IMMUNE RESPONSES INDUCED IN CHICKENS BY CAPSULAR EXTRACT OF P. multocida ISOLATED FROM FARM RAT

Received 19 February, 2021 Revised 22 March, 2021 Accepted 23 March, 2021


INTRODUCTION
Fowl cholera (FC), also known as avian cholera, avian pasteurellosis, avian hemorrhagic septicemia, is a contagious disease affecting domesticated and wild birds (Xiao et al., 2016). Fowl cholera is caused by Pasteurella (P.) multocida, a Gram-negative, non-motile, non-spore forming rod, occurring singly or in pairs, and occasionally in chains or filaments (Ievy et al., 2013). About 25% to 35% mortality in chickens of Bangladesh, is due to FC (Choudhury et al. 1985). Pathogenicity or virulence of P. multocida in relation to FC is complex and variable depending on the strain, host species and variation within the strain or host and conditions of contact between the two (Glisson et al., 2008). Based on capsular antigen, P. multocida strains are categorized as serogroup A, B, C, D, and E, and based on lipopolysaccharide (LPS) antigens, further classified into 16 (1 to 16) serotypes (Kwaga et al., 2013). In Asian countries, P. multocida type A:1, A:3, and type D are found to be responsible to cause FC, where the serotype A:1 causes 80% mortality in chickens (Ranjan et al., 2011). The ability of P. multocida to invade and reproduce in the host is enhanced by the presence of capsule that surrounds the organism (Boyce et al., 2000). Loss of ability of a virulent strain to produce capsule results in loss of virulence (Harper et al., 2006). Snipes et al. (1987) also recorded that the capsular form of P. multocida were more virulent than that of non-capsulated forms because the capsulated form of bacteria might easily overcome the host's humoral defense.
Fowl cholera is considered as one of the major enzootic bacterial diseases of poultry, causes severe economic loss in Bangladesh (Biswas et al., 2005). It occurs mainly in chickens, turkeys, ducks, geese, and quill (Parvin et al., 2011), however, the causative agent, P. multocida, is also recovered from poultry farm rodents (Curtis et al., 1980;Curtis, 1983;Snipes et al., 1988;Snipes et al., 1990). Curtis et al. (1980) isolated 41% (14 out of 34 rat-carcasses) P. multocida from 11 poultry farms. Besides, Snipes et al. (1988) reported 48 isolates of P. multocida of various somatic types from different species of mammals and bird in California, USA, and characterization of the isolates revealed that few were as same as the virulent P. multocida isolates of turkeys. Moreover, Curtis (1983) recorded the transmission of P. multocida infection from the brown rat (Rattus norvegicus) to domestic poultry.
Vaccination against FC is practiced as preventive measures in Bangladesh like other countries of the world to reduce the incidence of the disease (Kardos and Kiss, 2005). Two types of FC vaccines are available in Bangladesh, which are produced locally and reported to give better immunity (Rana et al., 2010). Alum precipitated formalin killed FC vaccine is produced in the Livestock and Poultry Research and Production Centre (LPVRPC; formerly known as Poultry Biologics Unit) of the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh, while the oil-adjuvant FC vaccine is produced in Livestock Research Institute (LRI), Mohakhali, Dhaka, under the Department of Livestock Services (DLS), Government of People's Republic of Bangladesh (Bag et al., 2015). But, those which are imported or prepared from field cases of FC are much more expensive and there is a chance of being severe infection if sufficient measures are not taken (Akhtar et al., 2016). It is scientifically established that immune responses are influenced by various factors, such as breed and rearing region (Rana et al., 2010), age of the hosts (Dick and Avakian, 1991), and the isolate used as vaccine seed (Akhtar et al., 2016). In this study, the vaccine was prepared with capsular extract of P. multocida which was isolated from the rats wandering in and around poultry farm as an alternative way to improve the immune status of chickens. The rat isolate of P. multocida can be considered as local isolates for vaccine seed development against FC in poultry (Snipes et al., 1988). Therefore, the present study is focused on the immune responses induced in chickens by newly developed vaccine with capsular extract of P. multocida isolated from poultry farm rats through the different routes of vaccination to determine the efficacy of that vaccine against a challenge infection with virulent duck isolate of P. multocida PM-38, Serotype-1 (X-73).

