Research Article
Open Access
Effects of Anticoccidial Drugs on Gross and
Histopathological Lesions Caused by Experimental
Rabbit Coccidiosis
Kennedy O Ogolla1*, Paul O Okumu1, Peter K Gathumbi1 and Robert M Waruiru1
1Department of Veterinary Pathology, Microbiology and Parasitology, University of Nairobi, P.O. Box 29053-00625, Kangemi, Nairobi,
Kenya.
*Corresponding author: Kennedy O. Ogolla, University of Nairobi, PO Box 29053-00625, Kangemi, Nairobi, Kenya, Tel: +254710143604.Email address:
@
Received: 8 June, 2018; Accepted: 22 June, 2018; Published: 27 June, 2018
Citation: Ogolla KO, Okumu PO, Gathumbi PK, Waruiru RM (2018) Effects of Anticoccidial Drugs on Gross and Histopathological Lesions Caused by Experimental Rabbit Coccidiosis. SOJ Vet Sci 4(3): 1-10. DOI:
10.15226/2381-2907/4/3/00158
Abstract
Effects of commonly used anticoccidial drugs in treating lesions caused by intestinal and hepatic coccidiosis have not been described for anticoccidial-drugs used in Kenya. The objective of this study was to describe the effects of four anticoccidial drugs on gross and histopathological
lesions caused by artificially-induced mixed Eimeria infection with inoculant dose of E. flavescens (20%), E. perforans (21%), E. intestinalis (9%), E. coecicola (4.2%), E. media (11.2%), E. piriformis (10.6%), E. stidae (16%), and E. Magna (8%). In a controlled laboratory trial, 60 weaner rabbits were randomly allocated to treatment groups A, B, C, D, E and F. Each group had 10 rabbits. Groups A and C served as uninfected-untreated and infecteduntreated control groups, respectively. Groups B, D, E and F were experimentally infected and treated with amprolium, diclazuril, sulfachloropyrazine, and trimethoprim-sulfamethoxazole, respectively. On day 30 post treatment, 3 rabbits from each treatment group were selected randomly and humanely euthanized for gross and microscopic lesion examination. Diclazuril and sulfachloropyrazine treatment groups had significantly minimal to no macroscopic and microscopic lesions. This was consistent with a high efficacy of the drugs in reversing intestinal and hepatic lesions of coccidiosis in rabbits. Rabbits from amprolium, trimethoprim-sulfamethoxazole and infected-untreated control groups presented with severe intestinal and hepatic gross lesions characterized by extensive hepatomegaly, numerous raised hepatic multinodular lesions and marked congestion of the intestines that indicated mild to no effect of the drugs in reversing hepatic and intestinal lesions. Microscopic lesions in rabbits treated with amprolium and trimethoprim-sulfamethoxazole had marked desquamation of intestinal and bile duct epithelium, distended and thickened bile duct, numerous coccidian parasites at different stages of development in duct epithelium and mature oocysts in the intestinal and bile duct lumens; as was in positive control group. Additionally, rabbits from the three treatment groups recorded higher intestinal and hepatic histological lesion scores.
Keywords: Amprolium; Diclazuril; macroscopic; pathology; rabbits; Sulfachloropyrazine.
Introduction
Rabbit coccidiosis is a protozoan infection caused by Eimeria
spp. parasites [1]. Two forms of coccidiosis affect rabbits
resulting in mild to severe macroscopic (gross) and microscopic
(histopathologic) lesions [2]. One form is hepatic coccidiosis
caused by Eimeria stiedae that targets the liver resulting in high
morbidity and mortality depending on infective dose [1]. The
other form is intestinal coccidiosis caused by several Eimeria
species which have varied pathogenicity and target sites along
the intestinal tract [1]. Most pathogenic of these are E. intestinalis
and E. flavescens; E. magna, E. media, E. irresidua, E. perforans,
E. piriformis, E. exigua and E. vejdovskyi have moderate to mild
pathogenicity [3]. Mixed infection by both forms are common
with weaner rabbits being most susceptible to the infection [4,
5]. Transmission of both forms of coccidiosis is mainly by fecaloral
route through consumption of feed and water contaminated
by sporulated oocysts [6]. Most cases of hepatic coccidiosis
present with gross and histological lesions characterized by
hepatomegaly, raised multinodular lesions on the liver surface,
distended gallbladder, dilated biliary tree, distended bile duct,
atrophy of biliary epithelium, hepatocyte necrosis among others
[7-14]. Gross and microscopic intestinal lesions of intestinal
coccidiosis have been described in numerous studies [1, 11,
14, 15]. Several anticoccidial drugs are available for preventing
and treating these lesions [16]. Diclazuril, a derivative of
benzeneacetonitrile, is indicated for prophylactic and curative
use in rabbits at 5ppm and 10ppm, respectively [17]. It acts by
blocking the excretion of oocysts which interrupts the life cycle of
Eimeria spp. [18]. Similarly, sulphonamides such as sulfadiazine,
sulfamethazine, sulfamerazine and sulfaquinoxaline have been
used in treatment and prevention of rabbit coccidiosis for
decades [19]. Other anticoccidial drugs that have been widely
used on clinical coccidiosis under various dosages with varied
efficacies include amprolium, salinomycin, maduramycin,
monensin, clopidol, narasin, robenidine and lasalocid [16]. Most
coccidiostats are rarely effective against coccidiosis once rabbits
present with clinical signs [1]. Consequently, they are effective
when administered on day of exposure to the oocyst [20]. Majority
of coccidiostats inhibit metabolic pathways of merozoites,
meronts and sporozoites thus interrupting completion of the life
cycle [16]. Efficacious therapeutic anticoccidials are able to either
ameliorate and/or eliminate these signs and lesions [20, 21].
