Efficacy of a Recombinant Vaccine Against Pasteurellosis in Chickens and Ducks

| Pasteurellosis or fowl cholera (FC), is an economically-important poultry disease caused by Pasteurella multocida serotype A. In ducks, it is caused by Riemerella anatipestifer. Outbreaks of FC is frequently reported, suggestive of inefficacious vaccine. In this study, recombinant cells carrying OMP36 gene of Pasteurella multocida A:1 was prepared as a killed vaccine to test against pasteurellosis in chickens and ducks. A total of 325 chickens and 125 ducks were selected and divided to 4 groups based on the types of immunization. Groups 1, 2, 3, and 4 were administered with the recombinant vaccine, control E. coli without insert, commercial FC vaccine, and sterile PBS, respectively. A booster dose was administered after 2 weeks. Each group were then further divided to 4 subgroups based on birds and challenge strain. A challenge trial was conducted against P. multocida A:1, A:3, or A:1,3 for the chickens; and P. multocida A:1 and R. anatipestifer for the ducks. Clinical signs were observed and serum samples were collected weekly for determination of IgG antibody levels. Birds with severe clinical signs were euthanized for necropsy. Following immunization of chickens, IgG increased at week 1 and continued to increase after booster vaccination at week 2, and peaked at week 4 for both recombinant and commercial vaccines. The recombinant vaccine stimulated higher level of antibody in chickens. Following challenge, the recombinant vaccine provided excellent homologous (21/25; 84%) and crossprotection against P. multocida serotype A:1,3 (23/25; 92%) but provided low cross-protection against P. multocida serotype A:3 (11/25; 44%). In ducks, the recombinant vaccine provided moderate protection (14/25; 56%) compared to the excellent protection provided by the commercial vaccine (23/25; 92%). All euthanized birds showed typical clinical signs and gross pathology of FC. In conclusion, the recombinant vaccine is suitable against pasteurellosis in chicken, being more superior compared to the commercial vaccine. However, it provides poor protection against R. anatipestifer infection in ducks.


Advances in Animal and Veterinary Sciences
December 2019 | Volume 7 | Issue 12 | Page 1135 or chronic respiratory infection by these pathogens (Homchampa et al., 1997). Sulpha drugs, semisynthetic penicillin, tetracycline, and erythromycin are administered in food or drinking water to control the disease (Huang et al., 2009). However, the usage of antimicrobials in animal production raises global concern pertaining to incidence of anti-microbial resistance (Stella et al., 2018).
Immunisation has also been used to control FC particularly in layers and breeders. Currently, commercially-available vaccines are in the form of bacterin, and only induce immunity to homologous serotypes (Homchompa et al., 1997). On the other hand, the use of live vaccine confers immunity against heterologous serotypes, but may revert to cause infection (Christensen et al., 2008). Furthermore, anaphylactic shock and short duration of protection are among the limitations of immunization using bacterin (Gershwin, 2015;Dellagostin et al., 2017). Hence, it is necessary to design a safe vaccine capable of conferring protection against homologous and heterologous challenges.
Recent vaccine development uses the outer membrane protein (OMP), which is part of the bacterial cell wall considered as a potent candidate for vaccine (Poolperm et al., 2018;Anun, 2001). The vaccine is prepared as recombinant cells that carry selected gene to provide better protection against challenge by P. multocida (Ibrahim et al., 2000). The objective of this study was conducted to compare the efficacy of a recombinant cell carrying OPM 36 gene of P. multocida A:1 against FC in chickens and ducks.

experimental animalS
A total of 325 Cobb chickens and 125 White Pekin ducks of 14 days old with no history of immunization against P. multocida and R. anatipestifer were selected. The chickens or the ducks were originated from one source. The birds were previously immunized against Marek's, Newcastle, and Gumboro diseases (Prabakaran, 2003). At the animal facility, they were kept in pens and were provided with commercial feed and drinking water. They were acclimatized for a week, while oral swabs were collected the following week for screening of P. multocida. The experiment was started when P. multocida was absent from all birds at which the birds were at 28 days old. All animal experiment was approved by an ethical review committee.