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 preimmunizing titre of the sera. Primary vaccination was given at the dose rate of 1 ml through intra-muscular (IM) and subcutaneous (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 postvaccination 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 37 0 C, each plate were examined for identification of organisms and subcultured for pure culture and then stored at 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 37 0 C 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 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 slides with one or two drops of 1:1000 acriflavine solution in distilled water. The reaction was read just after mixing. Isolates which 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 37 0 C 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 37 0 C 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 56 0 C 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 37 0 C 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 37 0 C 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 56 0 C 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 ×10 7 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 37 0 C 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 5.6×10 7 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

Statistical analysis
The effect of vaccination on experimental birds in terms of PHA titre and protection capacity of vaccinated birds against challenge infection was subjected to analysis of Geometric mean with standard error, as per method of Zar (2014). The PHA titres were analyzed by t-test to determine the protective capacity of vaccinated birds against challenge exposure (Zar, 2014).

Morphological, cultural and biochemical properties
For staining, smears were prepared from heart blood and liver impression of experimentally infected mice and chickens. Gram's stain revealed the presence of Gram negative, cocco-bacillary or rod shaped and generally occurred single or in paired and bi-polar organisms. Cowan (1985) and Cheesbrough (1985) also recorded similar staining characteristics of P. multocida. In blood agar and nutrient agar plate, the organisms produced small whitish colonies, which was opaque, circular and translucent in appearance. No hemolysis was observed in blood agar media. In nutrient broth, the P. multocida produced diffused turbidity. The selected P. multocida organism fermented dextrose, sucrose and mannitol but not maltose and lactose, and produced acid (indicated by the change of yellow colour of the medium) without gas, which was also observed by Cheesbrough (1985).

Results of pathogenicity test in mice
Experimental inoculation of isolated P. multocida in mice produced characteristic changes in different visceral organs. Following inoculation, mice died within 24 hours and there was a marked septicaemic lesions consisting of white necrotic foci and hemorrhages in lungs, trachea, liver and spleen. Glavits and Magyar (1990) found that mice and rabbits died of acute septicemia after intranasal infection with P. multocida isolated from acute case of FC. Mortality of mice indicated that the organisms were highly potent.

Result of acriflavine test
Slimy precipitation, no precipitation or partial flocculation was considered positive for capsule and complete flocculation were considered negative for capsule. Both the vaccine and challenge isolates were found positive for capsule in this study. Furian et al. (2014) also reported the same findings for acriflavine test.

Results of passive haemagglutination (PHA) test
Humoral immune response following vaccination of capsular extract FC vaccine was measured by PHA test. Choudhury et al. (1987) used the same method to measure the serum antibody titres following administration of FC vaccine. Prevaccination PHA titres of sera samples of all vaccinates and control birds was found to be at a mean of <4.

Results of protection test
All the control and experimentally vaccinated chickens that were challenged with virulent vaccine strain of P. multocida at the dose rate of 5.6×10 7 CFU/ml at IM route and manifested the following clinical findings and postmortem lesions: Clinical findings having post challenge At 6 hours of post inoculation (PI), the non-vaccinated chickens, which served as control, did not show any clinical sign. The clinical signs first observed at 12 hours of PI were characterized by dullness and depression. There were dullness, depression and slight rise of body temperature (42.5 0 C) and increased respiratory rates (30-

Postmortem lesions
During postmortem examination of unvaccinated control chickens, septicaemic conditions, blood vascular congestion and hemorrhagic enteritis were the important lesions in control chickens. Hemorrhage in the lung, abdominal fat and intestinal mucosa was common. Liver was swollen and sometimes congested and multiple necrotic foci were observed throughout the dorsal surface. These findings were agreed with the reports of Panna et al. (2015). Heart was enlarged and edematous, and trachea and lungs were hemorrhagic (Zahoor and Siddique, 2006). Choudhury et al. (1985) also reported the similar types of postmortem lesions in birds that died after experimental challenge infection. After two weeks of challenge exposure, a few of the selected survivors of each challenged group were sacrificed and the organism was reisolated, as per recommendation of Choudhury et al. (1987).

Survivability at challenge experiment
Vaccinated chickens which were exposed to challenge infection survived. The survivability rate of vaccinated chickens of group A was 100% and that of group B was 80% (Table 2). But all chickens of control groups failed to survive when challenged with virulent isolate of P. multocida.

CONCLUSION
To prevent and to 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.