CC BY
0Efficacy of Combined Vaccine against Salmonellosis and
Infectious Coryza in Poultry
Ibrahim, H.M.1*, Wafaa, R. Abd El-Aziz1, Halaa, El Sawy1, Sayed, R.H.2 and Gina, M. Mohammed2
1 Veterinary Serum and Vaccine Research Institute (VSVRI), Abbasia, Cairo, Egypt.
2 Central Laboratory for Evaluation for Veterinary Biologics (CLEVB), Abbasia, Cairo, Egypt.
"Corresponding author's Email: Dr.hazemibrahim@gmail.com
Received: 06 Aug 2017 Accepted: 11 Sept 2017
ABSTRACT
In the present study, efficacy of two prepared combined vaccines against salmonellosis and infectious coryza in poultry has been studied. Two vaccines were prepared using Salmonella Typhimurium and Enteritidis combined with Avibacterium paragallinarum serovars A, B, and C. one vaccine was adjuvanated with aluminium hydroxide gel and the other adjuvanated with montanide ISA71. The two vaccines were assayed in six weeks old Specific Pathogen Free (SPF) white Lohman layer chickens by injecting two doses of each vaccine 3 weeks apart. These chickens were challenged with either Salmonella virulent strains or Avibacterium paragallinarum different serovars 3 weeks post second dose. Antibody titers in sera of chickens against different antigens were higher in groups vaccinated with montanide oil vaccine than those vaccinated with aluminium hydroxide gel vaccine as detected by different serological tests; ELISA, micro-agglutination test and haem-agglutination inhibition test. Protection rate against challenge test were 80% and 85% for Salmonella and (80%; 90%, and 70%) and (90%; 100%, and 90%) to Avibacterium paragallinarum serovars A, B, and C respectively for combined vaccine adjuvanated by aluminum hydroxide gel and montanide ISA71. The protection rate was 15% against Salmonella Typhimurium and Enteritidis and 0% against infectious coryza among the unvaccinated chicken group.it could be concluded that producing a vaccine from locally isolated Salmonella and Avibacterium (Haemophilus) paragallinarum strains adjuvanated with montanide ISA71 is recommended to aid in controlling avian salmonellosis and Infectious coryza at the same time. Key words: Aluminum hydroxide gel, Chicken, Infectious coryza, Salmonellosis, Vaccine.
JWPR
Journal of World's Poultry Research
2017, Scienceline Publication
J. World Poult. Res. 7(3): 145-153, Sept 25, 2017
Research Paper, PII: S2322455X1700018-7 License: CC BY 4.0
INTRODUCTION
Salmonella is a persistent pathogen in the environment, able to easily survive and proliferate. The most commonly isolated serovars worldwide from various animal sources continue to be Salmonella Enteritidis and Salmonella Typhimurium which, besides producing gastroenteritis, are found in asymptomatic carriers in a wide variety of animal species. Of these, Salmonella Enteritidis is the most prevalent one followed by Salmonella Typhimurium (52.3% and 23.3% of the cases, respectively) (Lopez-Martin et al., 2016). Salmonella has
remained to be one of the most frequently detected causative agents in the food-borne outbreaks reported (26.6% of outbreaks). Eggs and egg products are frequently associated with Salmonella outbreaks. Salmonella Enteritidis and to a lesser extent, Salmonella Typhimurium are associated with egg-related outbreaks (EFSA, 2004).
Avian Infectious Coryza is a serious respiratory tract infection of domestic fowls caused by an opportunistic pathogen Avibacterium paragallinarum having an economic implication on the poultry industry and ornamental bird's population (Priya et al., 2012).
nBBRerinipm Ibrahim HM, Abd El-Aziz WR, El Sawy H, Sayed RH and Mohammed GM (2017). Efficacy of Combined Vaccine against Salmonellosis and Infectious Coryza in Poultry. J. World Poult. Res., 7 (3): 145-153.
Infectious Coryza is a contagious bacterial disease of poultry; it is a common bacterial disease in the commercial poultry (Gayatri et al., 2010). It mainly affects the upper respiratory tract of chickens. The meat of the affected chicken is condemned if it is infected with A. paragallinarum (Blackall et al., 2005).
