EFFECT OF EXPOSURE PRE-WEANING BUNNIES TO SPORULATED EIMERIA OOCYSTS ON IMMUNITY AND GROWTH TRAITS OF RABBITS

EFFECT OF EXPOSURE PRE-WEANING BUNNIES TO SPORULATED EIMERIA OOCYSTS ON IMMUNITY AND GROWTH TRAITS OF RABBITS

M.N.F. Hassanein⁽¹⁾ and Mary B. Ayoub⁽²⁾

(1) Animal Production Research Institute (APRI)- Rabbit Research Department, Giza, Egypt.

 (2) Animal Health Research Institute (AHRI)- Parasitology Department, Giza, Egypt.

This experiment aimed to determine if spraying intestinal sporulated Eimeria oocysts in an empty nest box will give natural immunity to growing bunnies against serious invocation comparing to the effect of rabbit ration pellets admixed with anti-coccidian powder under Egyptian practical conditions. Twenty five coccidia free New Zealand White (NZW) does with their offspring were randomly divided equally into 5 groups (G1 to G5) with mean weight of 455.2±10.69 gram/bunny. At day 25 of bunnies’ age, 15 ml of saline contain equal mixed of four intestinal  Eimeria spp. oocysts (40, 400 or 4000 per bunny) were sprayed in empty nest boxes of groups 1, 2 and 3 respectively (vaccination). At day 35 bunnies of all five groups were challenged orally with 5 ml of saline contained 5000 of equal mixed inoculation Eimeria spp. oocysts (challenge), the experiment ended at day 45 of bunnies’ age. All groups were fed to ad libitum with a rabbit commercial pellet ration, but only group 5 ration was supplemented with anti coccidian powder.

Results obtained indicated that post vaccination high oocysts excretions were observed combined with mortality rates and diarrhea in G1, G2 and G3.

 Post challenge high oocysts excretions were observed combined with high mortality rates and diarrhea in G1, G2 and G4 (G4 was the highest), while G3 and G5 had no oocysts excretions.

Feed intake and feed conversion between days 35-45 of age had significant differences between some groups where G5 was the best.

Conclusively, the results make us to advice to keep using the anti-coccidian in ration until we solve the problem of mortality post vaccination and to do more studies to confirm or deny our present results under the Egyptian conditions.

Key words: Rabbits, Eimeria, oocysts excretion, mortality, weight

In rabbit industry, getting infected by intestinal Eimeria species (coccidosis) causes digestive disorders, in some cases severe intestinal morbidity and mortality symptoms occurs, Leysen et al. (1989) noted coccidia is responsible for major losses in rabbit industry. The clinical condition generally observed is characterized by reduced in both food intake and body weight, also, diarrhea over the first four weeks. The condition may progress to death, which is attributed to the hepatic lesions that are also associated with the infection (Gomez-Bautista et al., 1986).

Drouet-Viard et al. (1997b) noted coccidiosis mostly affects young rabbits just after weaning (5 to 6 week old). Prevention of this disease must therefore be initiated before weaning. 'Precocious lines', derived from field species display good immunogenicity, though not pathogenic when administered at the right dose. Also, Homouda (1995) mentioned that the infection rate may reach 100 percent of these feedlots and up to 50% of the animals may develop clinical coccidiosis with a mortality rate of 30 percent.

Licois et al. (1995) demonstrated the immunogenicity of a developed earlier line of E. magna in young weaned rabbits. Suckling in standard breeding conditions is not susceptible to infection before the age of 20 days, and they are fully sensitive at about 30 days old corresponding to weaning age (Coudert et al., 1991). In order to be effective, vaccination should therefore be performed between 20 and 30 days old. Drouet-Viard et al. (1997a) found that age of 25 days was the best age to vaccinate the bunnies and full protection was obtained.

