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Research Detail

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Mashuda Akter
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Mosaraf Hossain
Department of Environmental Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Kamrul Hassan
Department of Microbiology and Parasitology, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh.

Ravi Yadav
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Fahima Morsheda
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Shahidur Rahman Khan
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Mahbubul Pratik Siddique
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

An experiment was conducted to investigate the immune response induced in chickens by capsular extract of Pasteurella multocida isolated from rats wandering in and around the poultry farms. The rat isolate of P. multocida was isolated and identified by cultural, morphological, and biochemical characteristics, followed by capsular extract preparation and experimental vaccine development. The isolated P. multocida was found Gram-negative, non-motile, non-spore forming rod occurring singly or pains and occasionally as chains or filaments in Gram’s-staining method. The isolates consistently produced acid from dextrose, sucrose and mannitol but not fermented maltose or lactose. The Capsular antigen was extracted and confirmed by acriflavine test. Finally, an experimental fowl cholera vaccine was prepared. Primary vaccination was performed at the dose rate of 5.6×107 CFU/ml through intramuscular and subcutaneous routes in birds of groups A (10 birds) and group B (10 birds) and group C (10 birds) were control birds. Secondary vaccination was similarly performed after 15 days of primary vaccination in groups A and B. The levels of pre-vaccination and post-vaccination sera were determined by the passive haemagglutination test. The passive haemagglutination antibody titre was recorded on 15 and 35 days of post-vaccination in groups A and B. It was demonstrated that experimental capsular extract fowl cholera vaccine conferred 100% protection (p<0.01) against challenge infection and found to be safe. It could be suggested that after a thorough field trial, the experimentally prepared capsular extract FC vaccine using rat isolate of P. multocida may be used side by side with conventional FC vaccine.

 

  Immune responses, Chickens, P. multocida, Capsular extract, Farm rat
  Bacteriology Laboratory of Department of Microbiology and Hygiene, Faculty of Veterinary Science, (BAU), Mymensingh.
  
  
  Animal Health and Management
  Chicken

To investigate the immune response induced in chickens by capsular extract of Pasteurella multocida isolated from rats wandering in and around the poultry farms.