VaccineS
Recombinant OMP 36 kDa gene of P. multocida serotype A:1 was previously prepared, inserted into pET 32 KL/LIC vector, and transformed into Escherichia coli BL21(DE3) strain before they were grown in Luria-Bertani broth with induction using isopropyl-β-D-thiogalactoside (IPTG). The culture was incubated at 26 o C for 24h. The culture was harvested for determination of bacterial concentration (Pommerville, 2014). It was then washed with PBS, and killed using 0.5% formalin at 4 o C for 18h. The culture was washed three times and re-suspended in PBS to a concentration of 3.0x10 8 cells/ml.
The control non-inserted vaccine was similarly prepared but without the OMP 36 gene insert. Other control vaccine used for chickens was commercial alum-precipitated vaccine containing 3.0x10 8 cfu/ml of P. multocida A:1 (MVP, Malaysia). For ducks, a commercial alum-precipitated vaccine containing 3.0x10 8 cfu/ml of P. multocida serotype A:1 and R. anatipestifer (MVP, Malaysia) was used. Sterile PBS was used as negative control.

bacterial inoculumS
Isolates of P. multocida serotypes A:1, A:3, A:1, 3 and R. anatipestifer from previous outbreaks of FC were obtained from stock cultures kept at -80°C. They were thawed at room temperature for 1h, and subsequently cultured onto blood agar and incubated at 37 o C for 24h. Fifteen colonies were selected from each serotype and cultured in brainheart infusion (BHI) broth, and incubated at 37 o C for 24h. The culture was adjusted to a final bacterial concentration of 1.0x10 9 cfu/ml.

experimental DeSign immuniZation anD cHallenge
The chickens and ducks were divided to 4 groups based on the types of immunization. Group 1 were immunized with the recombinant vaccine, Group 2 were with E. coli without insert, Group 3 with commercial FC vaccine, and Group 4 with sterile PBS. The immunization was done at the dose of 0.5ml, intramuscular. Two weeks after the first immunization, booster dose of respective vaccine was administered with similar dose and route. Two weeks after the booster dose, the birds were then further divided into subgroups based on the challenge strains and were challenged with the inoculums prepared (Table 1).

blooD Sampling anD eliSa
Blood samples were collected via the brachial vein at weekly interval. The serum was subjected to determination of IgG level against P. multocida A:1 using ELISA as previously described, with minor modification (Sthitmatee et al., 2008;Solano et al., 1983). Briefly, microplates were coated with 50µl of antigen; 3.0x10 6 cfu/ml P. multocida serotype A:1 in carbonate-bicarbonate buffer (pH 9.6), and incubated at 4 o C overnight. The plates were then washed, filled with 200µl of blocking buffer [PBS-0.5% Tween 20(PBS-T)-3% BSA] and incubated at 37 o C for 1h. After washing, 50µl of diluted serum samples in blocking buffer

Advances in Animal and Veterinary Sciences
December 2019 | Volume 7 | Issue 12 | Page 1136 (1:500) were added, and incubated at 37 o C for 1 h. The plates were washed, and filled with 50µl of diluted goat anti-chicken IgG peroxidase conjugate (i-DNA, USA) in blocking buffer (1: 10,000) and incubated at 37 o C for 1 h. Following washing, 100µl of TMB solution substrate buffer was added and incubated at 37 o C for 30 min. The reaction was stopped by adding 2N sulphuric acid. Absorbance value was measured at 450 nm wavelength. The absorbance values were plotted to compare between different groups. clinical SignS, SurViVal rate, anD poSt-mortem examination All birds were observed for a period of 2 weeks. Birds with severe clinical signs of FC were euthanized by slaughtering (Grandin, 1994), and were considered succumbed to the infection. Any surviving birds were euthanized at the end of the experiment. Survival rate was calculated for each group. Immediately after euthanasia, liver, lung, pericardial fluid, heart and spleen samples were collected for bacterial isolation.

bacterial iSolation
The abovementioned samples were cultured on blood agar and incubated at 37 o C for 24h. Pasteurella multocida and R. anatipestifer was identified using Gram stain and biochemical tests (Carter and Cole, 2012;Peter et al., 1996), and subsequent confirmation of P. multocida (Shivachandra et al. 2006) and R. anatipestifer (Kardos et al., 2006) were made with PCR.

StatiStical analySiS
Data of IgG antibody levels in the serum samples were tabulated and analyzed using One Way ANOVA and comparison of mean by Duncan test. Data on percentage of animal that died after challenge were tabulated and analyzed using Chi-square.