However, the effectiveness of these anticoccidials in reversing the
gross and histological lesions caused by intestinal and hepatic
coccidiosis has not been studied in details. Joyner et al. [22]
demonstrated how growth, oocyst shed, and serum activities
respond to treatments with robenidine, sulphaquinoxaline,
clopidol and methyl benzoquate. Additionally, Peeters et al.
described activities of robenidine, salinomycin, and clopidolmethylbenzoate
combination against rabbit hepatic coccidiosis
[23]. The aim of the present study was to describe the effects of
amprolium, trimethoprim-sulfamethoxazole, sulfachloropyrazine
and diclazuril in reversing (resolving) the macroscopic and
microscopic lesions caused by experimentally-induced rabbit
coccidiosis.
Materials and Methods
Design of Experiment
A total of 60 weanling California white and New Zealand
white rabbit breeds were randomly allocated to treatment groups
A, B, C, D, E and F each consisting of 10 rabbits. Groups A and
C served as negative (not infected and not treated) and positive
(infected but not treated) controls, respectively. Rabbits in groups
B, C, D, E and F were experimentally infected with 120,000 mixed
sporulated oocysts of E. perforans (21%), E. flavescens (20%),
E. intestinalis (9%), E. coecicola (4.2%), E. media (11.2%),
E.piriformis (10.6%), E. stidae (16%), and E. Magna (8%) prepared
as described by Ryley et al. [24]. The inoculant was administered
orally using a syringe. Rabbits were then treated when oocyst
counts per gram of feces reached 500,000 o.p.g and/or when
clinical signs of coccidiosis were observed. Group B was treated
with amprolium administered at 1000ppm for 7 consecutive days.
Group D was treated with diclazuril (Diclosol 1%) at 10 ppm for
48 hours. Group E was treated with sulfachloropyrazine for six
days as follows: 1st, 2nd, 3rd, 5th, 7th,
and 9th at 2000ppm. Group F was treated with trimethoprim-sulfamethoxazole combination
administered at 1000ppm for 7 consecutive days. All drugs
were administered in drinking water. The rabbits were clinically
observed on a daily basis for 20 days after treatment. On day
20 post treatment, three rabbits were randomly selected from
each treatment group for necropsy. The rabbits were euthanized
by sodium pentobarbital (Euthatol®) at 100mg/kg via intracardiac
route and systemic necropsy performed according to the
guidelines provided by the University of Nairobi.
Gross and Histopathological Examination
Gross lesions were examined at autopsy and tissue samples
collected from the liver, duodenum, ileum, caecum and colon for
microscopic examination, and lesion scoring. Histopathology
samples were preserved in buffered formalin (10%) and
routinely processed [25]. Thin sections 4-5 μm were prepared
and stained using haematoxylin and eosin [26]. All the slides
were examined using a light microscope and lesion comparisons
made in treatment groups. Histopathological lesions were scored
according to a set criteria: marked (41-100% of tissue involved),
moderate (21-40% of tissue involved), mild (11-20% of tissue
involved) and minimal (0- 10% of tissue involved) by recording
the nature and extent of lesion and its frequency of occurrence
in randomly selected sites in the tissue [27]. Specific intestinal
and hepatic lesions scored at various magnification powers are
as shown in Table 1.
Table 1: Lesion scoring criteria used and specific liver and intestinal lesions scored in the experimental efficacy trial.
