Research Article

 

Efficacy of a Recombinant Vaccine Against Pasteurellosis in Chickens and Ducks

 

Didik Handijatno1, Nur-Aliyah Ahmad2, Sabri Md Yusoff3, Annas Salleh2, Mohd Zamri-Saad2

1Department of Microbiology, Faculty of Veterinary Medicine, Airlangga University,60115 Surabaya, Indonesia; 2Department of Veterinary Laboratory Diagnosis, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Malaysia; 3Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Malaysia.

 

Received | August 23, 2019; Accepted | November 12, 2019; Published | November 26, 2019

*Correspondence | Annas Salleh, Department of Veterinary Laboratory Diagnosis, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Malaysia; Email: annas@upm.edu.my

Citation | Handijatno D, Ahmad N, Yusoff SM, Salleh A, Zamri-Saad M (2019). Efficacy of a recombinant vaccine against pasteurellosis in chickens and ducks. Adv. Anim. Vet. Sci. 7(12): 1134-1139.

DOI | http://dx.doi.org/10.17582/journal.aavs/2019/7.12.1134.1139

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

 

Copyright © 2019 Handijatno et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

INTRODUCTION

 

Pasteurellosis, also known as fowl cholera (FC) in chickens is caused by Pasteurella multocida. All serotypes of P. multocida have been previously isolated from avian hosts (Christensen et al., 2008), except capsular types E and serotypes 8 and 13. The capsular type A has been observed to be the most frequently isolated from cases of severe FC (Christensen et al., 2008). In ducks, infections by P. multocida or Riemerella antipestifer (previously in the genus Pasteurella), or both are sometimes known as FC. The disease leads to great economic loss in poultry and duck industries, mainly due to the high mortality (Baksi et al., 2018). This high mortality is the result of acute septicaemia 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 OPM36 gene of P. multocida A:1 against FC in chickens and ducks.

 

MATERIALS AND METHODS

 

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 26oC 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 4oC for 18h. The culture was washed three times and re-suspended in PBS to a concentration of 3.0x108 cells/ml.

 

The control non-inserted vaccine was similarly prepared but without the OMP36 gene insert. Other control vaccine used for chickens was commercial alum-precipitated vaccine containing 3.0x108 cfu/ml of P. multocida A:1 (MVP, Malaysia). For ducks, a commercial alum-precipitated vaccine containing 3.0x108 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 37oC for 24h. Fifteen colonies were selected from each serotype and cultured in brain-heart infusion (BHI) broth, and incubated at 37oC for 24h. The culture was adjusted to a final bacterial concentration of 1.0x109 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; 3.0x106 cfu/ml P. multocida serotype A:1 in carbonate-bicarbonate buffer (pH 9.6), and incubated at 4oC overnight. The plates were then washed, filled with 200µl of blocking buffer [PBS-0.5% Tween 20(PBS-T)-3% BSA] and incubated at 37oC for 1h. After washing, 50µl of diluted serum samples in blocking buffer (1:500) were added, and incubated at 37oC 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 37oC for 1 h. Following washing, 100µl of TMB solution substrate buffer was added and incubated at 37oC 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.

 

Table 1: Summary of the chicken and duck groupings, immunization, and challenge

 

Groups	Immunization1	Birds	Challenge strain
Group 1	Recombinant	Chicken	P. multocida A:1
		Chicken	P. multocida A:3
		Chicken	P. multocida A:1,3
		Duck	P. multocida A:1 + R. anatipestifer
Group 2	E. coli no insert	Chicken	P. multocida A:1
		Chicken	P. multocida A:3
		Chicken	P. multocida A:1,3
		Duck	P. multocida A:1 + R. anatipestifer
Group 3	Commercial	Chicken	P. multocida A:1
		Chicken	P. multocida A:3
		Chicken	P. multocida A:1,3
		Duck	P. multocida A:1 + R. anatipestifer
Group 4	PBS	Chicken	P. multocida A:1
		Chicken	P. multocida A:3
		Chicken	P. multocida A:1,3
		Chicken	none
		Duck	P. multocida A:1 + R. anatipestifer
		Duck	none
Group 5	PBS	Chicken	none
		Duck	none

 

1Birds were immunized 2 weeks apart with respective vaccine (0.5ml, IM).

 

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 37oC 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.

 

RESULTS

 

Survival rate in chickens

Chickens immunized with the recombinant vaccine showed 84% (21/25), 44% (11/25) and 92% (23/25) survival rates following challenged by P. multocida A:1, A:3 and A:1,3, respectively. The recombinant vaccine provided significantly (p<0.05) lower protection against P. multocida A:3 compared to P. multocida A:1 and A:1,3. In comparison, chickens immunized by the commercial vaccine showed significantly (p<0.05) lower level of protection at 64% (16/25) and 44% (11/25) against P. multocida A:1 and A:3, respectively but higher (p>0.05) level of protection at 80% (20/25) against P. multocida 1,3. The E. coli with no insert provided significantly (p<0.05) low homologous and heterologous protections at 40% (10/25), 32% (8/25) and 44% (11/25) against P. multocida A:1, A:3 and A:1,3, respectively. Only few chicken administered with PBS and challenged with P. multocida A:1, A:3, or A:1,3 survived the challenge trial (Table 2).

 

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 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.

 

Table 2: Summary of immunization and survival rate after challenge trial in chickens and ducks.

 

Groups	Immunization1	Birds	Challenge strain	Survival rate %
Group 1	Recombinant	Chicken	P. multocida A:1	84a
		Chicken	P. multocida A:3	44b
		Chicken	P. multocida A:1,3	92a
		Duck	P. multocida A:1 + R. anatipestifer	56b
Group 2	E. coli no insert	Chicken	P. multocida A:1	40c
		Chicken	P. multocida A:3	32c
		Chicken	P. multocida A:1,3	44b
		Duck	P. multocida A:1 + R. anatipestifer	20c
Group 3	Commercial	Chicken	P. multocida A:1	64b
		Chicken	P. multocida A:3	44b
		Chicken	P. multocida A:1,3	80a
		Duck	P. multocida A:1 + R. anatipestifer	92a
Group 4	PBS	Chicken	P. multocida A:1	8d
		Chicken	P. multocida A:3	4d
		Chicken	P. multocida A:1,3	16d
		Chicken	none	100a
		Duck	P. multocida A:1 + R. anatipestifer	0d
		Duck	none	100a

 

1Birds were immunized 2 weeks apart with respective vaccine (0.5ml, IM); a,b,c,dDifferent superscript letters signify significant (p<0.05) difference.

 

 

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 post-immunization, 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.

 

 

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. These birds were observed with gross lesions including haemorrhage musculature, hepatic congestion (Figure 4), haemorrhagic trachea, air sacculitis, ventricular congestion, and hydropericardium.

 

 

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 OMP36 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 OMP36 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 OMP36 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 mauscript, NAA and AS involved in revision of manuscript, MZS and SMY designed and supervised the study.

 

Conflict of Interest

 

The authors declare that there is no conflict of interest regarding the publication of this article.

 

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