Design of the experiment- The entire experiment was conducted in the Bacteriology Laboratory of Department of Microbiology and Hygiene, Faculty of Veterinary Science, (BAU), Mymensingh. The research work was accomplished in three steps. Firstly, isolation and characterization of P. multocida isolated from the rats wandering in and around poultry farm; secondly, preparation of capsular extract to be used as FC vaccine and finally, vaccination and evaluation of the prepared capsular extract FC vaccine. A virulent isolate of P. multocida collected from the rats wandering in and around the BAU poultry farm was used for the preparation of capsular extract FC vaccine. The vaccine was prepared according to the procedures described by Choudhury et al. (1987). A total of 30 Fayomi chickens of either sexes of 8 weeks age were selected for this study. The selected chickens were divided into three groups namely A, B and C, containing 10 chickens in each groups, A and B groups were used for trial vaccine while group C served as unvaccinated control. Pre-vaccination sera were collected to determine the pre- immunizing titre of the sera. Primary vaccination was given at the dose rate of 1 ml through intra-muscular (IM) and sub-cutaneous (SC) route in each chickens of group A and B, respectively. Booster dose was given with the similar dose and route to the chickens after 15 days of primary vaccination. The study was carried out in the animal shed of the Department of Microbiology and Hygiene with provision of a nutrient diet and ventilation following strict bio-security. Post-vaccination sera were collected at 15 and 35 days post-vaccination. The degree of antibody level of Pre-vaccination and post- vaccination sera were determined by PHA test. Protection test was carried out in each vaccinated and control groups after 15 days of secondary vaccination. Collection of samples- Rats were collected from wandering in and around the BAU poultry farm. Rat’s feces / swab were collected aseptically in sterilized petri dishes and test tubes with the help of sterilized inoculating loop. Each sample was cultured on individual petri dishes containing respective media. After 24 hours incubation at 370C, each plate were examined for identification of organisms and subcultured for pure culture and then stored a refrigerator for further study. Culture of P. multocida organisms- P. multocida organisms were cultured according to the standard method described by Cowan (1985). The collected organisms were inoculated in Blood agar (BA), Nutrient agar (NA) and Nutrient broth (NB) enriched with yeast extract and beef extract for better growth. The inoculating media was incubated at 370C in bacteriological incubator for characteristic colony formation. Subsequent subculture was done for getting pure culture. Gram’s staining and Carbohydrate fermentation reaction- The representative bacterial colonies were characterized morphologically using Gram’s stain and biochemically using basic five sugars according to the method described by Merchant and Packer (1967). Acriflavine test of capsular antigen- The test was carried out following the methods suggested by Furian et al. (2014) for the identification of antigenic variants and colonial characteristics. A number of colonies were picked up from the growth on BA plates. Heavy suspensions of organisms were prepared on a clean grooved glass slide with one or two drops of 1:1000 acriflavine solution in distilled water. The reaction was read just after mixing. Isolates that indicated reactions like slimy precipitation, no precipitation or partial flocculation in acriflavine test were considered as positive. Maintenance of stock culture of the organisms- Nutrient agar slants were used to maintain the stock culture. The P. multocida organisms were inoculated in slant by streaking and were incubated at 370C for 24 hours. Finally, sterile mineral oil was overlaid and kept the slant at room temperature for future use. Pathogenicity of the P. multocida isolate- In mice model: Pathogenicity test was done as described by Merchant and Packer (1967). Adult mice were used to observe the entero-pathogenicity of the selected isolated strains of Pasteurella spp. Adult mice were examined clinically and those found free from clinical symptoms were selected from the experiment. Mice were divided into 2 groups, test group (n=5) and control group (n=3). Then the strains of Pasteurella spp. were first grown on BA media. A small colony from BA media was added to the 5 ml to NB. The broth was incubated at 370C for 24 hours. A dose of 0.5 ml of culture was injected i/m to test group and 0.5 ml of sterile NB was injected i/m to control group of mice and kept in separate cages. All the mice were reared in the laboratory animal shed and given diet, nourishment kept under observation for 24 hours and clinical signs and symptoms at every 6 hours interval were recorded. In Fayoumi chickens: The fayoumi breed of poultry were divided into 2 groups (n=3) and control group (n=3). Each bird of group 1 was injected with 1 ml of bacterial suspension intramuscularly. The bird of group 2 served as control. All the birds were allowed to rear on same feed and environmental condition under observation for 24 hours and were observed for clinical signs and symptoms at every six hours interval. Preparation of capsular antigen- The capsular antigen of P. multocida was prepared according to the method suggested by Siddique et al. (1997). The fresh subculture of P. multocida was diluted with PBS and heated at 560C for 30 minutes in hot water bath to assist the removal of the capsular antigen. After heating, the suspension was centrifuged at 4500-6000 rpm for 20 minutes