Grade/
Score |
Grade description |
Focal and multifocal lesions |
diffusely distributed lesions |
various Eimeria stages in lamina propia and enterocyte (X400 mag)
(10 sites examined) |
various Eimeria stages in liver ducts
(X400 mag)
(10 sites examined) |
Multifocal lesions coalescing in liver architecture
(X40 mag)
(5 sites examined) |
Desquamated epithelium/
Enterocytes
(X400)
(10 sites examined) |
Desquamated duct epithelium
(X400 mag)
(10 sites examined) |
1 |
Minimal |
<10% of tissue involved |
<10% of tissue involved |
<10% of the tissue is involved |
<10% of tissue is involved |
<10% of tissue is involved |
2 |
Mild |
11-20% of the tissue involved |
11-20% of the tissue involved |
11-20% of tissue involved |
11-20% of tissue involved |
11-20% of tissue involved |
3 |
Moderate |
Between 21 to 40% of tissue parts affected |
Between 1 to 40% of tissue parts affected |
Between 21 to 40% of tissue parts affected |
Between 21 to 40% of tissue parts affected |
Between 21 to 40% of tissue parts affected |
4 |
Marked |
Between 41to 100% of tissue parts affected |
Between 41 to 100% of tissue parts affected |
Between 41 to 100% of tissue parts affected |
41-100% of tissue section involved |
41-100% of tissue section involved |
Animal Welfare
Rabbits were housed in clean cages in a room with uniform
environmental quality and fed on coccidia-free commercial
pellets. Water was provided to the rabbits ad libitum. The
experiment was approved by the University of Nairobi Ethics and
Animal Use Committee.
Data Analysis
Data obtained from the study was entered in MS excel 2016
spreadsheet. One or two way ANOVA of Genstat was used to
perform analysis of variance. Significant differences of means
of different treatment groups were illustrated by Bonferroni
multiple comparison test to control overall significance levels as
described in Genstat statistical analysis program (GenStat 15th
Edition). Means, Standard Error of Means (SEM) and significance
difference at 0.05 were provided.
Results
External Examination and Macroscopic (Gross)
Findings
From external examination, most of the rabbit carcasses
from amprolium, trimethoprim-sulfamethoxazole and infecteduntreated
positive control groups had rough hair coats, matted
perineal area, appeared ‘pot-bellied’, were emaciated and
dehydrated (Figure 1). Icteric mucous membrane was seen in
two rabbits from positive control and amprolium treatment
groups. However, few rabbits from these treatment groups did
not present with any overt gross finding at external examination.
One rabbit carcass from sulfachloropyrazine group had rough
hair coat with slight dehydration but the rest from the group
and those from diclazuril treatment group had no significant
findings at external examination. Rabbits from amprolium (B),
trimethoprim-sulfamethoxazole (F) and infected-untreated (C)
treatment groups presented with severe hepatic and intestinal
gross lesions at necropsy. Intestines from these three treatment
groups presented the following lesions: severe congestion of
the duodenum, ileum and caecum (Figures 2 and 3); dark-gray
Figure 1: A rabbit allocated to the diclazuril treatment group (D) with
diarrhea (matted perineal area), rough hair coat and distended abdomen
(pot-belly) on day 1 of treatment.
semi-solid luminal content (Figure 4), some with blood stains
and dark-necrotic foci on the caecum (Figure 2). Some sections of
the intestines had hyperemia and echymotic hemorrhages on the
serosal surface. Ballooned sections of the caecum (BC) and ileum
were also observed (Figure 2). Intestinal mucosa of duodenum,
jejunum and ileum was discoloured with numerous hemorrhagic
spots. Intestines from diclazuril (D) and sulfachloropyrazine (E)
treatment groups had minimal to no gross lesions (Figure 5), with
most appearing similar to intestines from uninfected-untreated
control group (A). Treatments by amprolium and trimethoprimsulfamethoxazole
drugs were ineffective in reversing the gross
lesions caused by hepatic coccidiosis. Livers from amprolium and
trimethoprim-sulfamethoxazole treatment groups had lesions
indistinguishable from those manifested by infected but not
treated control group. These lesions included discoloured and
enlarged livers with markedly dilated bile ducts and gallbladder
that contained thick yellowish-white contents that was viscous to
solid in consistency (Figures 6, 7 and 8). The entire biliary tree was
distended with yellowish-white thick-flowing content (Figures
7 and 8). Livers from treatment groups B, C and F had clearlydemarcated
raised yellowish-white nodular lesions (0.5-1cm in
diameter) covering the entire liver surface (Figure 6). Some of
these multinodular lesions occasionally coalesced to form larger
linear nodules (Figure 6). Additionally, these livers had fibrinous
exudates on their surfaces (Figure 6), were firm in consistency
and were congested. Wet smears of the intestinal mucosa and
livers revealed both mature and immature eimerian instead
of eimerian stages. Livers from sulfachloropyrazine treatment
group were only slightly enlarged with few tiny yellowish-white
lesions on the surface which were for most cases not raised. The
gallbladders from sulfachloropyrazine treatment group were
slightly distended with greenish-yellow slightly viscous content
(Figure 8E) indicating satisfactory efficacy of sulfachloropyrazine
in reversing these lesions, close to the free-flowing contents of
the negative control and diclazuril treatment group which had
superior efficacy in this trial (Figure 8D). Livers from diclazuril
treatment group had no significant lesions and were the same in
size with those from uninfected-untreated control group (Figure
4).
