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Detection of the Antibody Titer of Goatpox Vaccine Developed by BLRI in the Khulna Region, Bangladesh

M.M.I. CHOWDHURY
Corresponding author and Assistant Director
Prime Minister office, The people Republic of Bangladesh

N. ARA
Assistant Professor
Department of Pharmacy, University of Development Alternative, Dhaka, Bangladesh

N. SULTANA
Lecturer, Department of Microbiology, Faculty of Biological Science and Technology, Jessore science and Technology University, Jessore - 7408, Bangladesh

M.N.K. AZAM
Biotechnology and Genetic Engineering Department, University of Development Alternative, Dhanmondi, Dhaka, Bangladesh

K.M. HOSSAIN
Professor, Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna – 9208, Bangladesh.

Abstract/ Executive Summary:

Goatpox is a highly contagious viral disease of goats. The disease is caused mainly by Goatpox virus (GPV) and occasionally by Sheep pox virus (SPV) which is enveloped by double-stranded DNA viruses, classified in the genus Capripoxvirus of the family Poxvirade. The objective of the research work was to conduct an efficacy trail of Goatpox vaccine BLRI-4505 on selected goats of Khulna region developed by Bangladesh Livestock Research Institute (BLRI). BLRI developed GPV vaccine was injected into 30 goats of selected farmers in a village of Khulna, to observe the physiological condition and the effects of the vaccine. Rectal temperatures of the experimental animals were recorded for before vaccination and seven day after vaccination. Blood was collected on before vaccination (0-Day), 15th and 30th day of the post-vaccination period. Separated serum was used for C-ELISA (Enzyme Immune Slide Assay) to determine the antibody titre of vaccinated goats. Hyper-immune serum (several time vaccinated goats) was collected from BLRI experimental goats. Hyper-immune serum was used as a standard. Temperature fluctuations of the vaccinated goats were normal and no uneven effects were found. OD value of antibody titre was satisfactory level and increased in day 30 than day-0 and day- 15. This experiment reveals that the newly developed GPV vaccine is effective against Goatpox virus in Bangladesh and few more trials in the intensive and semi-intensive goat populated areas would be better about the efficacy result of the vaccine.

Keywords: BLRI, Capripoxvirus, Goat, Goat pox, Immunogenicity

Location: Virology Laboratory, Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka, Bangladesh

Start Date: 00-04-2006

End Date: 00-06-2006

Program Area: Animal Health and Management

Commodity: Vaccine, Goat

Objectives:

The objectives of the study were:
1. To detect the antibody titer of the Goatpox vaccine developed by BLRI in the Khulna region.
2. To study the physiological changes of goats after vaccination of newly developed vaccine.
3. Compare antibody titer of post-vaccination among the experimental animals.

Methodology:

The study was conducted in Virology Laboratory, Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka, during the period of April to June 2006. A detailed outline of materials and methods are given below. Design of the Experiment The whole experiment consists of two parts; the first one is vaccination of the goats and observes their physiological condition to determine the side effect of the vaccine, the second part was the assessment of GPV antibody in goats. The GPV vaccine BLRI-4505 was supplied from Livestock Research Institute (BLRI) Savar, Dhaka. The vaccine was given through the subcutaneous route. In the first experiment, trial is given in 30 goats. The serum samples were collected 15th and 30th day of post-vaccination. The degree of antibody level of hyperimmune serum and post-vaccination sera were determined by Competitive- Enzyme-Linked Immunosorbent Assay (C-ELISA). For determining the side effect of the vaccine, the experiments were conducted in farmer’s level at Austtagram, Kapalipara, and Daulatpur in Khulna. Experimental animals: For the determination of the antibody of GPV vaccine, the experiment was conducted on goats at farmers’ level at Austtagram, Kapalipara, Daulatpur in Khulna and the total no. of goat for trial was 30. Vaccination: The vaccine was given sub-cutaneously in the cervical lymph node region. Each of the experimental animals was vaccinated at same day. Blood collection In this experiment, the sera were collected at 15th and 30th day of post-vaccination. The sera of post-vaccination were tested by C-ELISA for the confirmation whether the animal was vaccinated or not. Blood was collected from Jugular vein puncture using syringe. The blood was left to clot overnight in clot boxes and serum was decanted into sterile tubes and kept in icebox for transportation to the laboratory. For the detection of GPV antibody all materials, chemicals, etc. were supplied by BLRI, Savar, Dhaka which are described below. * ELISA Reader: Computerized immuno-scan microplate reader with an interference filter of 450 nm was used for the reading of test samples. * Pipettes: Multi Channel pipette variable Range from 5-50μl and 50-250μl quality tips, reagent troughs and single-channel pipette, variable range from 5-50 μl and 200-1000 μl quality tips were used. * Glassware, plastic ware: Beaker (20-4000m1), flasks (50-1000ml), graduated cylinders (10-2000m1), graduated pipettes (1-20m1.) with suitable bulbs, storage bottles with closures (1-100m1.), dilution tubes (2-4ml.), racks were used. * Timer: Timer was used for preferably count down type with an audible alarm. * Marker pens (water-proof) and adhesive labels: Marker pens and adhesive were used for labeling the samples. For C-ELISA following reagent and sample were used: Goat pox virus antigen stock: One ml Goat pox antigen was supplied with the kit in glass vial. Anti-species conjugate stock: One ml freeze-dried horseradish peroxides (HRPO) conjugate, rabbit anti-mouse immuno-globulin were used. HRPO conjugate stock was further sub-divided into 500μl aliquots in 1 ml cryopreservation vials and used at l: 1000 dilution for the test. Control serum stocks: Freeze-dried strong positive hyperimmunized serums were supplied in glass vials at 1 ml volumes. The antibody stock was stored -20°C for further use in cryo-vials. At the time of the test cryovial containing antibody thawed at 37°C in water bath and used at 1:1000 for the test. At the time of the test cryo-vial containing antibody was thawed at 37 °C in water bath and used at 1:1000 dilutions for the test. Chromogen stock: Constituted 3.7 mg ortho-phenyl diamine (OPD) in tablet form and was used as the chromogen. One OPD tablets was dissolved in 75ml of locally produced deionized water just before the substrate or chromogen incubation step and aliquots in 6 ml prepared and stored at -20°C for further use. Substrate stock: We prepared 3% (w/v) / 882mM hydrogen peroxide for the test and hydrogen peroxide was supplied in tablet form and then one hydrogen peroxide tablet was placed in the empty container supplied and dissolved with 10 ml locally produced distilled water which constituted at 3% solution. The prepared substrate was stored at 40°C for further use. Coating buffer: We prepared 0.01M phosphate-buffered saline (PBS), pH 7.4 ±0.20 and then one tablet was dissolved in one liter of locally produced distilled water and pH was checked. Then prepared coating buffer was labeled and stored at 4°C for the period of two weeks as stock solution. Blocking buffer: We prepared 0.01M phosphate buffer saline, pH 7.4±0.20 plus 0.1% (V/V) Tween 20 plus 0.3%( v/v) normal bovine serum (C). Blocking buffer was prepared in 100ml volume of PBS containing 100μl, 200 μl and 300μl normal bovine serum (C). The prepared blocking buffer was labeled and stored at 4°C for two weeks. Washing buffer: We prepared 0.002 M phosphate-buffered saline (pH 7.4±0.20) and then 1 liter of locally produced deionized water and used as 1:4 dilution in distilled water. Stopping solution: 1 M sulfuric acid was prepared in 1 liter volume containing 55ml of concentrated sulfuric acid in 945ml of locally produced distilled water. The procedure of C-ELISA Test: The test procedures consisting of several steps of these procedures are described below. Coating of GPV antigen: Standard GPV antigen (5μl) was taken on a glass plate with 12 holes, air-dried and fixed with acetone for 30 minutes. Addition of blocking buffer: By fixing the glass plate, 20μl of blocking buffer was added in each hole.

Source of Information: J. Innov. Dev. Strategy 6(1):73-80(April 2012)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

This study is undertaken with a view to the efficacy trail of BLRI-4505 goat pox vaccine in Khulna region. For this reason, 30 goats were selected in farmers level in Austtagram Kapalipara, Daulotpur; Khulna. Animals are vaccinated and blood is collected on 15 and 30 days of post-vaccination. The temperature of animals was recorded. Serum samples of goats are examined in BLRI, Savar virology laboratory. Antibody titre was detected by C- ELISA. Antibody titre presents as an OD value. The temperature rise after vaccination is in minimal range. The antibody produced in response to vaccine in 15 day later is quite satisfactory in compare to hyperimmune serum. The antibody titre of day 30 is rising than days 15. The death of one vaccinated animals is unknown, the animals may be affected before vaccination. The post vaccinated animals did not found any exceptionable side effects to be released as a field level. The antibody titre of the vaccine is high as compare to hyper-immune serum. To control Goatpox and save the valuable goat and sheep wealth of the country, proper vaccination in the endemic zone is dire necessary. BLRI-4505, Goatpox vaccine will be effective to solve the problem because this vaccine is developed from our local isolates of Goatpox virus. The side effect of the vaccine was not found as well as antibody response was also good. Therefore, this vaccine can be released after conducting few more trials in the intensive and semi-intensive goat populated areas of the country.

Knowledge Management: Journal