Combined vaccines have the advantage of protection against more than one disease at the same time, besides, reducing vaccination expenses, decreasing the stress of vaccination for different vaccines, number of vaccination performed and saving time. So this study evaluates the efficacy of a prepared combined vaccine against salmonellosis and infectious coryza using two different adjuvants; aluminium hydroxide gel and montanide ISA 71.
MATERIALS AND METHODS
Bacterial strains
Salmonella Typhimurium and Salmonella Enteritidis
These two strains are local field isolates kindly obtained from Department of Bacterial Sera and Antigens, Veterinary Serum and Vaccine Research Institute (VSVRI), Abbasia, Cairo, Egypt. These strains were used for preparation of vaccines under test.
Avibacterium paragallinarum
The reference strains Avibacterium paragallinarum strain W (serovar A-1) and Modesto strain (serovar C-2) were obtained from MSD Animal Health/Intervet International bv., Boxmeer, The Netherlands; and reference strain 0222 (serovar B-1) was obtained from Dr. R.B. Rimler, USDA National. Animal Disease Center, Ames, Iowa, USA. Local field strain (A) has been originally isolated by Anaerobic Vaccines Research Department, VSVRI from an outbreak of Infectious Coryza in a laying flock in Egypt, confirmed using species level and serotype using serological tests with standard antisera against reference serovars.
Experimental birds
SPF one day old chicks. Forty chicks were used for safety testing of the prepared vaccines.
SPF white Lohman layer chickens. A total number of 150, six weeks old SPF white Lohman layer chickens were obtained from SPF Farm at Koom Osheem Fayuom province, Egypt. They were housed in batteries with the network floor. All birds were ascertained first to be free from Salmonella and coryza (organism and antibodies). They were fed on free balanced rations, and used for evaluation of prepared vaccines.
Vaccine preparation
Two combined vaccines were prepared according to Blackall et al. (1992) and Charles et al. (1994). Briefly, ST and SE were cultured on specific media. Equal volumes of each culture (adjusted to contain 1*108 CFU/ml) were mixed together and inactivated by adding 0.5% Formalin. Also cultures of Avibacterium paragallinarum serovars A, B and C were prepared (adjusted to contain 1*106 CFU/ml) and equal volume of each serotype were mixed and inactivated by adding 0.5% Formalin and 0.01% (w/v) of thimerosal was added as a preservative agent. Then the above cultures were combined together and divided into 2 parts; one part adjuvanated with 20% (v/v) aluminum hydroxide gel and the other part with Montanide ISA-71 (30:70 v/v).
Experimental design
A total of 150, six weeks old SPF white Lohman layer chickens were divided into three groups 50 chicks per each. Group 1 contained fifty chickens were vaccinated with the prepared combined aluminium hydroxide gel vaccine in a dose of 0.5 ml S/C. Group 2 contained fifty chickens were vaccinated with the prepared combined montanide ISA71 vaccine with dose of 0.5 ml S/C. Group 3 contained fifty chickens injected 0.5 ml S/C with normal saline, left as a control group.
Birds in group (1) and group (2) were boostered with the same vaccine (by the same route and dose) 3 weeks after first immunization. Serum samples were obtained regularly before immunization, weekly for 3 weeks after the 1st vaccination and every 2 weeks post boostering for 22 weeks. Then pooled and stored at -20 °C till used for following up the induced antibodies.
Quality control testing of the prepared experimental vaccines
Sterility test. The prepared vaccines were tested to be free from any external contaminant (aerobic and anaerobic bacteria, fungus and mycoplasma) according to OIE (2016).
Safety test. Safety of the prepared vaccines was monitored through the injection of a double field dose (1 ml) of the vaccine subcutaneously in each of 20 one day old SPF chicks. The chicks were observed daily for two weeks for any signs of local reactions, clinical signs or deaths.
Determination of immune response to the prepared vaccines Serological evaluation of humeral immune response of the vaccinated chickens against Salmonella Typhimurium and Salmonella Enteritidis
Micro-agglutination test (MAT)
Antibody titer in vaccinated and unvaccinated chickens was followed up on regular intervals post vaccination applying Micro-agglutination test (MAT) using sonicated antigen, according to the method described by Thaxton et al. (1970) and Brown et al. (1981).
ELISA
The developed humoral immune response against ST and SE in the vaccinated chickens was measured by ELISA in the sera using Salmonella antibody test kit (BioChek Poultry Immunoassays cat # CK117 for S. enteritidis and CK118 for S. typhimurium) according to Haider et al. (2007). Calculation of the antibody titers in ELISA were performed in relation to S/P ratio according to the following formulae:
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Calculation of Antibody Titer Logi0 Titer=1.13(Log s/p) +3.156.