Figure 2: Gross intestinal lesions from trimethoprim-sulfamethoxazole
treatment group (F) showing marked congestion and hyperemia (white
arrow), necrotic caecal parts (black arrow) and ballooned section of the
caecum (BC)
Figure 3: Amprolium treatment group (B) showing ballooned section
of the ileum (black arrow head) and caecum (black arrow), extensive
congestion and hyperemia of the ileum (white arrow), jejunum (white
arrow head) colon (white arrow with broken ends) caecum (black arrow
with curved end).
Figure 4: Gross intestinal lesions: D- duodenal section from diclazuril
treatment group with the normative pink colour compared with the
highly congested and necrotic duodenal part containing dark luminal
content from the positive control group (C)
Figure 5: Gross intestinal lesions, a shiny, healthy pink caecum and colon
from negative control group
Figure 6: Rabbit livers from diclazuril (D) and positive control (C) treatment
groups. Normal liver from diclazuril treatment group and one with
marked hepatomegaly, coalesced nodular lesions (arrow) and fibrinous
strands on liver surface from infected but not treated group (C) at the
end of experiment
Figure 7: Livers from trimethoprim-sulfamehtoxazole (F) and amprolium (B) treatment groups. Livers from amprolium
treatment group had more extensive hepatomegally compared to those from trimethoprim-sulfamethoxazole treatment
groups though the extent of hepatic injury was severe in both groups.
Figure 8: Hepatic lesions at the end of the efficacy trial: D, greenish-yellow contents from a normative gallbladder from diclazuril
treatment group(black arrow), and gallbladder and liver parenchyma with the normal dark appearance (white arrow). C, thick whitish-yellow contents from incised
gallbladder with numerous multinodular lesions from infected untreated group. E, slightly distended gallbladder dark appearance and fading liver
lesions from sulfachloropyrazine treatment group.
Histopathologic (Microscopic) Lesions and Mean
Lesion Scores
Histological examination of the intestines revealed severe
desquamation of epithelium (Figure 9A), extensive atrophy
of villi lining (enterocytes of villi epithelium) (Figure 9B),
marked hyperplasia of the submucosal goblet cells (Figures 9C
and 9D) and immature developmental stages of Eimeria spp.
parasites within enterocytes (Figures 9E and 9F) in treatment
groups B, C, E and F. Severe microscopic hepatic and intestinal
lesions scores were recorded by amprolium(B), trimethoprimsulfamethoxazole(
F) and infected-untreated(C) treatment
groups. Mean lesion scores are presented in Table 1 (intestinal)
and 2 (hepatic). Other lesions observed less frequently but not
scored were congestion, capillary haemorrhages and dilation of
blood vessels, red blood cells within the lumen of the intestines,
fusion of the villi, precipitates of protein in the intestinal lumen,
oedema of the muscularis and necrosis of enterocytes.
Hepatic lesions were characterized by marked fibrosis and
hyperplasia of the peribiliary (periductal) parts (Figures 10A &
10B) with leucocytic cell infiltration around the bile ducts (Figure
10E), formation of new ductules around the ducts (Figure 10D),
severe distention of the bile duct accompanied by flattening and
desquamation of their duct epithelium (Figure 10C), hyperplasia
of remaining biliary epithelium with varied developmental
stages of Eimeria (thin walled ovoid oocysts, macrogametocytes
and microgametocytes) within biliary epithelium (Figures 10D,
10E and 10F), atrophy and necrotic degenerative changes of the
hepatocytes and multiple coalescing lesions in treatment groups
B, C and F as presented in Figure 10. The enlarged bile ducts were
lined by pronounced columnar epithelial cells that formed several
papillary fronds which extended to the lumen of the duct (Figure
11A). There were areas with oocysts granulomas (Figure 11B).
Bile ducts lumen were filled and distended with almost mature
stages of Eimeria oocyst which resulted in pressure atrophy on
the neighbouring hepatocytes (Figure 11E). These lesions were
severe in treatment groups B, C and F with only few seen in
treatment group E, fewer in D but absent in the negative control
group (A). Liver lesions that were less frequently observed were
congested and dilated blood vessels (Figure 11B), haemorrhages
and bile pigmentation in the liver parenchyma (Figure 11C and
11D), and precipitation of protein in the duct lumen. Livers from
diclazuril treatment group had no significant lesions (Figure
11F). Mean lesion scores are summarized in Table 3.
