysts used in this study were derived from a rural chicken suffering from
natural clinical coccidiosis. Following evisceration at post mortem, the caeca
and intestines were separated, sliced open longitudinally and their contents
washed into a beaker using tap water. The washings were centrifuged and the
sediment re-mixed with saturated sodium chlorine solution (NaCl) to float the
oocysts (Anonymous, 1977). Thereafter, traces of salt
and colouring matter were removed by washing the sediments several times with
water through the process of centrifugation. The harvested oocysts were multiplied
in three chicks following oral infection. The chicks were routinely monitored
daily for the development of clinical coccidiosis and the presence of Eimeria
oocysts in their faeces. Thereafter, pure cultures of Eimeria oocysts
were obtained through floatation as earlier described. Oocysts were sporulated
for 72 h in 2% potassium dichromate solution and the species identified using
their morphology, sporulation time, predilection site and the pathological lesions
produced in chicken (Anonymous, 1977; Soulsby,
1982). Faecal oocyst counts were determined by the modified McMaster technique
using saturated sodium chloride solution as the floating medium (Anonymous,
1977). The oocysts were stored in 2% potassium dichromate solution that
was changed weekly until used. The infecting oocysts consisted of a mixture
of Eimeria tenella (26.85%), E. acervulina (61.54%) and E.
Phytochemical analysis of the aqueous stem bark extracts: The stem bark
of each plant was analysed for phytochemical constituents including tannins,
saponins, alkaloids, anthraquinone derivatives, terpenes, steroids and cardiac
glycosides as described by Sofowora (1984).
Determination of acute toxicity of the extracts: Thirty-five pullet chicks, separated into seven groups (A-H) of five chicks each were used to determine the acute toxicity of the aqueous stem bark extract of K. senegalensis. The birds in groups A-G were, respectively treated intraperitoneally with graded doses (50, 100, 200, 400, 800, 1200 and 1600 mg kg-1) of the extract in distilled water while chicks in group H (Control) were given only distilled water equivalent to the largest volume of the extract by the same route. Two new sets of thirty-five pullet chicks each were used to repeat the same procedure using the extracts of B. paradoxum and A. senegalensis, respectively.
The birds were monitored during a 24 h period for signs of toxicity and death.
Dead birds or those sacrificed after 24 h were subjected to necropsy for lesions.
The median lethal dose (LD50) was determined using the arithmetic
method of Karber as modified by Aliu and Nwude (1982).
Determination of chronic toxicity of the extracts: Thirty pullet chicks
divided into six groups (A-F) of five chicks each were used to determine the
chronic toxicity of K. senegalensis in chicks. The birds in groups A-E
were orally treated, respectively with various concentrations (100, 500, 1000,
1500, 2000 mg kg-1) of the water extract of K. senegalensis
daily for 28 days. Group F served as control and was orally treated with distilled
water equivalent to the largest volume of the extract administered. Two other
sets of thirty pullet chicks each were used to repeat the experiment using B.
paradoxum and A. senegalensis, respectively. Clinical signs, mortality
and gross and histopathological findings were used to assess the effects of
the various extracts on the chicks.
In-vitro anti-coccidial effect of the extracts: The in-vitro
anti-coccidial efficacy trials were conducted by observing the effect of the
plant extracts on the sporulation of Eimeria oocysts. Fresh faecal samples
were collected from infected birds and the oocyst counts determined (Anonymous,
1977). Various dilutions of the respective extracts of K. senegalensis,
B. paradoxum or A. senegalensis (100, 250, 500, 1000, 1500 mg
mL-1) in distilled water were placed in separate Petri dishes labelled
appropriately. A known number of oocysts (100 oocysts) were added to each Petri
dish and the set up was left at ambient temperature and monitored every 6 h
to observe sporulation of the oocysts over a 72 h period. The number and percentage
sporulation of the oocysts were then determined and recorded. These were used
to determine the percentage inhibition of oocyst sporulation. Similar observations
were made for oocysts sporulated in Amprolium and potassium dichromate solutions
which served as controls.
In-vivo anti-coccidial effect of the extracts: A total of 30 chicks divided into 6 equal groups (A-F) were used for this study. Groups A-E were each orally infected with 120,000 sporulated oocysts (the infection dose was based on the results of an earlier pilot study). The birds were monitored daily for the presence of oocysts in the faeces to determine patency and oocyst counts. On the first day that oocysts were detected in the faeces, the birds in groups A, B and C were, respectively treated with 100 mg kg-1 body weight of the respective extracts of K. senegalensis, B. paradoxum and A. senegalensis. The birds in Group D were at the same time treated with amprolium at 10 mg kg-1 while group E remained untreated. All treatments were given orally. Birds in group F were uninfected and untreated and used as healthy controls.
