Dept. of Poultry Diseases,

Animal Health Research Institute, Zagazig.

laboratory evaluation of selected disinfectants on Gumboro disease virus

(With 2 Tables)

By

H.A.A. Bayoumie and I.A.A. Mohamed*

* Dept. of Avian and Rabbit Med., Fac. of Vet. Med., ZagazigUniv.

(Received at 19/11/2008)

التقييم المعملى لبعض المطهرات المختارة ضد فيروس الجامبورو

هشام أحمد عبد البديع محمد بيومى ، عماد عزمى عوضين محمد

تم اجراء هذه الدراسة لتقييم فاعلية مرکبات اليود والجلوترالدهيد معمليا ضد فيروسات مرض الجامبورو. تم عزل فيروس الجامبورو من طيور بلدى ساسو عمر 35 يوم. تم تقييم فاعلية المطهرات عن طريق حساب عيارية الفيروس قبل وبعد المعاملة بالمطهر سواء فى وجود مواد عضوية أو فى غير ذلک وقد اختبرنا ثلاثة تخفيفات للمطهرين محل الدراسة. وتم قياس ترکيز أيون الهيدروجين فى ترکيزات المطهرات المستخدمة. من خلال هذه الدراسة لاحظنا قصور فى طريقة الحساب المتبعة. لحساب الانخفاض الحادث فى عيارية الفــيروس بعد المعاملة بالمطهرات فقد وجدنا فرضاً أنه لو کانت عــيارية الفـــيروس  10⁶EID₅₀/0.1 mlأن هذه العيارية قد انخفضت بعد المعاملة بالمطهرات إلى0.1 ml / EID₅₀ 10². فإن الجزء الذى انخفض فى عيار الفيروس يتم حسابه على أنه 10⁴EID₅₀/0.1 ml ولکن اذا ما حسبنا المقابل الحسابى للوغاريتم للعيارات السابقة لوجدنا1000000 وحدة فيروس للعيار 10⁶EID₅₀/0.1ml , 100 وحدة فيروس للعيار 10²EID₅₀/0.1 ml , 10000 وحدة فيروس للعيار 10⁴EID₅₀/0.1 ml وعند طرح 100 من 1000000 يکون الناتج 999900 وحدة فيروسية وهذا الناتج لا يعبر عنه العيار 10⁴EID₅₀/0.1 ml و أن هذه النتيجة سوف تؤدى الى خطأ فى الحساب ولذلک لجأنا الى حساب انخفاض عياريه الفيروس عن طريق حساب المقابل للوغاريتم وطرح قيمة الانخفاض فى عدد الوحدات الفيروسية لحساب کفاءة التطهير. من خلال هذه الدراسة وجدنا أن ترکيز أيون الهيدروجين فى محلول اليود 1 : 50 کان 6.2 وأن هذا التخفيف قد أدى إلى انخفاض عياريه الفيروس بنسبة 99.93% بعد 15 دقيقة وبنسبة 99.97 بعد 60 دقيقة وعند التخفيف بنسبة 1: 100 کان ترکيز أيون الهيدروجين 6.5. وقد أحدثت هذا الترکيز انخفاض فى عياريه الفيروس بنسبة 99.7% بعد 15 دقيقة و 99.99% بعد 60 دقيقة. أما التخفيف     1 : 200 فقد کان ترکيز أيون الهيدروجين له 6.7 وقد أحدث انخفاض فى عيارية الفيروس بنسبة 99.6% ، 99.8، 99.9% بعد 15 ، 30 و60 دقيقة على التوالي. وقد حصلنا على نتائج مقاربة لهذه عند استخدام مواد عضوية بنسبة 40% وبالرغم من اختلاف هذه النتائج عن نتائج الآخرين الذن أشاروا إلى تأثر مرکبات اليود بالمواد العضوية.  فقد وجدنا أن ترکيز أيون الهيدروجين فى المحلول بعد اضافة المواد العضوية قد تحولت إلى القلوية وهو السبب وراء هذا التأثير على فيروس الجامبورو لتأثرالأخير بالوسط القاعدى وهذه النتيجة لن تکون موجودة فى عائلات فيروسية أخرى. ويمکن أيضا أن نضع فى الاعتبار إجراء التجربة تحت الظروف المعملية وهو ما يختلف کثيرا عن الظروف الحقلية. أما عند استعمال المطهرات التى تحتوى على مرکب الجلوترالدهيد فقد لاحظنا فاعلية المرکب عند تخفيفه بنسبة 100:1 , 200:1 الا أن الترکيز الاخير کان أکثر أماناً على أجنة البيض المستعملة کنموذج بيولوجى للتقييم وأن قدرته على خفض عيارية الفيروس محل الدراسة کانت مماثلة للتخفيف 100:1بنسبة 99.9% وأحدث المطهر نتائج مماثلة عند استعماله فى وجود المواد العضوية ويمکن أن نعزى السبب فى ذلک إلى زيادة ترکيز أيون الهيدروجين تجاه القلوية وهذا الأخير له قدرة على التاثير على فيروس الجامبورو.

