Indian Journal of Animal Research

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Research Article

Efficacy of Immunization of Japanese Quail (Coturnix coturnix japonica) Against the Challenge with Different Eimeria Species

M.A. Elmorsy1,2,*, M. Das1, S.K. Senapati1, G.R. Jena1, S. Mishra3, S.K. Panda1, A.K. Kundu1, D. Kumar3
1College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubaneswar-751 003, Odisha, India.
2Department of Poultry and Rabbit Diseases, Faculty of Veterinary Medicine, Mansoura University-35516, Egypt.
3ICAR-Directorate of Poultry Research, Bhubaneswar-751 003, Odisha, India.

ABSTRACT

Background: Coccidiosis is one of the most dangerous diseases infecting Japanese quails. This work was conducted to study the efficacy of immunization of Japanese quail against the challenge with different Eimeria species causing coccidiosis and to optimize the dose of live sporulated oocysts that give immunity without adverse effects.

Methods: Dropping samples were collected from different Japanese quail pens of the Central Poultry Development Organization, Bhubaneswar, Odisha, India. Isolation, identification, single oocyst isolation and propagation were done for each species. On the 2nd day of age, Japanese quails were divided into 10 groups and orally inoculated with low doses, 100 and 1000 live sporulated oocysts, of each isolated Eimeria species separately. Four weeks post-immunization, the quails were challenged with a high dose of the respective species. Immunization efficacy was assessed regarding clinical signs, mortality rate, weight gain, feed consumption, feed conversion ratio, oocysts output, lesion scores and hematological parameters.

Result: All collected samples were Eimeria positive. Three Eimeria species were isolated and identified as E. bateri, E. uzura and E. tsunodai. Experimental results revealed that the immunized quails, either by 100 or 1000 oocysts of any isolated species, showed better results compared to the challenged, non-immunized controls. The 100-oocysts dose gave better results than those of 1000-oocysts dose without significant differences. These results indicated the effectiveness of immunization as an important tool in the management of Japanese quail coccidiosis.

INTRODUCTION

Quail production can be regarded as a branch of the modern poultry industry. Meat and egg production are the most common reasons for raising these birds (Sreeranjini et al., 2010).

Coccidiosis was regarded as a limiting factor for the quail industry as a result of the endogenous stages of the parasite those associated with intestinal lesions. The economic impact of coccidiosis is attributed to the reduction in animal production attributes as denoted by depressed growth rate, higher feed conversion ratio and increased mortality (Peek and Landmanab, 2011).

Because of the increasing problem of drug resistance to anticoccidial drugs, the search for anticoccidial vaccines has become an important topic. Therefore, it is necessary to establish a potential standard method for providing protection by live multivalent vaccines against clinical coccidiosis. There is no specific evidence that chickens are immune to coccidiosis other than by challenging with virulent parasites. Therefore, the major consideration in the assessment of vaccine efficacy is the clinical status of the birds following virulent challenge, particularly growth rate and feed conversion ratio (Williams and Catchpole, 2000). Bobwhite quails immunized by 100 and 1000 oocysts of E. lettyae provided good protection against challenge concerning various parameters (Gerhold et al., 2010). Attenuation of the virulence of coccidia in the live vaccines is largely done by the size of the dose and by the means of administration (Mcdougald and fitz-coy, 2013).

Although many previous studies were conducted on vaccination against coccidiosis of chicken and some Eimeria species of bobwhite quail, studies on immunization of Japanese quail against coccidiosis are limited. Thus, the present study was established to determine the efficacy of immunization of Japanese quail by low doses of live sporulated oocysts of each Eimeria species separately against a high dose challenge and to optimize the dose that stimulates immunity without adverse effects.