Discussion
In this study, diclazuril and sulfachloropyrazine
(sulphonamide) were efficacious in reducing gross and
histopathology lesions of rabbit coccidiosis. Samples from rabbits
treated with the two drugs recorded low to no histopathological
lesion scores close to those recorded by negative control group
(Tables 2 and 3). On the other hand, amprolium and trimethoprimsulfamethoxazole
treatment groups recorded poor efficacy against
lesions from rabbit coccidiosis. No significant difference (p>0.05)
was seen between gross and microscopic lesion scores recorded
by samples from rabbits under amprolium and trimethoprim
Figure 9: Microscopic characteristics of the intestinal lesions-Hematoxylin and Eosin stain. A, desquamated intestinal epithelium. B, disintegration of intestinal mucosa x400 (amprolium group). C, hyperplasia of goblet cells x400 (trimethoprim-sulfamethoxazole group). D, hyperplasia of goblet cells within the submucosa x40. E, oocysts within the intestinal lumen x400. F, several oocysts within the intestinal lumen x40.
Table 2: Mean intestinal microscopic lesion scores quantifying the effects of the anticoccidials on coccidial lesions.
Treatment group |
Caeco-colon lesion scores |
Duodenal lesion scores |
Jejunum-ileum lesion scores |
|
Epithelial desquamation |
Eimeria stages in intestinal tissue and lumen |
Epithelial desquamation |
Eimeria spp. stages in intestinal tissue and lumen |
Epithelial desquamation |
Eimeria stages in intestinal tissue and lumen |
Negative control (A) |
1.33 ± 0.33a |
1.00 ± 0.00a |
2.33 ± 0.67ab |
1.00 ± 0.00a |
3.0 ± 0.00a |
1.00 ± 0.00a |
Amprolium (B) |
3.67 ± 0.33bc |
3.67 ± 0.33b |
4.00 ± 0.00b |
4.00 ± 0.00b |
4.00 ± 0.00b |
4.00 ± 0.00b |
Positivecontrol (C) |
4.00 ± 0.00c |
4.00 ± 0.00b |
3.67 ± 0.33b |
4.00 ± 0.00b |
4.00 ± 0.00b |
4.00 ± 0.00b |
Diclazuril (D) |
1.67 ± 0.67ab |
1.00 ± .00a |
1.67 ± 0.33a |
1.00 ± 0.00a |
2.67 ± 0.33a |
1.00 ± 0.00a |
Sulfachloropyrazine (E) |
3.00 ± 0.58abc |
3.00 ± 0.58b |
3.00 ± 0.00ab |
3.33 ± 0.33b |
3.00 ± 0.00a |
2.33 ± 0.88ab |
Trimethoprim-sulfamethoxazole (F) |
4.00 ± 0.00c |
4.00 ± 0.00b |
4.00 ± 0.00b |
4.00 ± 0.00b |
4.00 ± 0.00b |
4.00 ± 0.00b |
P value |
0.001 |
<0.001 |
0.001 |
<0.001 |
<0.001 |
<0.001 |
Aggregated mean lesion scores of three rabbits per treatment group. Values without similar superscript in a column are significantly different at 0.05
Table 3: Mean hepatic microscopic lesion scores quantifying the effects of the anticoccidials on coccidial lesions.
Treatment group |
Hepatic Lesion Scores |
Epithelial desquamation |
Duct distension |
Eimeria spp. stages in liver tissues |
Hepatocyte necrosis |
Periductal/
peribiliary fibrosis |
Multiple coalescing lesions |
Negative control (A) |
1.33 ± 0.33a |
1.67 ± 0.33a |
1.00 ± 0.00a |
1.00 ± 0.00a |
1.00 ± 0.00 |
1.00 ± 0.00a |
Amprolium (B) |
4.00 ± 0.00b |
4.00 ± 0.00b |
4.00 ± 0.00b |
4.00 ± 0.00c |
4.00 ± 0.00 |
4.00 ± 0.00b |
Positive control (C) |
4.00 ± 0.00b |
4.00 ± 0.00b |
4.00 ± 0.00b |
4.00 ± 0.00c |
4.00 ± 0.00 |
3.67 ± 0.33b |
Diclazuril (D) |
1.00 ± 0.00a |
1.33 ± 0.33a |
1.00 ± 0.00a |
1.00 ± 0.00a |
1.0 0 ± 0.00 |
1.00 ± 0.00a |
Sulfachloropyrazine (E) |
3.33 ± 0.67b |
2.33 ± 0.33a |
3.67 ± 0.33b |
2.67 ± 0.33b |
2.00 ± 0.00 |
1.67 ± 0.33a |
Trimethoprim-sulfamethoxazole (F) |
4.00 ± 0.00b |
4.00 ± 0.00b |
4.00 ± 0.00b |
4.00 ± 0.00c |
4.00 ± 0.00 |
4.00 ± 0.00b |
P value |
<0.001 |
<0.001 |
<0.001 |
<0.001 |
- |
<0.001 |
Values without similar superscript in a column are significantly different at 0.05. Aggregated mean lesion scores of three rabbits per treatment group.