All the birds in the various experimental groups were monitored daily for signs
of disease including inappetance/anorexia, diarrhoea, bloody faeces, anaemia
and death during a 28 days period. Faecal oocyst counts and live body weights
of the birds were determined daily while the PCV was determined every 2 days
for each experimental group. Dead birds and those humanely sacrificed at the
end of the study were subjected to necropsy for lesions. The microhaematocrit
method was used to determine the packed cell volume of the experimental birds
(Coles, 1974) while the laboratory triple beam balance
was used to determine live body weights.
Histopathology: In each case, tissue samples of the heart, lungs, lymph
nodes, kidney, intestine, liver and spleen were obtained at necropsy, fixed
in 10% buffered formalin, embedded in paraffin wax, sectioned at 5 μm thick
and stained with Haematoxylin and Eosin as described by Drury
and Wallington (1976). Stained tissue sections were examined under the light
microscope for presence of lesions.
Data analysis: Data obtained from the study were summarized as Means±standard
deviations and statistical differences between the means determined by the analysis
of variance (ANOVA) and the paired students t test at 5% level
of significance (GraphPad Software Inc., 1998).
Phytochemical constituents: The results revealed that the three plants
contained varying concentrations of tannins, terpenes and steroids (Table
1). However, only A. senegalensis contained saponins while K.
senegalensis and B. paradoxum contained cardiac glycosides and alkaloids.
Anthraquinones were present in only A. senegalensis and B. paradoxum.
Acute toxicity: Following the administration of graded doses of the extracts of K. senegalensis to the birds, no clinical signs of toxicity were observed in the groups treated with the 50-400 mg kg-1 concentrations of the extract; slight depression in the group given 800 mg kg-1 and depression, inappetance and limping in those treated with higher concentrations (1200 mg kg-1 and 1600 mg kg-1). No deaths were noted in any of the treated groups (Table 2). Lesions were not observed in any of the treated groups except those given the 1,600 mg kg-1 concentration showed had areas of epithelial necrosis and haemorrhages of the intestinal mucosa, slight hepatic and splenic congestion. The LD50 of the extract could not be determined due to the absence of mortalities among the treated groups.
constituents of Khaya senegalensis, Annona senegalensis and
|-: Negative, +: Mildly positive, ++: Moderately positive,
+++: Copiously positive
pattern of four weeks old chicks following single or prolonged (28 days)
administration of K. senegalensis, B. paradoxum and A.
|*Number of birds per group = 5
No signs of toxicity were observed in the birds given 50-200 mg kg-1 of B. paradoxum. However, depression, limping, drooping and anorexia were noted in the groups given 400 mg kg-1 or higher concentrations of the extract; the severity increasing with concentration of the extract. Deaths occurred only in the groups given 800 mg kg-1 or higher concentrations of the extract (Table 2). Lesions were noted only in the groups given 1200 and 1600 mg kg-1 and these included haemorrhages, epithelial and glandular tissue necrosis and villous atrophy in the intestine. The intraperitoneal LD50 of the extract was 1260 mg kg-1.
Depression and weakness were noted in the birds given 50 and 100 mg kg-1 concentrations of A. senegalensis. At higher concentrations (200 mg kg-1 and above) there were varying degrees of depression, weakness, anorexia, droopiness and ruffled feathers; the severity increasing with concentration of the extract. Deaths were recorded only among the birds given 200 mg kg-1 or higher concentrations of the extract. The lesions observed were serosal haemorrhages and epithelial necrosis of the intestine in the birds given the 200 mg kg-1 concentration of the extract. At the 400 mg kg-1 and higher concentrations, there were varying degrees of muscular and hepatic congestion and the intestines had areas of haemorrhages, epithelial necrosis and villous atrophy. The intraperitoneal LD50 was 270 mg kg-1.
Chronic toxicity: The groups treated with K. senegalensis did not manifest any morbidity or mortality except in the group treated with 2,000 mg kg-1 concentration of the extract that were slightly depressed. However, lesions of varying degrees of severity were recorded in the various groups treated with the extract. There was myocarditis and the intestines showed villous atrophy with epithelial and glandular necrosis. The kidneys showed interstitial nephritis with mononuclear cellular infiltration and renal coagulative and tubular necrosis. The liver had focal areas of necrosis and mononuclear cellular infiltration (Fig. 1) while the lungs were congested with some degree of serous pneumonia. The severity of the lesions was concentration dependent being most severe in those chicks given the highest concentration of the extract.
No clinical manifestations of toxicity were noted in the birds treated with various concentrations of B. paradoxum except in the groups given 1,500 and 2,000 mg kg-1 concentrations of the extract that became depressed, anorectic and reluctant to move 6-8 h after the first treatment. Mortalities (20-100%) and lesions were noted only in those groups treated with 500 mg kg-1 or higher concentrations of the extract (Table 2). The lesions in the heart included superficial mononuclear cellular infiltration of the epicardium (Fig. 2) while in the liver there were focal areas of hepatic congestion and necrosis with mononuclear cellular infiltration. In the intestine, there was glandular and epithelial necrosis with villous atrophy and mononuclear cellular infiltration of the mucosa (Fig. 3). The clinical signs and lesions were concentration dependent being most severe in the birds that received the highest concentration of the extract.