MATERIALS AND METHODS

Samples collection and parasite isolation and identification
 
Dropping samples were collected from the litter of different pens housing different ages of Japanese quail at the Central Poultry Development Organization (CPDO), Bhubaneswar, Odisha, India. Samples packed in icebox were sent to laboratory for examination by direct smear method (Urquhart, 2003) and by floatation technique under the light microscope according to Duszynski and Wilber (1997). Positive samples were prepared and oocysts were collected by concentration according to the method of Soulsby (1968). Freshly collected oocysts were suspended in a 2.5% freshly prepared potassium dichromate solution and incubated for few days (3-7 days) at room temperature for sporulation as described by Long (1971). Eimeria oocysts were harvested according to the methods described by Shirley (1995) and preserved in potassium dichromate solution (2.5%) at 4-8°C. Identification was done using light microscope (40X and 100X) besides morphometric identification by calibrated ocular micrometer according to Henddrix and Robinson (2006). Identification was done according to the identification guides described by Berto et al., (2013).
 
Experimental birds
 
1-day-old Japanese quail chicks were purchased from CPDO hatchery for isolation and propagation of single Eimeria species and for the experimental trial. The quails were kept in the College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubaneswar, Odisha, India. Quail chicks were kept in brooding cages with their floors covered by a linoleum layer and wood shaving litter in good sanitary conditions to avoid coccidian contamination. Quails received a non-medicated starter ration and ad libitum drinking water. All quails were reared following the directions for the use of experimental animals of the Institutional Animal Ethical Committee of the College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology.
 
Single oocyst isolation and propagation
 
This was done according to the method of Khalafalla and Daugschies (2010) with some modifications. Briefly, clean small petri dishes were divided into very small areas (1×1 mm2) by using a marker pen and marked with different numbers. A first layer of pre-warmed 5% nutrient agar was spread over the divided plates and kept for 10 min at 4°C to solidify. A thin layer of oocysts suspension (100 oocysts / mL) was spread over the first layer. The plates were examined microscopically. A separate single sporulated oocyst was cut from the agar and placed into a small gel capsule, then introduced into the esophagus of 7-days-old Japanese quails followed by few drops of water. Quails were bred in 3 separate groups (one group for each species) to avoid cross-contamination between the different isolates.  On day 6 post-infection, intestines and ceca were collected and examined for post mortem lesions. Dropping samples plus intestinal and cecal contents were collected separately and examined by direct smears. The isolated oocysts of different species were compared morphologically with the identification guides previously described by Berto et al., (2013) and then preserved for further experimental work. The isolated species were propagated through coccidiosis free Japanese quails, reared in separate sterilized cages to avoid cross-contamination.
 
Experimental trial
 
The experiment was done on January 2021 with a duration of 37 days. It was conducted at the Department of Veterinary Clinical Medicine, College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology.

On the 2nd day of age, quails were randomly divided into 10 groups of three replicates each, 10 birds per replicate. Birds were treated by oral inoculation (intra esophagus) by a small pipette. Immunization was done on the 2nd day of age. It was conducted by oral inoculation (intra esophagus) of 0.2 mL inoculum containing 100 or 1000 oocysts of each Eimeria species separately suspended in sterilized distilled water. Challenge was done 4 weeks post-immunization (day 30 of age). It was conducted by oral inoculation (intra esophagus) of 0.5 mL inoculum containing the challenge dose of the respective Eimeria species suspended in sterilized distilled water. Quails were grouped as follows:
 
Group (1): Negative control group (non-infected, non-treated): inoculated by a sham-dose of 0.5 mL of sterilized distilled water on day 30 of age.
Group (2): E. bateri positive control group: Quails were challenged with 1x105 sporulated oocysts of E. bateri.
Group (3): Quails were inoculated with 100 sporulated oocysts of E. bateri and then challenged with 1x105 sporulated oocysts of E. bateri.
Group (4): Quails were inoculated with 1000 sporulated oocysts of E. bateri and then challenged with 1x105 sporulated oocysts of E. bateri.
Group (5): E. uzura positive control group: Quails were challenged with 1x105 sporulated oocysts of E. uzura.
Group (6): Quails were inoculated with 100 sporulated oocysts of E. uzura and then challenged with 1x105 sporulated oocysts of E. uzura.
Group (7): Quails were inoculated with 1000 sporulated oocysts of E. uzura and then challenged with 1x105 sporulated oocysts of E. uzura.
Group (8): E. tsunodai positive control group: Quails were challenged with 4x104 sporulated oocysts of E. tsunodai.
Group (9): Quails were inoculated with 100 sporulated oocysts of E. tsunodai and then challenged with 4x104 sporulated oocysts of E. tsunodai.
Group (10): Quails were inoculated with 1000 sporulated oocysts of E. tsunodai and then challenged with 4x104 sporulated oocysts of E. tsunodai.
 