summary

The efficacy of iodine and glutraldehyde containing compounds against infectious bursal disease virus (IBDV) was assayed by comparing the virus titer before and after exposure to each disinfectant. The test was conducted at room temperature in presence or absence of organic matter “40% foetal bovine serum”. The two tested disinfectants were effective against IBDV under all test conditions and at the dilutions used 1:50, 1:100 and 1:200. In this paper a new method for calculation of viral regression after disinfection, was devised as it gives a more reasonable calculation.

Key words: Chicken, gumboro disease, disinfectant

Introduction

            Infectious bursal disease is one of the widely spreaded acute, highly contagious diseases of young chickens that had lymphoid tissue target with special predilection for the bursa of fabricius (Lukert and Saif, 2003).

 Disease prevention depends on proper disinfection of poultry premises, proper management and specific active immunization (Bayoumie, 1997).

            Faragher (1972) stated that contaminated poultry premises is the main source for IBDV infection, this is helped by the very stable physicochemical properties of IBDV (Benton et al., 1967).

            The short life span of broilers (30-35 day) is insufficient to generate active immunity. For this, disease prevention strategies should run parallel to ensure successful poultry operation (Bayoumie, 1997).

            Few scientific researches about chemical disinfection of IBDV are available. The present work evaluates commercially available iodine, and glutraldehyde containing disinfectant on IBDV in presence or absence of organic materials after different reaction times.

Materials and methods

A-Materials:

1- Samples for isolation of IBDV:

Samples for IBDV isolation were collected from a native saso chicken flock 35 day old exhibiting 80% morbidity and 50% mortality. The affected flock was showing signs and post mortem lesion specific for IBD as described by Lukert and Saif (2003). Severely hemorrhagic and inflammed bursae were collected for virus isolation (Rosenberger et al., 1998).

2- IBDV live vaccine:

            An intermediate (D-78) live vaccine strain kindly obtained form Prof. Dr. S.Assily, Poultry Vaccine Dept., Serum & Vaccine Res. Inst. El-Abbassia.

3- IBDV reference antigen and antisera:

            IBDV “serotype 1” reference antigen and antisera were obtained from the international marketing center, Cairo, Egypt.

4- Test system:

            Embryonated chicken eggs (ECE) from a small native breeder flock that received influenza vaccination only were used for virus isolation, viral propagation, viral identification and testing disinfection potency.

5- Foetal bovine serum:

            Foetal bovine serum produced by “life technologies” obtained from Bardisi medical. This serum was virus and mycoplasma tested, and it was heat inactivated at 56°C for 30 min before use.

6- Tested disinfectants:

a- Glutraldehyde containing compound, (62.5 gm/liter) was tested as antiviral agent against IBDV, at dilutions 1:50, 1:100 and 1:200.

b- Iodine containing compound 2.5% active iodine w/v. was similarity tested.

7- Water diluents:

            Under ground water from a commercial poultry farm was used after being autoclaved.

8- Source of organic matter (OM):

            40% solution of foetal bovine serum in autoclaved distilled water was prepared, and was used as a source for organic matters as adapted by Ismail et al. (1976).

B- Methods:

1- Sample preparation for viral assay:

            Collected bursae were grounded. AGPT against reference IBDV antigen and antisera was performed (Beard 1980), positively reacting bursae, were further processed for virus isolation (Rosenberger et al. 1998, Senne, 1998). The CAM route was chosen for virus isolation, specific mortality and PM lesion were recorded as previously described by Hitchner (1970). Examination for heat resistance (56°C for 5 hrs) was performed as described by Benton et al. (1967). Virus neutralization was performed according to Thayer and Beard (1998).

2- pH measurement:

            pH of tested concentration for both disinfectants in presence or absence of (OM) was determined using an electrical pH meter (Jenway 3510).