Antibody titer = AntiLog
Serological evaluation of humeral immune response of the vaccinated chickens against Avibacterium paragallinarum serovars A, B, and C
Haemagglutination inhibition test
Antibody response in vaccinated and unvaccinated chickens was followed up on regular intervals post vaccination applying Haemagglutination Inhibition (HI) test using sonicated antigen, according to the method described by Blackall et al. (1990).
Enzyme-Linked Immunosorbent Assay (ELISA)
It was done according to Ryuichi et al. (2012) for Avibacterium paragallinarum serovars (A, B, and C). Optical Density (OD) was measured at 490 nm by using a micro plate reader (DYANA Tech., USA). The S/P ratio was calculated and expressed as ELISA titer.
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Efficacy test (Challenge)
Challenge by Avibacterium paragallinarum serovars A, B and C
All challenge was done by intra sinus inoculation with 0.1 ml overnight broth culture of Avibacterium paragallinarum serovars A, B and C challenge dose containing 1x106 CFU/ml. Clinical signs of Infectious Coryza were recorded from day-1 to day-7 after inoculation. The presences of any nasal discharge and facial edema in challenged chickens were recorded. A protected chicken was defined as a chicken that had shown no clinical signs.
Challenge by Salmonella Typhimurium and Salmonella Enteritidis strains
Via administrating the vaccinated chickens 3 weeks post boostering dose by a dose of 1 ml virulent ST and SE broth culture containing 1x108 CFU /ml (OIE, 2016).
Fecal shedding
Shedding of Salmonella was detected in the fecal samples collected from challenged vaccinated and non-vaccinated chicks up to 4 weeks post challenge.
Statistical analysis
The level of protection present in the vaccinated groups were analyzed and compared with parametrical correlation using Student's T test (significant difference at P < 0.05) (Snedecor and Cochran, 1980).
Ethical approval
All animal procedures were approved by the Animal Ethics Committee at Veterinary Serum and Vaccine Research Institute (VSVRI), Abbasia, Cairo, Egypt.
RESULTS
Safety and sterility of prepared vaccines
Both of two vaccines were found to be safe and sterile.
Humeral immune response of the vaccinated chickens against Salmonella Typhimurium and Salmonella Enteritidis
Table 1 and 2 illustrated results of MAT and ELISA which are parallel to each other as the antibody titers started rising 2 weeks post first vaccination and reached peak sixth week post boostering. It was clear that MAT and ELISA titer for combined montanide ISA 71 vaccine was higher or double the titer of combined aluminium hydroxide gel vaccine for both antigens. The obtained results shown in tables 1 and 2 were analyzed statistically using Student's T test and it was found that there is a significant difference at P < 0.05 between group 2 (vaccinated with combined montanide ISA71 vaccine) and group 1 (vaccinated with combined aluminium hydroxide gel vaccine).
Humeral immune response of the vaccinated chickens against Avibacterium paragallinarum serovars A, B, and C
Results of Haem-agglutination Inhibition (HI) text and ELISA as shown in table 3 and 4 were in accordance to those of Salmonella organisms of both vaccines. As antibody titers start raising two weeks post first vaccination and reached peak six weeks post boostering. The obtained results in tables (3 and 4) were analyzed statistically using Student's T test and it was found that
there is a significant difference at P >0.05 between group 2 (vaccinated with combined montanide ISA71 vaccine) and group 1(vaccinated with combined aluminium hydroxide gel vaccine).
Concerning ELISA titers for Avibacterium paragallinarum serovars (A and C) in both vaccines as shown in table 4, wee paralleled with that of HI, also there was a statistically significant difference in ELISA titer between both vaccines.
Table 1. Measurement of antibody against Salmonella Typhimurium and Enteritidis in sera of vaccinated and unvaccinated
layer chickens using microagglutination test.