 Pakandl (2009) mentioned that vaccination trials were preformed by Drouet-Viard et al. (1997b) with a precocious line of E. magna. Vaccination either per os (oral solution)or using spray dispersion of oocysts into nest boxes gave satisfactory results. He added that development of efficacious methods, alternative to continuous medication with anti-coccidiosis drug, of rabbit coccidiosis control, is a challenge for future research work.

Therefore, the aim of this work is to determine if spraying intestinal sporulated Eimeria oocysts in an empty nest box will give natural immunity to growing bunnies against serious invocation comparing to the effect of rabbit ration pellets admixed with anti coccidian powder, under Egyptian practical conditions.

MATERIALS AND METHODS

The practical part of this study was carried out in a private farm in Shubrament Region, Giza Governorate, while all laboratory procedures were carried out at the Animal Health Research Institute (AHRI), Parasitology Department, Giza, Egypt.

Animals:

Twenty five coccidia free New Zealand White (NZW) does rabbits with their offspring were randomly divided equally into 5 groups (G1 to G5) with 26 to 28 bunnies per group with mean weight 455.2±10.69 gram. Doe rabbits were housed individually in wire cages 45x55x35 cm, the nest box measures were 35x35x35 cm. At day 35 bunnies were weaned by transferring the doe to another cage and bunnies of each cage were weighed.

Feeding:

Bunnies of groups 1 to 5 were fed to ad libitum with a rabbit commercial pellet ration, but only group 5 ration was supplemented with anti coccidiosis powder approved and used in Europe (European Medicines Agency). Free access to fresh water through watering nipples was granted.

Parasites:

The Eimeria spp. oocysts for vaccination and challenge inoculation were obtained from naturally infected rabbits and recently sporulated, it contained a mixture of E. intestinalis (very pathogenic), E. magna (moderate pathogenic) and  E. coecicola and E. media (non or slightly pathogenic) as classified by Lebas et al. (1997).

The oocysts were kept at 4°C in 2.5% potassium dichromate solution and were classified and identified according to Soulsby (1988). The method used for counting the number of oocysts per ml. was described by Caudert   et al. (1995).

Vaccination and challenge:

• At day 25 of age bunnies of groups 1, 2 and 3 were vaccinated with equal mixed Eimeria spp. oocysts by spraying 15 ml of saline contain 40, 400 and 4000 oocysts per bunny of groups 1, 2 and 3 respectively (the bunnies were out of the box) (Drouet-Viard et al., 1997a).
• At day 35 bunnies of all groups were challenged orally with 5 ml of saline contained 5000 of equal mixed inoculation Eimeria spp. oocysts.          Vaccination and challenge step were summarized at Table 1:

Output oocysts:

The output oocysts were measured from bunnies feces (collected by butting a plastic net under the cages) 6-9 days post vaccination and challenge inoculation according to Drouet-Viard et al. (1997b).

Productive performance:

Bunnies’ numbers and weights at days 25 (vaccination), 35 (challenge) and 45 (the end) were recorded. Also, feed intake between days 35-45 was recorded.

Bunnies’ means of weight gain and mortality rate% between days 25-35, 35-45 and 25-45 were calculated.  Feed intake, feed conversion between days 35-45 were also calculated.

Statistical analysis:

Data were statistically analyzed using Least Square Means, according to SAS (1996) using the following model:

                               Y_ij = µ + T_i + F_j + e_ijk

Where: Y_ijk = Observation of the i^th doe rabbit, µ  = Overall mean, common element to all observations, T_j   = Effect of treatment group (i=1..5), F_j    = Feed without or with anti-coccdian powder (j= 1 & 2), e_ijk   = Random error component assumed to be normally distributed.

Differences among means were tested by Duncan's multiple range test (Duncan, 1955). P.S.:  the effect of F_j   (Feed without/with anti-coccidian powder) was considered in the statistical analysis model just to remove its effect on the standard error.