The extract of A. senegalensis did not produce any signs of toxicity in the chicks except for slight depression and inappetence noted 2-3 h after the first treatment with the 1,500 and 2,000 mg kg-1 concentrations of the extract. There were no deaths in any of the treated groups (Table 2). However, lesions were evident in those chicks treated with 1,000 mg kg-1 and higher concentrations of the extract and these included focal areas of hepatic and intestinal epithelial necrosis and haemorrhages as well as mononuclear cellular infiltration of the intestinal submucosa. There was tubular necrosis and mononuclear cellular infiltration into the interstitial tissues of the kidney (Fig. 4). These lesions were concentration dependent being most severe in the group given the highest concentration of the extract.
||Photomicrograph of the liver showing focal necrosis and mononuclear
leucocytic infiltration in a chick treated with aqueous stem bark crude
extract of Khaya senegalensis at 1000 mg kg-1 for 28
days. H and E x400
||Photomicrograph of the heart showing thickening by fluid exudation
(a) and mononuclear cellular infiltrates (b) in a chick treated with aqueous
stem bark crude extract of Butyrospermum paradoxum at 2000 mg kg-1
for 28 days. H and E x400
In-vitro anticoccidal effects: Compared to the untreated control
group (incubation in 2% potassium dichromate solution) oocyst sporulation was
not inhibited in any way by incubation in K. senegalensis stem bark extract
(Table 3). However, the various concentrations of B. paradoxum
and A. senegalensis produced varying degrees of inhibition in oocyst
sporulation; the former plant extract producing the greater effect of the two.
The percentage inhibition was concentration dependent, being highest at the
highest concentrations of the extracts used.
||Photomicrograph of duodenum showing epithelial necrosis (arrowed)
leading to loss of villi, glandular necrosis (a) with mononuclear cellular
infiltration (b) in a chick treated with aqueous stem bark crude extract
of Butyrospermum paradoxum at 1500 mg kg-1 for 28 days.
H and E x200
||Photomicrograph of kidney showing focal area of tubular necrosis
with mononuclear cellular infiltration (arrowed) in a chick treated with
aqueous stem bark crude extract of Annona senegalensis at 2000 mg
kg-1 body weight for 28 days. H and E x400
The inhibition of oocyst sporulation produced by the two extracts was similar
to that produced by amprolium at the 1.2 mg mL-1 concentration used
in the study.
In-vivo anticoccidial effects: Oocysts were first detected in
the faeces of all the infected birds by day 4 post infection (Fig.
5). In the untreated group, oocyst numbers rose rapidly to attain peak counts
by day 8 post infection before being reduced to lower numbers maintained to
the end of the study.
vitro anti-coccidial effect of the plant extracts on the sporulation
of Eimeria oocysts
|*Culture in potassium dichromate was used as control and standard
(0% reduction in oocyst sporulation)
||Oocyst counts of chicks infected with mixed Eimera
species and treated with the extracts or amprolium and their controls
Treatment with amprolium or the three extracts checked the rise in oocyst out
put so that lower peaks were recorded in these groups with an eventual elimination
of the oocysts from the faeces by day 11, 14 and 18, respectively post treatment
with amprolium, A. senegalensis and B. paradoxum but not with
K. senegalensis or the untreated group during the remaining part of the
||Packed cell volume of chicks infected with mixed Eimera
species and treated with the extracts or amprolium and their controls
||Live weight of chicks infected with mixed Eimera species
and treated with the extracts or amprolium and their controls
The Packed Cell Volume (PCV) of the healthy control birds remained within their preinfection levels without any significant changes throughout the study period (Fig. 6). On the other hand, the PCV of all the infected groups became reduced from day 4 post infection. The decline in PCV was greatest in the infected/untreated group followed respectively in descending order by those groups treated with the extracts and amprolium. Attempt at return to preinfection values in all the infected groups was successful from day 12 post infection and only in the groups treated with amprolium and A. senegalensis.
The chicks in all the experimental groups gained weight during the study (Fig.
7). However, weight gain was highest in the healthy control group followed,
respectively in descending order by those treated with amprolium A. senegalensis,
K. senegalensis and B. paradoxum. The untreated group gained the
least weight while the healthy control group gained significantly more weight
than any of the infected but treated groups.