Figure 10: A Microscopic characteristic of hepatic lesions stained with Hematoxylin and Eosin. A, distended bile duct containing numerous oocysts in the lumen (Double-headed white arrow), marked periductal (peribiliary) fibrosis (Black arrow head) and desquamated duct epithelium (arrow) x40 from a rabbit treated with amprolium. B, Severe desquamation of duct epithelium (Arrow), and periductal fibrosis (arrow head) x100 in an infected-untreated rabbit. C, Distended ducts (DD) having flattened epithelium with minimal (arrow head) to no projections
into the lumen (white arrow) filled with oocysts (black arrow),
peribiliary fibrosis (PF), formation of new ductules (Bent arrow) and
an area of hepatocyte necrosis and degeneration (HD) x100 in a rabbit
from trimethoprim-sulfamethoxazole group. D, oval to circular large
macrogametocytes within duct epithelium (White arrow), a round small
microgametocyte (arrow head) at x1000 in a rabbit from amprolium
treatment group. E, Different developmental stages of Eimeria within
the duct epithelium with infiltration of inflammatory cells (Arrow) x400
in a rabbit from amprolium group. F, different developmental stages of
Eimeria with varied shapes and sizes (DDS) x400 from trimethoprimsulfamethoxazole
treatment.
Figure 11: Microscopic hepatic lesions stained with Hematoxylin and
Eosin. A, Numerous papillary branches in the bile duct arising from extensive
proliferation of columnar epithelial cells of the biriary system
x 40 (White arrow) in a rabbit from trimethoprim-sulfamethoxazole
group. B, Congestion (arrow), peribiliary fibrosis (arrow heads) x100 in
rabbit from sulfachloropyrazine group. C, bile pigmentation x400 (black
arrow) (trimethoprim-sulfamethoxazole group). D, bile pigmentation
within the hepatic parenchyma (thick arrow) and yellowish-brown foci
of necrosis (thin arrow) at x400 from trimethoprim-sulfamethoxazole
group. E, numerous mature oocysts in the lumen of two ducts with desquamated
epithelium (double-headed arrow) x400 (amprolium group).
F, liver with relatively normal hepatic architecture (NHA) from diclazuril
treatment group x40.
sulfamethoxazole treatments and those of infected-untreated
control group. In our study, the artificial inoculation was induced
through oral route using syringes as previous studies have
shown that experimental infections induced by alternative routes
such as intraperitoneal and intravenous only results in mild
infections [28]. Pathogenesis and pathology following ingestion
of sporulated oocysts has been described in several studies
[1, 6, 29]. In the current study, only diclazuril used at 10 ppm was
able to completely resolve gross and microscopic lesions of both
hepatic and intestinal coccidiosis. Hepatic and intestinal samples
from rabbits treated with diclazuril recorded lowest microscopic
lesion scores close to those of negative control group. Similarly,
samples from diclazuril treatment group had minimal to no
gross lesions indicative of its high efficacy in treating clinical
coccidiosis which agree with previous studies in rabbits and in
poultry [30, 31]. Treatment with sulfachloropyrazine recorded
satisfactory efficacy in ameliorating gross and microscopic
lesions of coccidiosis with only minor ones that included healing
fibrotic non-raised foci and fibrinous strands at the end of the
study. Microscopic lesion scores organized fibrinous strands
recorded by diclazuril and sulfachloropyrazine treatment groups
had no significant difference (p>0.05). Prophylactic and curative
efficacy of other sulphonamides against chicken and rabbit
coccidiosis have been reported [16, 32, 33]. Studies have shown
that coccidiostats are best used prophylactically as they are not
effective against coccidiosis once clinical signs are presented [16].