by using coarse stone bids. The supernatant was considered as a source of capsular antigen. Preparation of capsular extract fowl cholera vaccine- This procedure was carried out according to the procedure described by Choudhury et al. (1987) with slight modification. In this study, laboratory mice were used for passages of isolates of P. multocida. For this, the isolates of P. multocida organisms were cultured in blood agar media and kept in bacteriological incubator at 370C for 24 hours and examined the purity of culture and subsequently subcultured in the same media for 24 hours. The isolated colonies were then incubated in nutrient broth added with yeast extract 0.5 gm per liter (0.5gm/L) and beef extract 2 gm per liter (2gm/L) and incubated at 370C for 24 hours for massive growth. Later on, formalin was added in the broth culture at the rate of 8 ml per liter (8ml/L), heated at 560C for 30 minutes in hot water bath to assist the removal of the capsular antigen. After heating, the suspension was centrifuged at 4500-6000 rpm for 20 minutes by using coarse stone bids. The supernatant was considered as a source of capsular antigen after performing acriflavine test. Then, CFU estimation and sterility test were performed according to the method described by Michael et al. (1979) and Choudhury et al. (1987), respectively. Vaccination of the chicken- This procedure was carried out according the procedure described by Choudhury et al. (1987). Fowl cholera vaccine was administered at the dose rate of 1 ml of 5.6 ×107 CFU through IM and SC route via thigh muscle in each selected group of A and B, respectively, at 9 weeks of age. Booster dose was given with the same dose and route after 14 days of primary vaccination in both groups. Collection of serum from immunized birds- For collection of serum from the immunized birds the procedure described by Siddique et al. (1997) were followed. Five ml of blood was collected from the wing vein of all vaccinated chickens of each group without anticoagulant and was poured gently in the sterile glass test tubes at 63 days (as pre-vaccinated sera), after 2 weeks of primary vaccination (at 77 days) and after 3 weeks of secondary vaccination (at 98 days). Protection test- A known highly virulent duck P. multocida, PM-38, Serotype-1 (X-73) which obtained from stock culture of the Department of Microbiology and Hygiene was inoculated in blood agar as subculture and incubated at 370C for 24 hours. After three subsequent mice passage the subculture so prepared as broth culture to add 3ml PBS. Then CFU of each culture was determined by plate count method and was also tested for purity by Gram’s staining method. For preparation of challenge dose, the procedure suggested by Choudhury et al. (1987) was followed. Both vaccinated and control group of birds were subjected to challenge, at 45 days of revaccination through IM inoculation following the procedure of Choudhury et al. (1987). The challenge inoculums contain 5.6×107 CFU. For the protection test, 10 birds from group A and B and 5 birds from group C were selected and challenged after 17 days of blood collection of secondary vaccination. Birds after challenge infection were observed daily up to one week for any clinical signs and symptoms of FC. The clinical findings of both the vaccinated and unvaccinated chickens were observed and recorded. Passive haemagglutination (PHA) test- The test was used to determine titres of antibodies in birds having been inoculated with the antigen containing P, multocida as per the method by Tripathy et al. (1970), Choudhury et al. (1987) and Siddique et al. (1997) but with slight modification. According to Tripathy et al. (1970), the pH of PBS, concentration of tannic acid solution, strength of Na2HPO4.12H2O and KH2PO4.2H2O, were 6.4, 1:25000, 0.15M, and 0.15M respectively, but those values in the present study were 7.2, 1:20000, 0.20M and 0.20M, respectively. Principle and method of the test- The sensitivity and specificity of PHA test procedure depends on the use of soluble antigen. In this case, capsular antigens (soluble antigen) of P. multocida are coupled to chemically modified erythrocytes (sheep erythrocyte), and then antigen-coated erythrocytes readily react with specific antibodies and results in haemagglutination. This method was carried out according to the method described by Tripathy et al. (1970). The endpoint was determined by observing the highest dilution at which cells are agglutinated. Agglutination was indicated by a flat deposition of a diffuse thin layer of clumping of RBC on the bottom of the wells. The results were recorded by deposition of a diffuse thin layer of clumping of RBC on the bottom of the wells which indicated HA positive and a compact buttoning with a clear zone indicated HA negative. The reciprocal of the highest dilution of sensitized tanned SRBC was considered as the titer of the serum. Statistical analysis- The effect of vaccination on experimental birds in terms of PHA titer and protection capacity of vaccinated birds against challenge infection was subjected to analysis of Geometric mean with standard error, as per the method of Zar (2014). The PHA titers were analyzed by t-test to determine the protective capacity of vaccinated birds against challenge exposure (Zar, 2014).

  Res. Agric., Livest. Fish.8(1): 117-124, April 2021
  DOI: https://doi.org/10.3329/ralf.v8i1.53273
Funding Source:
1.   Budget:  
  

To prevent and reduce the incidence of FC, it may be suggested that capsular extract FC vaccine prepared with isolated rats P. multocida may be used side by side with conventional FC vaccine after thorough field trial.

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