# Morphometric Identification, Gross and Histopathological Lesions ofEimeria Species in Japanese Quails (Coturnix coturnix japonica) in Zaria, Nigeria

Authors: H. A. Umar; I. A. Lawal; O. O. Okubanjo; A. M. Wakawa
Journal: Journal of Veterinary Medicine
 (2014)

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## Abstract

The objective of the study was to identify the species, gross and histopathological lesions ofEimeria in Japanese quails in Zaria. A total of 400 fresh faecal samples were collected and 10 quail birds were purchased from a quail farm. The faecal samples were processed using simple floatation technique. Oocysts shape indices of sporulated oocysts were determined. The intestines were observed for gross lesions and segments were analyzed using Giemsa stain and  Haematoxylin and Eosin stain and then observed microscopically for the developmental stages of the parasite. Four species of Eimeria were identified in the study. Eimeria bateri of shape index of 1.36 conformed to the guidelines used while the other three Eimeria species with shape indices of 1.48, 1.03, and 1.40 were not confirmed. The main gross lesion seen was nonhaemorrhagic ballooning of the caeca. Intestinal scrapping smear revealed a developmental stage of the parasite (merozoites) in the jejunum. Histopathology also revealed a developmental stage (schizont) of the parasite in the caecum and desquamation of the epithelial lining with areas of necrosis. Further study is required using molecular techniques to properly identify the unknown species of Eimeria that were detected in the study.


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## Body

## 1. Introduction

Quails are most susceptible to various diseases such as coccidiosis which is recognized as a serious parasitic disease problem limiting quail industry [1]. Quail production has become important in Nigeria. Descriptions ofEimeria date from the beginning of the last century, and ever since means for an appropriate characterization and identification of the species have been discussed. Several parameters can be used [2] and new methods have been developed [3–6]. Various species ofEimeriahave been isolated from the different species of quails such asE.tsunodai,E. uzura, andE. bateridescribed from Japanese quails [7] andE. lophortygisandE. okanaganensisdescribed from California quails, whileE. crustiandE. oreortygisare described from mountain quail [8],E. conturnicisandE. bateriare described from grey quail [9],E. coliniandE. lettyaeare described from bob white quail [10], and alsoE. tahamensisis described from Arabian quail [11]. The natural infection ofEimeria in quails is characterized as subclinical because of the mild and nonspecific clinical signs. Nevertheless, coccidiosis is considered as an important disease because the endogenous stages of the parasites and a high number of oocysts in feces are associated with intestinal lesions [7]. Therefore, the objective of the study was to identify the species and report the gross and histopathological lesions ofEimeriain Japanese quails in Zaria, Nigeria.


## 2. Materials and Methods

### 2.1. Study Area

This study was carried out in Zaria located in Kaduna State, located within latitudes 11°7′ to 11°12′ N and longitude 07°41′ E. It is a medium sized city with an estimated population of 408,198 [12]. It is divided administratively into Zaria and Sabon Gari LGAs [13]. It has an estimated land area of about 300 square kilometers and it is approximated that about 40–75% of its working population derive their principal means of livelihood from agriculture [14]. Zaria which is located in the North Guinea Savannah zone of Nigeria has an annual ambient temperature, ranging between 18.0
±
3.7°C and 31.8
±
3.2°C. The harmattan season (the cold-dry period of the year) lasts from November to February, while the hot-dry season lasts from March to May and the rainy season lasts from June to October [15, 16].


### 2.2. Sample Collection, Handling, and Processing

Forty fresh faecal samples were collected using polythene bags. Ten live quail birds were also gotten from the farm. The samples were labeled and transported to the Helminthology Laboratory in the Department of Veterinary Parasitology and Entomology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria. In the laboratory samples not examined immediately were refrigerated at 4°C to maintain the integrity of the oocyst [17].


### 2.3. Laboratory Examination

The faecal samples were examined for the presence of coccidia oocysts using the simple floatation technique as described by Urquhart et al. [18].Oocyst positive samples were diluted into 2.5% aqueous potassium dichromate (K2Cr2O7) and kept in Petri dishes for sporulation at room temperature. After sporulation, oocysts were recovered by centrifugation with saturated sugar solution as described by Duszynski and Wilber [17] and used in subsequent analysis.The shape indices (length/width) of the sporulated oocysts were determined by using the method for species identification as described by Harper and Penzorn [19].The calculated oocysts shape index values were then compared with the standard diagnostic guide provided by Teixeira et al. [20] to determine the species encountered in the study.