The livers and intestines from amprolium and trimethoprimsulfamethoxazole
(potentiated sulphonamide) treatment groups
had severe gross and microscopic lesions indicating their
inability to treat clinical coccidiosis. Gross lesions in these groups
were characterized by hepatomegaly, raised yellowish-white
multinodular lesions, distended biliary tree filled with viscous to
cheesy content, fibrinous strands on the liver surface, congested
and hemorrhagic intestines with ballooned sections consistent
with those described in previous studies [34, 35]. Hyperplasia
and cystic enlargement of the bile duct epithelium has been
reported to results in the yellowish-white nodular lesions on the
liver surface seen in our study [8, 28]. Severe microscopic lesions
described for treatment groups B and F were similar to those of
positive control group and have been described in other studies
[34-36]. Al-Naimi et al. attributed hyperplasia of the bile duct
epithelium to the multiplication of Eimerian parasites in duct
epithelium, the predilection site of the parasite. Studies have
shown that disruption of the continuity of bile ductile epithelium
often results in formation of occysts granuloma reported in our
study as the oocysts acts like foreign bodies [34-36]. Widespread
fibrosis seen in livers from treatment groups B, C and F increase
as the infection gets into chronic phase [1, 12 ,34]. Icterus was
only seen in few severely affected rabbits probably because the
main product of heme breakdown in rabbits is biliverdin as
opposed bilirubin [7]. Other lesions reported in our study and
that have been described in previous studies include deposition
of bile pigment, obstructive jaundice in hepatic parenchyma
and sinusoid dilatation [37, 38, 39]. The findings of this study
show that diclazuril and sulfachloropyrazine drugs can be
used therapeutically against rabbit coccidiosis once clinical
signs presents and have high efficacy in reversing associated
lesions. Therapeutic use of amprolium and trimethoprimsulfamethoxazole
is not effective in reversing coccidial lesions.
Acknowledgement
The authors acknowledge Regional Universities Forum
for Capacity Building in Agriculture (RUFORUM) for funding
this study and University of Nairobi for technical support.
Also acknowledged are Mr. John Mukiri who assisted with
histopathology work, Mr. Samuel Indeche (animal attendant)
and Mr. Richard Otieno (laboratory technologist who helped with
preparation of inoculum used in the study.
Declarations
Authors declare no conflict of interest
Data availability
- Pakandl M. Coccidia of rabbit: a review. Folia Parasitol. 2009; 56(3): 153–166.
- Eckert J, Taylor M, Catchpole J, Licois D, Coudert, P, Bucklar H. Identification of Eimeria species and strains. Morphological characteristics of oocysts. In: Eckert J, Braun R, Shirley M, Coudert P. 1995; 113-116.
- Coudert P, Licois D, Drouet-Viard F. Eimeria species and strains of rabbits. Biotechnology: Guidelines on techniques in coccidiosis research, Part. I: Eimeria and Isospora, Office for official publications of the European communities, Luxembourg. 1995;52-73.
- Gomez-Bautista M, Rojo-Vazquez FA, Alunda JM. The effect of the hosts age on the pathology of Eimeria stiedae infection in rabbits. Vet. Parasitol. 1987; 24(1-2): 47-57.
- Kvicerova J, Pakandl M, Hypsa V. Phylogenetic relationships among Eimeria spp. (Apicomplexa, Eimeriidae) infecting rabbits: evolutionary significance of biological and morphological features. Parasitol. 2008;135(4):443-452.
- Patton KWH, Gorham JR, Flatt RE. Domestic Rabbits Diseases and Parasites. Pacific Northwest Extension Publication Oregon, Idaho, Washington. 2008 ;310:19-24.
- Barriga OO, Arnoni VJ. Pathophysiology of hepatic coccidiosis in rabbits. Vet. Parasitol. 1981;8(3):201-210.
- Wang JS, Tsai SF. Prevalence and pathological study on rabbit hepatic coccidiosis in Taiwan. J. Life Sci. 1991;15(4): 240-243.
- Al-Rukibat RK, Irizarry AR, Lacey K, Kazacos KR, Storandt ST, De-Nicola DB. Impression smears of liver tissue from a rabbit. Vet. Clin. Pathol. 2001;30:57-61.
- Toulah FH, Al-Rawi MM. Efficacy of garlic extract on hepatic coccidiosis in infected rabbits (Oryctolagus cuniculus): histological and biochemical studies. J. Egypt Soc. Parasitol. 2007;37(3):957-968.
- Yakhchali M, Aliasghar T. Eimeriidosis and Pathological Findings in New Zealand White Rabbits. J Biol Sci. 2007;7: 1488-1491.
- Al-Mathal EM. Hepatic Coccidiosis of the Domestic Rabbit Oryctolagus cuniculus domesticus L. in Saudi Arabia. World Journal of Zool. 2008;3(1):30-35.
- Abu-Akkada SS, Oda SS, Ashmawy KI. Garlic and hepatic coccidiosis: Prophylaxis or treatment? Trop Anim Health Prod. 2010;42(7):1337-1343.
- Szkucik K, Renata P, Klaudiusz OS, Waldemar P. Occurrence of gastrointestinal parasites in slaughter rabbits. Parasitol Res. 2014;113(1):59-64.
- Sivajothi S, Sudhakara-Reddy B, Rayulu VC. Intestinal coccidiosis infection in domestic rabbits. Int J Biol Res. 2014; 2(2):48-50.
- Duszynski DW, Lee C. Chapter 10-Strategies for Management, Control, and Chemotherapy. The Biology and Identification of the Coccidia (Apicomplexa) of Rabbits of the World. 2013;253-262.