Weeks post vaccination Group (1) Group (2)* Control
Serovar Typhimurium Serovar Enteritidis Serovar Typhimurium Serovar Enteritidis Serovar Typhimurium and Enteritidis
0 0 0 0 0 0
2WPV 40 40 40 40 0
3WPV 40 40 80 80 0
Boostering
2 WPB 80 80 160 80 0
4WPB 160 160 320 320 0
6WPB 320 320 640 640 0
8WPC 320 320 320 320 0
10WPC 320 320 320 320 0
12WPC 160 160 320 320 0
14WPC 160 160 160 160 0
16WPC 80 80 160 160 0
18WPC 80 80 80 80 0
20WPC 40 40 40 80 0
22WPC 20 20 20 40 0
Group (1): SPF layer chickens vaccinated with combined aluminium hydroxide gel vaccine; Group (2): SPF layer chickens vaccinated with combined montanide ISA71 vaccine; Control: Unvaccinated group; WPV: Weeks post vaccination; WPB: Weeks post boostering; WPC: weeks post challenge; * Significant at P < 0.05; The antibody titer in MAT was expressed as Geometric Mean Titer (GMT)
Table 2. Measurement of antibody against Salmonella Typhimurium and Enteritidis in sera of vaccinated and unvaccinated
layer chickens using ELISA
Group (1) Group (2)* Control
Weeks post vaccination Serovar Typhimurium Serovar Enteritidis Serovar Typhimurium Serovar Enteritidis Serovar Typhimurium and Enteritidis
0 93 100 93 100 100
2WPV 975 850 1530 1443 112
3WPV 1453 1413 2553 2721 111
Boostering
2WPB 2189 2189 3517 3617 128
4WPB 2344 2544 3782 3982 123
6WPB 2763 2791 4543 4484 130
8WPV 2675 2547 3925 3855 143
10WPC 2320 2250 3845 3745 135
12WPC 2230 2130 3667 3686 156
14WPC 1970 1940 3253 3354 122
16WPC 1515 1465 2180 2370 129
18WPC 1325 1298 2020 2120 125
20WPC 1250 1110 1890 1970 123
22WPC 1140 1020 1680 1730 128
Group (1): SPF layer chickens vaccinated with combined aluminium hydroxide gel vaccine; Group (2): SPF layer chickens vaccinated with combined montanide ISA71 vaccine; Control: Unvaccinated group;WPV: Weeks post vaccination; WPB: Weeks post boostering; WPC: weeks post challenge; * Significant at P < 0.05
Table 3. Geometric mean of Haem-agglutinating Titer against Avibacterium paragallinarum serovars A and C in sera of
vaccinated layer chickens.
Group (1) Group (2)* Control
Weeks post vaccination Serovar A Serovar C Serovar A Serovar C Serovar A and C
0 0 0 0 0 0
2WPV 40.32 40.31 28.50 32 0
3 WPV 40.23 40.31 35.78 43.11 0
Boostering
2WPB 40.8 57.01 57.01 80.63 0
4WPB 50.79 57.01 71.83 90.50 0
6WPB 57.01 71.83 101.59 114.04 0
8WPB 57.01 71.83 101.59 114.04 0
10WPB 57.01 71.83 101.59 114.04 0
12WPB 50.79 57.01 90.50 101.59 0
14WPB 40.34 57.01 90.50 101.59 0
16WPB 40.87 50.79 90.5 101.59 0
18WPB 35.91 50.79 80.63 90.50 0
20WPB 28.50 40.31 80.63 90.50 0
22WPB 28.50 40.31 80.63 90.50 0
Group (1): SPF layer chickens vaccinated with combined aluminium hydroxide gel vaccine; Group (2): SPF layer chickens vaccinated with combined
montanide ISA71 vaccine; Control: Unvaccinated group; WPV: Weeks Post Vaccination; WPB: Weeks Post Boostering; *Significant at P < 0.05
Table 4. ELISA results (S/P ratio) of vaccinated and unvaccinated layer chickens against Avibacterium paragallinarum
serovars A and C.