### 2.4. Necropsy

The birds were euthanized and this was accomplished in accordance with guide for the care and use of laboratory animals [21].Segments of the small intestine (duodenum, jejunum, and ileum) and large intestine (caeca) were taken for gross and histopathological studies.The segments were examined macroscopically for gross lesions. Intestinal scraping smear was carried out to detect the presence of developmental stages of the parasite within the intestine using Giemsa stain as described by Dubey [22].Histological examination was carried out to confirm the presence of the developmental stages of the parasites and pathological lesions within the small and large intestine using Haematoxylin and Eosin stain as described by Mitchell et al. [23].


### 2.5. Statistical Analysis

Oocysts measurements were analyzed using the software Excel (Microsoft) for mean and standard deviation.


## 2.1. Study Area

This study was carried out in Zaria located in Kaduna State, located within latitudes 11°7′ to 11°12′ N and longitude 07°41′ E. It is a medium sized city with an estimated population of 408,198 [12]. It is divided administratively into Zaria and Sabon Gari LGAs [13]. It has an estimated land area of about 300 square kilometers and it is approximated that about 40–75% of its working population derive their principal means of livelihood from agriculture [14]. Zaria which is located in the North Guinea Savannah zone of Nigeria has an annual ambient temperature, ranging between 18.0
±
3.7°C and 31.8
±
3.2°C. The harmattan season (the cold-dry period of the year) lasts from November to February, while the hot-dry season lasts from March to May and the rainy season lasts from June to October [15, 16].


## 2.2. Sample Collection, Handling, and Processing

Forty fresh faecal samples were collected using polythene bags. Ten live quail birds were also gotten from the farm. The samples were labeled and transported to the Helminthology Laboratory in the Department of Veterinary Parasitology and Entomology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria. In the laboratory samples not examined immediately were refrigerated at 4°C to maintain the integrity of the oocyst [17].


## 2.3. Laboratory Examination

The faecal samples were examined for the presence of coccidia oocysts using the simple floatation technique as described by Urquhart et al. [18].Oocyst positive samples were diluted into 2.5% aqueous potassium dichromate (K2Cr2O7) and kept in Petri dishes for sporulation at room temperature. After sporulation, oocysts were recovered by centrifugation with saturated sugar solution as described by Duszynski and Wilber [17] and used in subsequent analysis.The shape indices (length/width) of the sporulated oocysts were determined by using the method for species identification as described by Harper and Penzorn [19].The calculated oocysts shape index values were then compared with the standard diagnostic guide provided by Teixeira et al. [20] to determine the species encountered in the study.


## 2.4. Necropsy

The birds were euthanized and this was accomplished in accordance with guide for the care and use of laboratory animals [21].Segments of the small intestine (duodenum, jejunum, and ileum) and large intestine (caeca) were taken for gross and histopathological studies.The segments were examined macroscopically for gross lesions. Intestinal scraping smear was carried out to detect the presence of developmental stages of the parasite within the intestine using Giemsa stain as described by Dubey [22].Histological examination was carried out to confirm the presence of the developmental stages of the parasites and pathological lesions within the small and large intestine using Haematoxylin and Eosin stain as described by Mitchell et al. [23].


## 2.5. Statistical Analysis

Oocysts measurements were analyzed using the software Excel (Microsoft) for mean and standard deviation.


## 3. Results

### 3.1. Oocyst Speciation

Table1 shows the oocyst dimensions and morphological characteristics of differentEimeria species identified in the study. TheEimeria species encountered in the study wereEimeria bateri(Figure 2) and three otherEimeriaspecies (Figures 3, 4, and 5) not covered by the diagnostic guidelines.Table 1
Morphological characteristics and speciation ofEimeria in Japanese quail.
Species
Oocyst size (μ)
Shape index
Morphology
Polar granule
Oocyst wall
Confirmed species
Length(Mean ± StE.)
Width(Mean ± StE.)
Range
(a)Eimeriaspp.
22.20± 0.58
16.38± 0.42
(18.20–25.48)(14.56–18.20)
1.36
Subspherical
Present
Double
Eimeria bateri
(b)Eimeriaspp.
22.36± 1.67
15.08± 0.95
(14.56–25.48)(10.92–18.20)
1.48
Ovoid
Absent
Double
Unknown
(c)Eimeriaspp.
16.64± 1.08
16.12± 0.74
(14.56–21.84)(14.56–18.20)
1.03
Ellipsoidal
Absent
Double
Unknown
(d)Eimeriaspp.
20.57± 0.40
14.74± 0.56
(18.20–21.84)(10.92–21.84)
1.40
Subspherical
Absent
Double
Unknown