RESULTS:

Oocysts excretion and mortality ratio%:

Oocysts excretion:

At day-35 of age (10 days post vaccination) oocysts excretions were 1.2×10⁶, 5.6×10⁶, 7.1×10⁶, ND (not detectable) and ND for groups 1 to 5, respectively. While, at day-45 of age (10 days post challenge) oocysts excretions were 2×10⁵, 3×10⁴, ND, 1.7×10⁷ and ND for groups 1 to 5, respectively (Table 2).

Mortality ratio,%:

Mortality rates between days 25-35 of age were 7.41%, 10.71%, 15.38%, 0.00% and 0.00% for groups 1 to 5, respectively. While, mortality between days 35-45 of age rates were 16.0%, 12.0%, 4.55%, 29.63% and 3.85% for groups 1 to 5, respectively (Table 2). Overall mortality rates between days 25-45 of age were 22.22%, 21.43%, 19.23%, 29.63% and 3.85% for groups 1 to 5, respectively.

Some growth performance traits and feed intake:

At day-35 of age (10 days post vaccination), mean bunny body weights of  the vaccinated groups (G1, G2 and G3) were lower to significantly lower comparing with non vaccinated groups (G4 and G5) means were 596.6, 576.4, 598.6, 609.4 and 617.0 grams, respectively. Also, the same remark was appeared in mean body weight gain trait (table 3).

At day-45 of age (10 days post challenge), significant differences between all groups in means of final body weight and weight gain. Means of final bunny weights were 700.2, 717.0, 769.0, 691.2 and 846.4 grams. While, means of body weight gain were 242.2, 264.8, 309.4, 250.0 and 405.2 grams for groups 1 to 5, respectively (Table 3).

Feed intake and feed conversion between days 35-45 of age had significant differences between some groups. Feed intake means were 484.7, 578.0, 765.5, 495.0 and 814.6 grams. While, feed conversion ratios were 4.37, 4.11, 4.43, 4.86 and 3.56 for groups 1 to 5, respectively (Table 3).  

DISCUSSION:

According to Coudert et al. (1995) who stated that intestinal coccidia can produce 1-5×10⁶ per digested oocysts, we can from the results of secreted oocysts estimate that only 1-2% of the sprayed oocysts were ingested.

In this study, only vaccination dose of 4000 oocysts (G3) gave an execration of 7.1×10⁶ oocysts which is lower than those recorded by Akpo et al (2012)who found that over (24±2.6)×10⁶ oocysts per animal, but this caused a mortality rate of 15.38% and that may be caused due to the noticeable diarrhea which occurred on vaccinated bunnies. On the other hand, this dose (4000 oocysts) gave a total protection when bunnies were challenged and no oocysts output were detected post challenge (table 2), these results are in agreement to that recorded by Drouet-Viard et al. (1994) who found that a dose of 3500 oocysts gave total protection to suckling vaccinated at 25, 27, or 29 days old. This result is believed that bunnies had acquired good protection and developed strong immunity against infection with coccidia.

Post vaccination, a high mortality rates and low body weight gains (tables 2 and 3) were observed accompanied with appearance of diarrhea in groups 1, 2 and 3 bunnies which caused economical losses especially in G3 comparing with groups 4 and 5, this result indicates that the possible reason is the vaccination and the increase of mortality rate came in harmony with the dose sprayed.

Also, higher mortality rates and lower body weight gain accompanied with appearance of diarrhea appeared again after challenge doses were given at day 35, but this time in groups 1, 2 and 4 comparing with groups 3 and 5 and caused again high economical losses especially in G4.

The previous results came in harmony with Leysen et al. (1989) who noted coccidia is responsible for major losses in rabbit industry. On the other hand, Akpo et al. (2012) recorded no mortality or diarrhea after vaccination.

Also, the previous results indicates that the vaccination given previously to the bunnies of group 3 protected them from the harmful effect of the challenge dose, which came in harmony with Drouet-Viard et al. (1997a), and the anti-coccidian powder which admixed with group 5 ration gave the same protection.