SurViVal rate in DuckS
Following immunization with the recombinant vaccine, 56% (14/25) of the ducks were protected against a challenge by the combination of P. multocida A:1 and R. anatipestifer. In contrast, the commercial vaccine provided significantly (p<0.05) higher rate of protection at 92% (23/25). Immunization by E. coli failed to provide protection when significantly (p<0.05) low (20%; 5/25) number of the ducks survived. None of the duck administered with PBS and challenged with P. multocida A:1 and R. anatipestifer survived the challenge (Table 2).

bacterial iSolation anD iDentification
The respective P. multocida was isolated from all organ samples of chickens, while R. anatipestifer was isolated from the lungs of ducks that succumbed to the challenge. All isolates of P. multocida were confirmed by PCR with the

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December 2019 | Volume 7 | Issue 12 | Page 1137 1044 bp band while R. anatipestifer with the 700bp band ( Figure 1). All organ samples from surviving chickens and ducks yielded no isolation of P. multocida or R. anatipestifer.

Serum igg antiboDy reSponSe
Prior to immunization, there was significantly (p<0.05) low level of IgG in both chickens and ducks of all groups. However, following the first immunization, the IgG level increased significant (p<0.05) among the chickens and ducks immunized with the recombinant and commercial vaccines, but not following immunization with E. coli (Figure 2). The antibody levels increased until week 4 postimmunization, particularly after the administration of booster dose on week 2. Further increase of the IgG level was observed on week 5 following challenge but started to decrease on week 6.

Figure 2:
IgG antibody against P. multocida A:1 response in chickens during the immunization and challenge trial.
The IgG level was not significantly (p>0.05) increased following immunization with E. coli and in the control unimmunized groups. However, slight increase in the levels of IgG was observed on week 5 following challenge (Figures 2, 3).

clinical SignS anD groSS patHology
All challenged chickens and ducks that were euthanized due to severe clinical signs of FC had abnormal respiratory sounds, greenish diarrhea, weakness, and anorexia.

DISCUSSION
Despite the availability of vaccines against FC, outbreaks were commonly reported globally, affecting both domestic and wild birds (Srinivasan et al., 2011;Hassan et al., 2017;Sellyei et al., 2017;Jaeger et al., 2018). This might be due to the poor usage of the currently available vaccines. This study investigates the attempts to use the OMP 36 gene of P. multocida serotype A:1, expressed in E. coli strain BL21 through pET 32 plasmid as recombinant vaccine against FC in chicken and duck.
Following immunizations, the antibody titers increased significantly until week 4 following activation of memory cells (Dick and Avakian, 1991;Tizard, 2018). It seemed that both recombinant and commercial vaccines produced similar antibody pattern in chickens and ducks. Nevertheless, the antibody pattern following booster with the recombinant vaccine in chicken was significantly (p<0.05) higher than that of the commercial vaccine, possibly due to the superior antigenicity of recombinant OMP 36 that was expressed more after induction with IPTG than the whole cell P. multocida in the commercial vaccine. On the other hand, antibody titers in ducks following exposures to recombinant and commercial vaccines showed no significant difference (p>0.05).
The impact of antibody response could be observed following challenge. The recombinant vaccine that produced higher antibody level was capable of providing better protection in chickens, particularly after challenged by P. multocida A:1 and A:1,3. However, the recombinant vaccine that did not significantly different from the commercial vaccine did poorly in ducks upon challenged by P. multocida A:1 and R. anatipestifer. The significant finding of this study is that the recombinant cells carrying OMP 36 gene were capable of providing cross protection against homologous and heterologous P. multocida infection in chicken. Similar observations were previously reported (Chung et al., 2005;Sthitmatee et al., 2008), particularly due to the fact that vaccines made from bacteria that were cultured in vivo or in vitro under iron limited condition (Ibrahim et al., 2000;Kedrak and Opacka, 2003).
The low protection provided by the recombinant vaccine in the ducks suggested that the IgG antibody against P. multocida A:1 conferred were not capable of providing cross protection against infection by R. anatipestifer. Hence, further research should focus to improve this aspect. Detection and insertion of antigenic gene from R. anatipestifer into the existing recombinant cells to produce bivalent recombinant vaccine should be considered. However, this may be a challenge since previous study utilizing OMPA and P45 genes of R. anatipestifer had failed to provide effective protection in ducks against R. anatipestifer infection (Huang et al., 2002).

concluSionS
The study showed that the usage of recombinant vaccine derived from OMP 36 kDa gene of P. multocida serotype A:1 insert into pET 32 plasmid and E. coli strain BL21 as expression host cell able to provide high protection against homologous (P. multocida A:1) and heterologous P. multocida A:1,3, but provided moderate cross protection against heterologous P. multocida serotype A:3 in chickens. This is more superior compared to the tested commercial vaccine. In ducks, the recombinant vaccine able to provide partial protection against P. multocida A:1 and R. anatipestifer, but significantly lower than the tested commercial vaccine.

AUTHORS CONTRIBUTIONS
DH conducted the study, data analysis and writing of ma-