Regular monitoring of birds was done for clinical signs and mortalities after inoculation and throughout the experiment (37 days). Dropping samples were collected between days 4-9 post-challenge to detect the effect of immunization on oocyst output through counting by McMaster-chamber as described by Conway and Mckenzie (1991).

On days 4, 5, 6 and 7 post-challenge, 3 birds from each group were sacrificed to examine intestines and ceca for lesion scores. The severity of lesions was evaluated according to Elmorsy et al., (2021).

Individual blood samples were drawn from each bird via wing vein in EDTA vials on day 7 post-challenge for hematological tests. Calculation of total erythrocyte and total leukocyte counts was done according to the method of Natt and Herrick (1952) and Campbell (1995) by using Natt and Herrick’s stain solution. Haemoglobin (Hb) percent was measured by using commercial kit HEMOCOR-D reagent (Crest Biosystems, Goa, India) (cyanmethemoglobin method) as described by Van Kampen and Zijlstra (1961). Packed cell volume (PCV), Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated using the procedures described by Jain (1986). Differential leucocyte count was done through blood smear stained with Wright-Giemsa stain according to the method of Benjamin (1985).

Birds and feed of each replicate were weighed on day 30 of age, prior to challenge and on day 7 post-challenge. Average feed intake, weight gain and feed conversion ratio were calculated.
 
Statistical analysis
 
Data were statistically analyzed by SPSS version 20 by one-way analysis of variance (ANOVA) using least significant difference (LSD) at P-value≤ 0.05 as described by Snedecor and Cochran (1981).

RESULTS AND DISCUSSION

Identification of isolated Eimeria species
 
Eimeria isolates were identified as E. bateri, E. uzura and E. tsunodai (Fig 1). The present study revealed that all collected fecal samples were Eimeria positive and this agreed with Gesek et al., (2014) who reported that coccidiosis is the most predominant parasitic disease in quails. Two Eimeria species were isolated from intestine (E. uzura and E. bateri) and one from cecum (E. tsunodai). These results agreed with those of Teixeira et al., (2004). The morphological characters of the isolated Eimeria species in our study were similar to those previously described by Berto et al., (2013).

Fig 1: E. bateri (left) (100X), E. uzura (middle) (40X) and E. tsunodai (right) (40X) sporulated oocysts in saturated salt solution.


 
Single oocyst isolation and propagation
 
Single oocyst isolation was successfully carried out for the three identified species. The group inoculated with E. bateri showed a mild decrease in the body weight with mucoid and watery diarrhea and this was similar to the findings of Norton and Pierce (1971). Oocysts and lesions were found only in the intestine, represented by ballooning in the duodenum and jejunum with watery intestinal contents (Fig 2).

​

Fig 2: Small intestine of a Japanese quail inoculated by E. bateri, during propagation, showed ballooning in the duodenum and jejunum (Arrows) with watery and foamy contents (score +2).



E. uzura inoculated group showed a mild decrease in the body weight with mucoid and watery diarrhea and this agreed with the results of Tsunoda and Muraki (1971) and Ruff et al. (1984). Oocysts and lesions were found only in the intestine as ballooning with mucoid watery contents in the duodenum and jejunum, congested parts in jejunum and ballooning in ileum.

E. tsunodai inoculated group showed a decrease in body weight, watery diarrhea on day 4 post-inoculation. Dropping was coffee-colored or tinged with blood on days 5 and 6 post-inoculation. These observations were similar to the findings of Tsutsumi and Tsunoda (1972). Oocysts and lesions appeared in the cecum and to some extent in the rectum. Lesions ranged from ballooning in the 2 ceca with foamy watery cecal contents to hardened fibrous cecal contents tinged with blood and thickened cecal mucosa.
 