3- Safety of disinfectant for the test system:

The tested dilutions of each disinfectant was inoculated in 5 ECE, daily mortality was recorded after neglecting the non specific mortality.

4- Testing Virucidal activity of the selected disinfectants in presence or absence of organic matters:

            Isolated IBDV was 10 fold serially diluted once using sterile saline and another in 40% foetal bovine serum. Selected test disinfectant were diluted 1:50, 1:100 and 1:200 using the autoclaved under ground water as diluent. A 0.5 ml of the viral saline suspension or viral serum suspension was added to 0.5 ml of the tested disinfectant dilutions. A reaction time 15, 30 and 60 min. was given for this mixture. Each dilution at every reaction time was inoculated in 5 ECE via allantioc sac route (AS) to ensure precise reaction time limits.

5- Calculation of disinfectant potency:

            Mortality of inoculated chicken eggs was daily recorded and was specifically confirmed through the examination of inoculated CAM in AGPT. Titer calculation was preformed according to Spearman and Karber Cunningham (1973). Antilog 10 of the obtained titer was calculated. Reduced virus titer (c) was calculated by subtracting the antilog of post disinfection titer (b) from the antilog of virus control (a). The percent of disinfection success (d) is obtained by dividing c/a.

6- Preparation of IBDV hyperimmune sera:

            Some of the fertile chicken eggs were used to hatch a day old chicks. The latter were eye inestillated repeatedly every four days with the live intermediate IBDV vaccine until birds became 60 day old, collected sera were examined with AGPT for IBDV precipitating antibodies.

Results

1- Clinical picture and necropsy findings in the field case:

            The examined native saso flock was 35 day old, showed 80% morbidity and 50% mortality. The PM lesion observed was, dehydrated darkened skeletal muscles with subcutaneous hemorrhage on thigh muscles; urate deposition in the urters; enteritis, the liver is enlarged with peripheral areas of infarction, hemorrhages at the juncture of proventriculus and gizzard, spleen is mildly enlarged with necrotic foci on its surface, and the bursae were enlarged and surrounded by gelatinous hemorrhagic fluid. Their plicae were also hemorrhagic.

2- Results of viral assay:

a- Virus isolation:

 Embryo mortality due to inoculated bursal homogenate is shown in Table (1). Precipitins specific for IBDV was observed after the 2^nd passage. The 3^rd passage was performed for further adaptation on test system. The PM lesion observed in inoculated eggs via CAM route was congestion of embryo, paleness of liver with mild thickening of CAM. Positive reaction of infected CAM in AGPT against reference IBDV antigen and antisera is a preliminary indication for successful isolation of IBDV.

b- Results of viral titration, resistance to heat inactivation and virus    neutralization:

Results are shown in Table (1)      

Table 1: Virological assay.

 3-Results of pH measurement and disinfection potency with iodine:

            The pH of diluted iodine 1:50 was 6.2 this dilution reduced virus titer 99.93% after 15 min. reaction and the reduction was 99.97% after 60 min. the pH of diluted iodine 1:100 was 6.5, disinfection success was 99.7% after 15 min. and 99.9% after 60 min., while the pH of diluted iodine 1:200 was 6.7 and the disinfection success was 99.7, 99.84 and 99.9% after 15, 30 and 60 min. respectively.

            The pH of diluted iodine 1:50 in 40% foetal bovine serum was 7.26 this dilution reduced virus titer 99.6, 99.8 and 99.9% at 15, 30 and 60 min. reaction times respectively, comparable results were obtained at dilutions 1:100 and 1:200 at the same test conditions (Table 2).

4- Results of pH measurement and disinfection potency with glutraldehyde containing compound:

            The disinfection potency of dilution 1:50 of the glutraldehyde containing compound couldn’t be tested because it killed all inoculated ECE in the safety trial. The pH measurement of dilutions 1:100 and 1:200 was 7.5 and 7.6 respectively and the disinfection success was 99.9% similar results of disinfection success was obtained in presence of organic matter at pH measurement 8.6 and 8.78 for dilutions 1:100 and 1: 200 respectively.

Table 2: Virucidal activity of iodine, glutraldehyde containing disinfectant in presence or absence of organic matters, with   different dilutions and reaction times.

  PD = Post disinfection titer EID₅₀/0.1 ml               ND = Not done       VP = Virus particle                                                                              DC = disinfection concentration