### 3.2. Gross and Histopathological Lesions

The fresh faecal sample was positive forEimeriaoocysts (Figure 1). The main gross pathology seen was the ballooning of the caeca which contains no bloody exudate in the lumen (Figure 6). Histopathologically, desquamation of intestinal mucosa was observed (Figure 9) and caecal necrosis (Figure 8). Developmental stages of the parasite especially the merozoites and schizont were seen in the intestinal epithelium (Figures 7, 8, and 9).Figure 1
Numerous unsporulatedEimeria oocyst from quails using simple floatation technique (×10).Figure 2
SubsphericalEimeria bateri oocyst with a polar granule using ocular micrometer (×40).Figure 3
OvoidEimeria oocyst using ocular micrometer (×40).Figure 4
EllipsoidalEimeria oocyst using an ocular micrometer (×40).Figure 5
SubsphericalEimeriaoocyst with no polar granule using ocular micrometer (×40).Figure 6
Ballooning of the caeca of a Japanese quail naturally infected withEimeria bateri.Figure 7
Jejunum of Japanese quail showing the developmental stage (merozoite) ofEimeriaspp. in the epithelium using Giemsa stain (×100) oil immersion.Figure 8
Histological section of the caecum showing the (a) developmental stage (schizont) ofEimeriaspp. and (b) area of necrosis H&E (×40).Figure 9
Histological section of duodenum showing the (a) developmental stage (schizonts) ofEimeriaspp. and (b) desquamation of the epithelium H&E (×40).


## 3.1. Oocyst Speciation

Table1 shows the oocyst dimensions and morphological characteristics of differentEimeria species identified in the study. TheEimeria species encountered in the study wereEimeria bateri(Figure 2) and three otherEimeriaspecies (Figures 3, 4, and 5) not covered by the diagnostic guidelines.Table 1
Morphological characteristics and speciation ofEimeria in Japanese quail.
Species
Oocyst size (μ)
Shape index
Morphology
Polar granule
Oocyst wall
Confirmed species
Length(Mean ± StE.)
Width(Mean ± StE.)
Range
(a)Eimeriaspp.
22.20± 0.58
16.38± 0.42
(18.20–25.48)(14.56–18.20)
1.36
Subspherical
Present
Double
Eimeria bateri
(b)Eimeriaspp.
22.36± 1.67
15.08± 0.95
(14.56–25.48)(10.92–18.20)
1.48
Ovoid
Absent
Double
Unknown
(c)Eimeriaspp.
16.64± 1.08
16.12± 0.74
(14.56–21.84)(14.56–18.20)
1.03
Ellipsoidal
Absent
Double
Unknown
(d)Eimeriaspp.
20.57± 0.40
14.74± 0.56
(18.20–21.84)(10.92–21.84)
1.40
Subspherical
Absent
Double
Unknown


## 3.2. Gross and Histopathological Lesions

The fresh faecal sample was positive forEimeriaoocysts (Figure 1). The main gross pathology seen was the ballooning of the caeca which contains no bloody exudate in the lumen (Figure 6). Histopathologically, desquamation of intestinal mucosa was observed (Figure 9) and caecal necrosis (Figure 8). Developmental stages of the parasite especially the merozoites and schizont were seen in the intestinal epithelium (Figures 7, 8, and 9).Figure 1
Numerous unsporulatedEimeria oocyst from quails using simple floatation technique (×10).Figure 2
SubsphericalEimeria bateri oocyst with a polar granule using ocular micrometer (×40).Figure 3
OvoidEimeria oocyst using ocular micrometer (×40).Figure 4
EllipsoidalEimeria oocyst using an ocular micrometer (×40).Figure 5
SubsphericalEimeriaoocyst with no polar granule using ocular micrometer (×40).Figure 6
Ballooning of the caeca of a Japanese quail naturally infected withEimeria bateri.Figure 7
Jejunum of Japanese quail showing the developmental stage (merozoite) ofEimeriaspp. in the epithelium using Giemsa stain (×100) oil immersion.Figure 8
Histological section of the caecum showing the (a) developmental stage (schizont) ofEimeriaspp. and (b) area of necrosis H&E (×40).Figure 9
Histological section of duodenum showing the (a) developmental stage (schizonts) ofEimeriaspp. and (b) desquamation of the epithelium H&E (×40).