Experimental trial
 
Positive control groups of each Eimeria species were severely affected and showed signs such as depression, ruffled feathers, huddle together, decrease in appetite and emaciated breast muscle. The fecal abnormalities ranged from soft to brownish foamy mucoid to watery diarrhea. Coffee-colored dropping, blood-tinged dropping and bloody diarrhea were seen in E. tsunodai positive control group. The immunized, challenged groups of each Eimeria species showed lower clinical signs and fecal abnormalities compared to the non-immunized, challenged groups.

The groups challenged with E. bateri had no mortalities. E. uzura positive control group showed 6.67% mortality, while both 100 and 1000-E. uzura oocysts immunized groups had 3.33% mortality. A significant decrease (P<0.001) in mortality occurred in 100 and 1000-E. tsunodai oocysts immunized groups by 3.33% and 6.67%, respectively, compared to E. tsunodai positive control group which possessed the highest mortality percent (20%) (Table 1).

Table 1: Means of mortality rate and lesion score throughout 7 days post-challenge 1.



The immunized groups by E. bateri or E. uzura showed an insignificant decrease (P>0.05) in the lesion score compared to that of their positive controls. While, a significant decrease (P<0.05) in the lesion score was seen in E. tsunodai immunized groups compared to E. tsunodai positive control group where bloody cecal core and bloody diarrhea were present. Both 100 and 1000-oocysts immunized groups of each Eimeria species showed the same lesion score (Table 1) (Fig 3-9). These observations were in agreement with those reported by Gerhold et al., (2010) in Bobwhite quail immunized against E. lettyae.

​

Fig 3: Mild congestion (arrow) in the duodenum of a Japanese quail challenged by 105 E. bateri oocysts (+ve control group) (5th day post-challenge).



​

Fig 4: Duodenum of a Japanese quail challenged by 105 E. bateri oocysts (+ve control group) showed mucoid contents in the lumen with mildly thickened mucosa (arrow) (score +2) (5th day post-challenge).



​

Fig 5: Mild ballooning in the jejunum and ileum (arrows) of a Japanese quail challenged by 105 E. uzura oocysts (+ve control group) (score +2) (7th day post- challenge).



​

Fig 6: Ceca of a Japanese quail challenged by 4×104 E. tsunodai sporulated oocysts (+ve control group) showed severe ballooning, hemorrhage and clotted blood causing bloody cecal core (score +3) (6th day post-challenge).



​

Fig 7: Small intestine of a Japanese quail immunized by E. bateri 100-live sporulated oocysts and then challenged by 105 E. bateri oocysts, 4 weeks later. All parts of intestine were normal except small parts of ileum (arrow) showed mild ballooning with watery foamy contents (score +1) (6th day post-challenge).



​

Fig 8: Small intestine of Japanese quail immunized by E. uzura 100-live sporulated oocyst and then challenged by 105 E. uzura oocysts, 4 weeks later. All parts of intestine were normal except small parts of jejunum (arrow) showing mild ballooning with watery foamy contents (score +1) (7th day post-challenge).



​

Fig 9: Ceca of a Japanese quail immunized by E. tsunodai 100-live sporulated oocysts and then challenged by 4×104 E. tsunodai oocysts, 4 weeks later, showed mild ballooning (arrows) with normal contents (score +1) (7th day post-challenge).



Concerning oocyst output, the immunized groups of each Eimeria species showed a dramatic highly significant decrease (P<0.001) in the oocyst output compared to their positive control groups. The 100-oocysts immunized groups had oocyst output insignificantly lower (P>0.05) than that of the 1000-oocysts immunized groups (Table 2). Similarly, Gerhold et al., (2010) reported that the immunized Bobwhite quail by 100-oocysts of E. lettyae produced around 99.7% fewer oocysts compared to the non-immunized, challenged control.

​

Table 2: Means of daily oocyst output, oocyst per gram feces (OPG), 4th - 9th day post-challenge1.



Regarding feed consumption, the immunized groups of each Eimeria species showed a significantly better feed consumption compared to their positive controls. The difference in feed consumption between the 100 and 1000-oocysts doses of different Eimeria species was insignificant (Table 3).