Group (1) Group (2)* Control
Weeks post vaccination Serovar A Serovar C Serovar A Serovar C Serovar A and C
0 0.031 0.023 0.044 0.021 0.002
2WPV 1.304 1.474 1.292 1.344 0.011
3 WPV 1.388 1.476 1.549 1.598 0.233
Boostering
2WPB 1.454 1.455 1.936 1.942 0.043
4WPB 1.474 1.519 1.975 1.936 0.022
6WPB 2.190 2.274 2.095 2.011 0.056
8WPB 2.130 2.235 2.164 2.274 0.044
10WPB 2.091 2.064 2.278 2.274 0.070
12WPB 1.782 1.940 2.087 2.164 0.033
14WPB 1.566 1.885 2.011 2.036 0.056
16WPB 1.431 1.850 1.975 2.011 0.099
18WPB 1.519 1.770 1.907 1.942 0.043
20WPB 1.472 1.549 1.869 1.936 0.065
22WPB 1.199 1.454 1.848 1.907 0.023
Group (1): SPF layer chickens vaccinated with combined aluminium hydroxide gel vaccine; Group (2): SPF layer chickens vaccinated with combined
montanide ISA71 vaccine; Control: Unvaccinated group; WPV: Weeks Post Vaccination; WPB: Weeks Post Boostering; *Significant at P < 0.05
Results of Challenge test
As shown in tables 5 and 6, the protection rates in chickens vaccinated either with combined aluminium hydroxide gel vaccine or combined montanide ISA71 vaccine were 80% and 85% for Salmonella organisms. On the other hand it was (80%, 90% and 70%) for combined aluminium hydroxide gel vaccine and (90%, 100% and
90%) for combined montanide ISA71 vaccine against Avibacterium paragallinarum serovars A, B, and C. Meanwhile, the protection rate was 15% against Salmonella Typhimurium and Salmonella Enteritidis and 0% against infectious coryza among the unvaccinated chicken group.
Fecal shedding of Salmonella Typhimurium and Salmonella Enteritidis from challenged chickens
Fecal shedding of Salmonella Typhimurium and Salmonella Enteritidis as shown in table (7), from chickens vaccinated with either combined aluminium hydroxide gel vaccine or combined montanide ISA71
vaccine in the 1st, 2nd and 3rd weeks post challenge were (25%, 12.5% and 12.5%) and (22.22%, 11.11% and 0%) respectively while in the 4th week the fecal shedding disappeared. Regarding the control unvaccinated birds the fecal shedding were 66.66%, 66.66%, 33.33% and 33.33% in the 1st, 2nd, 3rd and 4th weeks post challenge respectively.
Table 5. Protective Efficacy of combined vaccine against salmonellosis in SPF chickens challenged with virulent strains
VACCINE Serovar No. of inoculated Survived Protection %
chickens# chickens
Group (1) Typhimurium 10 8 80
Enteritidis 10 8 80
Group (2) Typhimurium 10 8 80
Enteritidis 10 9 90
Control Typhimurium 10 1 10
Enteritidis 10 2 20
*Protection % = (Survival birds/ total number of birds) x 100; Group (1): SPF layer chickens vaccinated with combined aluminium hydroxide gel vaccine; Group (2): SPF layer chickens vaccinated with combined montanide ISA71 vaccine;# Challenge with virulent Salmonella Typhimurium and Salmonella Enteritidis; Control: Unvaccinated group.
Table 6. Protective Efficacy of combined vaccine against infectious coryza in SPF chickens challenged by Avibacterium paragallinarum serovars A, B, and C
Survived
VACCINE serovar No. of inoculated chickens# chickens Protection %
A 10 8 80
Group (1) B 10 9 90
C 10 7 70
A 10 9 90
Group (2) B 10 10 100
C 10 9 90
A 10 0 0
Control B 10 0 0
C 10 0 0
Group (1): SPF layer chickens vaccinated with combined aluminium hydroxide gel vaccine; Group (2): SPF layer chickens vaccinated with combined montanide ISA71 vaccine; # Challenge with virulent Avibacterium paragallinarum serovars A, B, and C; Control: Unvaccinated group.
Table 7. Results of fecal shedding of Salmonella Typhimurium and Salmonella Enteritidis from layer chickens after challenge
Chicken groups No. of birds positive for isolation / total No. of living birds
1st week 2nd week 3rd week 4th week
Group (1) 2/8 (25%) 1/8 (12.5%) 1/8 (12.5%) 0/8 (0%)
Group (2) 2/9 (22.22%) 1/9 (11.11%) 0/9 (0%) 0/9 (0%)
Control 2/3 (66.66%) 2/3 (66.66%) 1/3 (33.33%) 1/3 (33.33%)
Group (1): SPF layer chickens vaccinated with combined aluminium hydroxide gel vaccine; Group (2): SPF layer chickens vaccinated with combined montanide ISA71 vaccine; Control: Unvaccinated group.