The results of the phytochemical analysis revealed that the aqueous extracts
of the stem barks of K. senegalensis, B. paradoxum and A. senegalensis
contain several active chemical components many of which are present in
each of the three plant extracts with a few being exclusively present in only
one or two of them. Many of the active chemical components obtained are known
to be toxic (Humphreys, 1988) and clinical manifestations
and lesions associated with their toxicity were noted in some of the birds given
either the single intraperitoneal or prolonged oral treatment of the extracts.
The dose-related nature of the manifestations and lesions suggest they are pharmacological
and a reflection of the variations in the concentrations of the various components
in each plant extract.
Among the three extracts tested K. senegalensis had the least number
and concentration of the active chemical components and at the concentrations
and duration of administration used in this study the extract was the least
toxic as it produced only mild clinical signs and lesions without mortality
even at very high concentrations. In an earlier study, Onyeyili
et al. (2000) administered up to 6,400 mg kg-1 of the
extract in rats without recording any severe toxic effects or deaths. On the
other hand B. paradoxum was most toxic, causing up to 80-100% mortality
when administered intraperitoneally or orally. Mbaya et
al. (2007) reported essentially similar clinical signs, pathological
lesions and mortality pattern in rats following intraperitoneal administration
of graded doses (400-3200 mg kg-1 body weight) of the ethanolic stem
bark extract of the plant. Anona senegalensis was also toxic but at concentrations
in excess of 200 mg kg-1 when given by the intraperitoneal route.
However, when administered orally, A. senegalensis extract became less
toxic and caused no mortality among the treated birds probably due to poor rate
of absorption of its toxic chemical components or their degradation to relatively
safer by-products by digestive enzymes in the gastrointestinal tract during
the long period of administration to the animals. Furthermore, the reduced toxicity
of the extract by the oral route agrees, in part, with the observations of Hashemi
et al. (2008) that water suspensions of herbal aqueous extracts were
not usually toxic to birds when administered by the oral route even at concentrations
of up to 2000 mg kg-1 body weight.
The susceptibility of animals to plant materials depend on the types of active
principles in the plant, the concentration given to the animal, the rate of
their metabolic conversion to metabolites in the liver and their subsequent
excretion from the body (Amna et al., 2011;
Abdelgadir et al., 2010). The liver is the major
organ for metabolism in the body (Baggot, 1984). Consequently,
the lesions observed in the liver may be as a result of damage caused in the
organ during the biotransformation of these active chemical components by the
organ. The excretion of some of the active components or their by-products through
the kidney and their subsequent toxicity to the organ may account for the severe
and widespread lesions noted in the kidney of some of the treated birds during
this study. The widespread nature of the lesions in several organs and tissues
including the lungs, heart, intestines and spleen suggest that the active components
were widely distributed in the body where they produced toxic effects especially
in the birds treated with B. paradoxum extracts.
Among the three extracts tested, B. Paradoxum exerted the greatest percentage reduction in oocyst sporulation but had the lowest in-vivo anti coccidial effect. These contrasting features show that it has relatively little contribution in the treatment and control of coccidiosis through the oral route as it appears to act most effectively during the pre-infective stage. It is possible that the active components responsible for the high anti-sporulation effect produced in vitro were denatured, degraded or inactivated by enzymatic action in the intestine following oral treatment. On the other hand, K. senegalensis produced no in vitro anti-sporulation effect but had mild effect in vivo suggesting that the active components responsible for the noted in vivo effects may be by-products of enzymatic action on some of the chemical components in the extract. The in vitro anti-sporulation effect produced by B. paradoxum and A. senegalensis was concentration dependent suggesting that they were pharmacologically mediated.
Compared to the healthy control group of birds, treatment of Eimeria infected birds with each of the three extracts resulted in substantial reduction in faecal oocyst out put by the birds. Annona senegalensis produced the highest effect followed respectively K. senegalensis and B. paradoxum and these effects were manifested through the appreciation of the packed cell volume, improvement in live weight gain and the suppression of oocyst out put and sporulation.
In conclusion therefore, the results of this study showed that the crude aqueous extracts of the stem barks of K. senegalensis, A. senegalensis and B. paradoxum contain many chemical components that may be toxic to four-weeks old chicks when administered orally or intraperitoneally. Khaya senegalensis exhibited a highest margin of safety while B. paradoxum was the most toxic by both routes of administration. The three extracts also produced anticoccidial effects mediated through substantial reduction or complete termination of oocyst production and sporulation as well as appreciation of the PCV and weight gain of Eimeria infected birds treated with the extracts. These effects were highest with A. senegalensis and K. senegalensis. The high toxicity of B. paradoxum precludes its possible usefulness as an anticoccidial agent. There is need for farmers who use these extracts in the traditional treatment of avian coccidiosis to be mindful of their levels of toxicity to avoid unproductive effects.
Dr. Kabiru Yahayah, the second author presented this research as part of an M.Sc. dissertation and shortly after, translated in an unfortunate automobile accident. This publication is a tribute to his efforts to do his best at all times.