​

Table 3: Means of feed consumption (g), weight gain (g) and FCR, 7 days post-challenge1.



The 100 and 1000-E. bateri oocysts immunized groups showed a significant increase (P<0.001) in the weight gain compared to the positive control group. The 100-E. bateri oocysts immunized group had a significantly higher (P<0.05) weight gain than that of the 1000-E. bateri oocysts immunized group. Both E. uzura and E. tsunodai immunized groups possessed a significantly higher (P<0.001) weight gain compared to their positive controls. The groups immunized by 100-oocysts of E. uzura or E. tsunodai showed an insignificant improvement in the weight gain compared to the 1000-oocysts immunized groups (Table 3).

Regarding FCR, both 100 and 1000-E. bateri oocysts immunized groups showed a significant improvement (P<0.001) in FCR compared to the positive control group. The 100-E. bateri oocysts immunized group had a significantly lower FCR than that of the 1000-E. bateri oocysts immunized group. Both E. uzura and E. tsunodai immunized groups showed a significant improvement (P<0.001) in FCR compared to their positive controls. The groups immunized by 100-oocysts of E. uzura or E. tsunodai showed an insignificant lower FCR than those of 1000-oocysts immunized groups (Table 3) (Fig 10).

​

Fig 10: Means of FCR in different treatments of Japanese quails experimentally challenged with 3 Eimeria species separately.



The improvement in the performance parameters caused by immunization may be attributed to the produced immunity resulting in suppression of parasite development and so lowering the intestinal lesions and disorders.

Concerning the hematological parameters, PCV values were significantly higher in the immunized groups of each Eimeria species compared to their positive controls except in case of the 1000-E. bateri oocysts immunized group where the increase was insignificant. There was an increase in Hb and WBCs values in the immunized groups compared to the positive controls. The positive control groups revealed a significant reduction in PCV and Hb values in comparison with the negative control group. MCHC value was significantly reduced in the E. uzura positive control group compared to the negative control group. RBCs, PCV, Hb and MCHC values were significantly reduced in E. tsunodai positive control group compared to the negative control group (Table 4) and this may be attributed to the hemorrhage and blood loose caused by this species. These negative impacts were reduced by immunization by E. tsunodai live oocysts, 100 and 1000-oocysts doses. In addition, the negative effects of coccidiosis on blood parameters may be due to the destructive effects caused by the endogenous developmental stages of Eimeria resulting in disturbances in the absorption of the essential nutrients and traces required for erythropoiesis and Hb synthesis as supported by Teixeira et al., (2004).

​

Table 4: Means of hematological results, 7 days post-challenge 1.



The differential leukocyte count revealed a significant increase in the number of lymphocytes in the immunized groups of each Eimeria species compared to the negative and positive controls. Scattered immunocytes (reactive lymphocytes) were found in the blood films of the immunized, challenged groups of each Eimeria species. Contrary, there was a significant increase in the number of heterophils in the positive controls compared to the immunized groups and the negative control group (Table 5). These observations can be explained by Campbell (1994) who mentioned that scattered reactive lymphocytes may be noticed in the peripheral blood smear after antigenic stimulation by an immunization or a disease and that the infectious agents like bacteria, fungi, or parasites are associated with leukocytosis and heterophilia. The degree of heterophilia usually indicates the severity of the initial inflammatory process.

​

Table 5: Means of differential leukocyte count, 7 days post-challenge 1.

CONCLUSION

We found that the immunization of Japanese quail by low doses of live sporulated oocysts of 3 Eimeria species separately gave excellent results, regarding all tested parameters, against the challenge by high doses. The 100-oocysts doses showed better results than the 1000-oocysts doses with insignificant differences. Therefore, further studies on coccidiosis vaccination in Japanese quail are needed such as the use of attenuated oocysts in vaccination, trials on different doses of live oocysts and vaccination of Japanese quails when reared in different systems.

ACKNOWLEDGEMENT

Many thanks to the Ministry of Higher Education of the Arab Republic of Egypt and to the Indian Council for Cultural Relations (ICCR) for providing the scholarship.

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