DISCUSSION
Avian salmonellosis is a large group of acute and chronic diseases of poultry caused by any one or more member of genus Salmonella. However, particular Salmonella Enteritidis is the most prevalent one followed by Salmonella Typhimurium (Capita et al., 2003).
Infectious coryza is an acute respiratory disease of chickens caused by the bacterium Avibacterium paragallinarum. The greatest economic losses associated with infectious coryza results from poor growth performance in growing birds and marked reduction (1040%) in egg production in layers (Blackall and Matsumoto, 2003).
Charoenvisal et al. (2017) examined efficacy of four commercial Infectious Coryza vaccines available in Thailand for protection rate against Thai field isolates serovar A, B, and C. The study revealed that the protection rate of Infectious Coryza vaccines depended on the strains isolated from each country.
So in this study combined vaccines have the advantage of protection against more than one disease at the same time, beside, reducing vaccination expenses, number of vaccination performed and saving time. The efficacy of a prepared combined vaccine against salmonellosis and infectious coryza using two different adjuvants; aluminium hydroxide gel and montanide ISA 71 was monitored in sera of vaccinated chickens using HI, MAT and ELISA. It was clear that antibody titers in sera of chickens for all tests were paralleled to each other in starting and increasing titer and also after boostering as illustrated in tables 1, 2, 3, 4 and 5 which agree with that obtained by Akeila et al. (2014). Who evaluated a combined vaccine against A. paragallinarum and S. Enteritidis and found that antibody titers reached the maximum levels at the 6th WPV in the vaccinated groups.
With serovar B vaccines, a HI test was not done as it is based on a hyaluronidase-treated antigen and formaldehyde-treated RBC and gave only very low HI titers following vaccination (as compared with serovar A or C vaccines) but the vaccinated birds were significantly protected against homologous challenge, These results correlate with other studies done by Yamaguchi et al. (1991).
The protection rates against Salmonella Typhimurium and Enteritidis as measured by challenge test were 80% and 85% in chickens vaccinated with combined aluminium hydroxide gel vaccine and combined montanide ISA71 vaccine are respectively, as shown in table 5.
Also the protection rates against Avibacterium paragallinarum serovars A, B, and C were 80%, 90% and 70% in chickens vaccinated with combined aluminium hydroxide gel vaccine and were 90%, 100% and 90% of the montanide ISA71 vaccine respectively (Table 6).
Meanwhile, the protection rate was 15% against Salmonella Typhimurium and Enteritidis and 0% against infectious coryza among the unvaccinated chicken group and these results agreed with by Akeila et al. (2014) who reported 73.3% and 93.3% protection rate against S. Enteritidis and A. paragallinarum, respectively in a combined vaccine containing both organisms.
The fecal shedding of Salmonella Typhimurium and Enteritidis in the 1st, 2nd and 3rd weeks post challenge in chickens vaccinated with combined aluminium hydroxide gel vaccine was 25%, 12.5% and 12.5% , while it was 22.22%, 11.11% and 0% for those vaccinated only with montanide ISA71 vaccine, respectively. The fecal shedding disappeared in the 4th week.
Regarding the control unvaccinated birds the fecal shedding were 66.66%, 66.66%, 33.33% and 33.33% in the 1st, 2nd, 3rd and 4th weeks post challenge and these result agreed with Nourhan et al. (2015) who found that fecal shedding of Salmonella organisms in vaccinated group of chickens with S. Kentucky reached 8.33% while the unvaccinated control group at 3 week post challenge revealed fecal shedding of 25 %. No shedding was detected at the fourth week post challenge in the vaccinated group, while there was 16.6% shedding in control unvaccinated group.
So, the SPF layer chickens vaccinated with combined vaccine against salmonellosis and infectious coryza adjuvanted with montanide ISA71 gave high immune response and protection which is capable of improving vaccine efficacy via the induction of a strong and long lasting immunity. Also it is an excellent adjuvant stimulating humoral and cellular responses. This product is recommended for producing a potent vaccine able to protect layer chickens against salmonellosis and infectious coryza.
CONCLUSION
From the above results it could be concluded that producing a vaccine from locally isolated Salmonella and Avibacterium (Haemophilus) paragallinarum strains adjuvanted with montanide ISA71 is recommended to aid in controlling avian salmonellosis and infectious coryza at the same time.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
All authors participated in making the design, performing the experiment, analyses of the data, and writing the paper.
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