- Vanparijs O, Hermans l, Van der Fl, Marsboom R. Efficacy of diclazuril in the prevention and cure of intestinal and hepatic coccidiosis in rabbits. Veterinary Parasitol. 1989;32(2-3):109-117.
- Giorgi M, Niccolini A, Soldani G, Martelli F. Pharmacokinetic Study of Diclazuril in Pre-Ruminant and Ruminant Lambs. ISRA J VET MED. 2010;65(2):62-67.
- Levine ND. Protozoan Parasites of Domestic Animals and of Man, second ed. Burgess Publishing Company, Minneapolis, Minnesota. 1973.
- H David Chapman,John R Barta,Damer Blake,Arthur Gruber,Mark Jenkins,Nicholas C.Smith, et al. A selective review of advances in coccidiosis research. Adv Parasitol. 2013; 83:93-171.
- US Department of Health and Human Services; Food and Drug Administration. Guidance for evaluating the effectiveness of Anticoccidial drugs in food-producing animals. Center for Veterinary Medicine, 2012.
- Joyner lP, Catchpole J, Berret S. Eimeria stiedai in rabbits: the demonstration of responses to chemotherapy. Res Vet Sci. 1983;34(1):64-67.
- Peeters JE, Chariier G, Antoine O, Mammerick M. Clinical and pathological changes after Eimeria intestinalis infection in rabbits. Zbl. Veterinary Medicie. B. 1984;31:9–24.
- Ryley JF, Meade R, Burst JH, Robinson TE. Methods in coccidiosis research: Separation of oocysts from faeces. Parasitol.1976,73:311-326.
- Kiernan JA. Histological and histochemical methods. Oxford: Pergamon Press.1981;201-230.
- Carlton W, Mc Gavin MD. Thompson's Special Veterinary Pathology. 2nd Edn. Mosby, St. Louis. 1995;46-47.
- Shackelford C, Long G, Wolf J, Okerberg C, Herbert R. Qualitative and quantitative analysis of non-neoplastic lesions in toxicology studies. Toxicol Pathol. 2002;30(1):93-96.
- Pakes SP, Gerrity LW. Protozoal Diseases. In: The Biology of the Laboratory Rabbit, Manning, P.J., D.H. Ringler and C.E. Newcomer (Eds.). Academic Press, Inc., San Diego,Calif.1994;205-229.
- Bhat TK, Jithendran KP, Kurade NP. Rabbit coccidiosis and its control: a review. World Rabbit Sci. 1996;4(1):37-41.
- Vereecken M, Lavazza A, De Gussem K, Chiari M, Tittarelli C, Zuffellato A, Maertens L. Activity of diclazuril against coccidiosis in growing rabbits: experimental and field experiences. World Rabbit Sci. 2012;20(4):223-230.
- El-Banna HA, El-Bahy MM, El-Zorba HY, El-Hady M. Anticoccidial Efficacy of Drinking Water Soluble Diclazuril on Experimental and Field Coccidiosis in Broiler Chickens. J Vet Med. 2005;52:287-291.
- Laha R, Hemaprasanth DA, Harbola PC. Comparative efficacy of sulphadimidine and combination of amprolium, sulphaquinoxalline in the control of natural coccidial infection in rabbits. INDIAN VET J. 1999;76:1013-1015.
- Hunduma A, Kebede B. Comparative Study on the Efficacy of Amprolium and Sulfadimidine in Coccidia Infected Chickens in Debre Zeit Agricultural Research Center Poultry Farm, Bishoftu, Ethiopia. SOJ Veterinary Sciences.2016; 2(1):1-5.
- AL- Naimi RAS, Khalaf OH, Tano SY, Al- Taee EH. Pathological study of Hepatic coccidiosis in naturally infected rabbits. QJVMS. 2012;11(1).
- Sivajothi S, Reddy BS, Rayulu VC. Study on impression smears of hepatic coccidiosis in rabbits. J Parasit Dis. 2016;40(3):906-909.
- Mehmoud AZ, Ibrahim MK. Granulomatous hepatitis in baldy rabbits associated with coccidial infection. Assuil. Veterinary Journal. 1989;21:55-58.
- Darzi MM, Mirms-Kamil SA, Nashirudddullach N, Munshi ZH. Pathological changes and local defense reaction occurring in spontaneous hepatic coccidiosis in rabbits (Oryctolagus cuniculus). World Rabbit Sci. 2007;15:23-28. doi:10.4995/wrs.2007.608
- Singla LD, Juyal PD, Sandhu BS. Pathology and therapy in naturally Eimeria stiedae–infected rabbits. J Protozool Res. 2000;10:185-191.
- Sanyal PK, Sharma SC. Clinicopathology of hepatic co