The low performance in means of bunny weight, weight gain and feed intake in challenged groups 1, 2 and 4 post challenge (days 35-45) may be caused as a result of low appetite and diarrhea occurred on sick bunnies which came in agreement with Leysen et al. (1989) and poor feed conversion resulted because the total feed intake was related to live bunnies at the time of challenge-included the feed consumed by dead bunnies.

Conclusion:

In this study, vaccination (spray the empty nest poxes with 4000 oocysts per bunny) reduced significantly the oocysts output after challenge inoculation and led to a correct growth. It gave the impression to be efficient, quick, and easy and avoid animal manipulation. However, the high mortality rate post vaccination led to high economical losses.

These results make us to advice to keep using the anti-coccidian in ration until we solve the problem of mortality post vaccination and advice for more studies to confirm or deny our present results under the Egyptian conditions.

REFERENCES

Akpo,Y.; Kpodékon, M.T., Djago Y., Licois D. and Youssao, I.A.K. (2012). Vaccination of rabbits against coccidiosis using precocious lines of Eimeria magna and Eimeria media in Benin. Veterinary Parasitology, 184 : 73– 76.

Caudert, P., D.Licois and J. Besnard (1991). Establishment of a specified pathogen free breeding colony (SPF) without hysterectomy and hand rearing procedures. Proceeding of the 4th Congress of the World Rabbit Science Association, 10-14 October 1988, Budapest, pp.: 137-148.

Coudert, P., D. Licois and Drouet-Viard,F. (1995). Eimeria and Isospora. Eimeria species of rabbit. In: J. Eckert, R. Braun, M. W. Shirley and P. Coudert (Editors). Biotechnology Guidelines on Techniques in Coccidiosis Research, Office for Official Publications of European Communities, Luxembourg, pp.: 52-73.

Drouet-Viard,F., Coudert, P., Licois, D. and Boivin, M., (1994). Essai de vaccination contre une coccidiosis à Eimeria magna à ĺaide d`une lignée précoce In: Vlémes Journées de la Recherche Cunicole,6-7 dés 1994, La Rochelle, Vol 1, 39-43.

Drouet-Viard F., Coudert, P, Licois, D. and Boivin, M., (1997a). Acquired protection of the rabbit (Oryctolagus cuniculus) against coccidiosis using a precocious line of Eimeria magna: effect of vaccine dose and age at vaccination. Veterinary Parasitology, 69: 197-201.

Drouet-Viard F., Coudert, P, Coudert, P. and Boivin,M., (1997b). Vaccination against Eimeria magna coccidiosis using spray dispersion of precocious line oocysts in the nest box. Veterinary Parasitology, 70: 61-66.

Duncan, D. B. (1955). Multiple range and multiple F-test. Biometrics, 11:1-42.

European Medicines Agency (1996).

http://www.ema.europa.eu/docs/en_GB/document_library/Maximum_Residue_Limits_-_Report/2009/11/WC500013726.pdf

Gomez-Bautista, M.; Garcia, M.V. and Rojo-Vazquez, F.A. (1986). The levels of total protein fractions in the serum of rabbits infected with Eimeria stiedai. Annales de Para-sitologie Humaine et Comparée, 61, no. 4, p. 393-400.

Homouda, I.A. (1995). Effect of some herbal growth promoter on calcium absorption in femals rabbit. "J. Agric. Sci., Mansoura Univ., 20 (7). 3199-3205.

Lebas, F., P. Coudert, H. de Rochambeau and R.G. Thébault (1997). The Rabbit, Husbandry, health and production. Food And Agriculture Organization Of The United Nations, Rome, 1997, pp: 100.

Leysen, E., A. Neirynck, W. Deleersnijder, P. Coudert, J. Peeters, H. Revets and R. Hamers (1989). Coccidia and intestinal coccidiomorphs: Idenyification of sporozoite surface antigens of Eimeria stiedai and Eimeria magna. Proceedings of the Fifth International Coccidiosis Conference on Coccidia and Intestinal Coccidiomorphs, 509-514.