## 4. Discussion

Quails are considered a branch of the modern poultry industry in Nigeria. The study showed that there were coccidia oocysts in the farm sampled from. In our study the identification ofEimeria species was done using the oocysts morphometric technique. FourEimeriaspecies were identified from Japanese quails in the study.Eimeria bateri with shape index of 1.36 conformed to the guidelines of Teixeira et al. [20] and Mohammad [24]. The other threeEimeria species with shape indices of 1.48, 1.03, and 1.40 could not speciate as they do not conform to any of the given species by the above authors.In spite of the shortcomings identified above, differences in morphology of the oocyst which were ovoid, subspherical, and ellipsoidal, presence and absence of polar granule can be observed. This means that other unidentified species ofEimeria exist in Zaria. Discrimination ofEimeria species using this technique has sort of limitations to be used as a single tool for diagnosis, meaning that results obtained with this method should be carefully interpreted [25, 26]. This is because the measurements of the oocysts may undergo variations due to changes in metabolism of the parasites or birds and even in the value of the shape morphometric indices of the oocysts that may overlap leading to misleading conclusions regarding the species [27]. Despite the limitations of morphometric techniques, there are some reports that indicated that oocyst morphometry could also be a sensitive method for the discrimination ofEimeria species of poultry in field trials as it shows high degree of agreement with the molecular methods [28–31]. The use of molecular techniques is the most specific and rapid way of diagnosingEimeria infection especially when it involves several species occurring concurrently [32–34]. However, in developing countries like Nigeria very few laboratories have the facilities and personnel to carry out these molecular techniques for routine diagnosis of coccidiosis. The study also showed that the gross lesions associated withEimeria infection in Japanese quails were limited to mainly ballooning of the caeca. The only clinical signs observed were diarrhea. The reports of previous works seem to be at variance with the present observation as regards the pathogenicity ofEimeria in quails. Mazurkiewicz et al. [35] reported a wide range of clinical signs such as lack of appetite, ruffled feathers, uncoordinated movements, inhibition of laying, and loss of weight in naturally infected young and mature quails reared at the laboratory. In young Japanese quailsinfected withEimeria bateri, mild loss of weight, anorexia, and softening of feces were observed and were considered mild and easy to overcome [36]. Tsunoda and Muraki [37] also reported low pathogenicity in Japanese quails experimentally infected with 1 × 105 oocysts ofEimeria uzura with signs limited to diarrhea and anemia with no mortality was reported. However, Ruff et al. [38] used pure and mixed cultures ofE. uzurato infect quails and reported mortality, lower weight gain, and poor reproductive performance. Several factors such as environment, differences in the parasite strain, and management system may be responsible for the discrepancies in the observation of the various works. The findings from this study show that merozoites are the main endogenous stages of the parasites found in the small intestine and schizonts in the large intestine. The absence of other pathogenic stages such as the gametocytes means that the pathology of the infected quails will be mild. Histopathological lesions were located in the villi. These observations resemble those of Norton and Peirce [36], Mazurkiewicz et al. [35], and Tsutsumi [9] as regards the site of infection. Desquamation of epithelial lining and caecal necrosis were also observed in the study.In conclusion, fourEimeriaspecies were identified in the study, onlyEimeria bateri speciated as it conformed to the guideline used. Due to the limitations of speciation using the oocysts morphometry technique, there is the need for further studies to be carried out using molecular techniques to properly identify the unknown species ofEimeria which were detected in the study. The study also showed that the gross and histopathological changes in intestinal tract pointed to the serious effect ofEimeriaspecies in quails. This observation has an important value since there is a paucity of information on the pathogenicity ofEimeria spp. in quails.



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*Source: 451945-2014-11-05.xml*