Licois, D., P. Coudert, F. Drouet-Viard and M. Boivin (1995). Eimeria magna: Pathogenicity, immunogenicity and selection of a precocious line. Veterinary Parasitology, 60:27-35.

Pakandl, M. (2009). Coccidia of rabbit: A review. Folia Parasitologica 56[3]:153–166.

SAS. (1996). SAS' Procedure Guide. Version 6.12, Forth Edition, SAS Institute, Inc., Car-ry, NC, USA.

Soulsby, E. J. L. (1988). Helminth, Arthopods and Protozoa of Domestic Animals. English Language Book Society, Baillere, Trindal.

تأثیر رش خلفات الأرانب قبل الفطام لحویصلات الکوکسیدیا المعدیة على المناعة وبعض صفات النمو

محمد نادر فکری السید حسانین ⁽¹⁾  ، ماری بشرى أیوب ⁽²⁾

(1) معهد بحوث الإنتاج الحیوانی – قسم بحوث الأرانب والرومی والطیور المائیة – الدقی – الجیزة - مصر

(2) معهد بحوث صحة الحیوان – قسم الطفیلیات – الدقی – الجیزة – مصر

کان الهدف من هذه التجربة هو تقییم تأثیر رش حویصلات کوکسیدیا معویة معدیة داخل بیوت ولادة خالیة من الخلفات وما إذا کانت ستعطی مناعة طبیعیة للخلفات ضد أی عدوى شدیدة وذلک مقارنة بالمناعة المعطاة بواسطة مضاد کوکسیدیا مضاف للعلف تحت الظروف العملیة المصریة.

تم استخدام 25 أنثى خالیة من الکوکسیدیا من سلالة النیوزیلندی الأبیض مع خلفاتها ، قسمت الإناث إلى خمس مجموعات متساویة بمتوسط وزن 455,2±10,69 جرام لکل خلفة.

فی الیوم 25 من عمر الخلفات تم رش 15 مل من محلول ملحی یحتوی على أعداد متساویة من حویصلات أربعة سلالات من الکوکسیدیا المعویة المعدیة بمعدل 40، 400، 4000 حویصلة / خلفة للمجموعات 1 ، 2 ، 3 على التوالی (التحصین) واستبقت المجموعتین 4 ، 5 لجرعة التحدی.

فی الیوم 35 من عمر الخلفات تم إجراء عملیة التحدی وذلک بتجریع کل خلفة 5 مل محلول ملحی یحتوی على 5000 حویصلة معویة معدیة من السلالات المستخدمة فی الرش. وانتهت التجربة فی عمر 45 یوم من عمر الخلفات.

تم تغذیة کل مجموعات الأرانب حتى الشبع على علیقة أرانب تجاریة خالیة من مضادات الکوکسیدیا ماعدا المجموعة الخامسة فتمت لها اضافة مضاد کوکسدیا للعلیقة.

بعد جرعة التحصین ظهرت إفرازات من بویضات الکوکسیدیا من خلفات المجموعات 1 ، 2 ، 3 مصحوبة بمعدلات نفوق وإسهال وکان أعلاها المجموعة الثالثة.

بعد جرعة التحدی ظهرت إفرازات من بویضات الکوکسیدیا من خلفات المجموعات 1 ، 2 ، 4 مصحوبة بمعدلات نفوق وإسهال وکان أعلاها المجموعة الرابعة.

اختلفت معدلات الغذاء المأکول والتحویل الغذائی بین المجامیع وکان أفضلها المجموعة الخامسة.

التوصیة: من هذه النتائج نوصى باستمرار إضافة مضادات الکوکسیدیا للعلیقة وإجراء مزید من الأبحاث لتأکید أو نفی هذه النتائج تحت الظروف المصریة.