Cross Reaction of Serum in Salmonella enteritidis-Vaccinated Chicken to Some Salmonella enterica Serotypes

Salmonella spp. has been recognized as the major cause of food-borne illness in humans worldwide causing remain relevant to public health. Poultry vaccination is one promising strategy to mitigate Salmonella infection in poultry and, in turn, in humans as well. The objective of this study was to assess the potential of cross-reaction of serum in Salmonella enteritidis-vaccinated chicken to some serotype of Salmonella enterica. Four female, Isa Brown layer chickens (20 weeks old), were vaccinated with S. enteritidis strain Sm24/Rif12/Ssq (intra vena) to induced the production of specific antibodies in serum. Crossreaction of serum in S. enteritidis-vaccinated chicken were assess with agar gel immunodiffusion test (AGID) with S. enteritidis, S. pullorum, S. typhimurium, S. typhi, and Escherichia coli antigens. Serum could react with S. enteritidis and all types of S enterica used in this study (S. pullorum, S. typhimurium, S. typhi), but could not react with E. coli. The potential of cross-reaction of serum in S. enteritidis-vaccinated chicken to some serotypes of S. enterica may play a role in reducing the infection caused by that serotype.


INTRODUCTION
Salmonella spp.has been recognized as the major cause of food-borne illness in humans worldwide causing remain relevant to public health (Yang et al., 2015).Although many different foods have been implicated in salmonellosis outbreaks, most cases are usually caused by the consumption of poultry and eggs (Okamura et al., 2012;Goh et al., 2015).Intense international trade of animals and products of animal origin facilitates the spread of these bacteria (Liebana et al., 2013), making salmonellosis an international public health concern that is responsible for serious economic losses to the poultry industry and governments worldwide (Plym and Wierup, 2006).The Salmonella enteritidis and S. typhimurium serotypes are the most frequent causes of food contamination leading to salmonellosis in humans (Gustavsson et al., 2015).On the other hand, S. pullorum caused a significant problem in commerciel egg and poultry production (Foley et al., 2011).Salmonella control within farms that breed and rear poultry is a significant public health issue (Dhanani et al., 2015).
Poultry vaccination is one promising strategy to mitigate Salmonella infection in poultry and, in turn, humans as well (Okamura et al., 2012).A variety of Salmonella strains are known to cause extraintestinal infections in poultry (Johnson et al., 2010).Salmonella can infect humans and are the main causes of food contamination (Gallati et al., 2013).Although these infections do not cause severe symptoms in poultry, the eggs and meat of infected animals can become a reservoir for infection of human consumers (Elsheimer-Matulova et al., 2015).These a symptomatic bird carriers play a major role in Salmonella propagation and in food contamination.Salmonella infect chickens via the fecal-oral route, colonize the alimentary tract, invade internal organs such as the liver and spleen, and finally spread to the reproductive tract.The S. enteritidis bacteria can be transmitted to the eggs, which can be transmitted to humans through consumption of these contaminated eggs, which is a major public health issue (Lee, 2015).
The vaccines are targeted for the most often reported serovars of human infections namely S. enteritidis and S. typhimurium.Vaccination has a limited effect on improving animal health and welfare and is used primarily for public health reasons.Vaccines can decrease public health risk caused by Salmonella in poultry products by reducing the colonisation of reproductive tissues as well as reducing faecal shedding (Jawale and Lee, 2014).
Cross-protection can enhance the clearance of pathogens through the acquired immune response.Several live vaccine strains have conferred high levels of protection against infection with heterologous serovars.A vaccine constructed for a single serovar of Salmonella may induce immunity against other heterologous serovars of Salmonella (Nandre et al., 2015).It has been demonstrated that Salmonella live vaccines can elicit cross immunity against members of the same Kauffmann-White scheme serogroup (Chacana and Terzolo, 2006).The objective of this study was to assess the potential of cross-reaction of serum in S. enteritidisvaccinated chicken to some serotypes of S. enterica.

Isolate Vaccine and Vaccination
The vaccine strain used in this study is commercial vaccine of S. enteritidis strain Sm24/Rif12/Ssq (AviPro®, PT.Lohmann Animal, Health Indonesia).Four female, Isa Brown layer chickens (20 weeks old), were used in this study.The chickens were reared in individual cage (40 x 40 x 80 cm) at 22°C with free access to water and feed.
The antibody production technique in chickens refer to Wibawan et al. (2010).Chickens were vaccinated in the first week with 1 mL live attenuated S. enteritidis vaccine (10 9 cfu/mL, intra vena).The vaccination was repeated in the second week and the third week with 1 mL antigen suspension (three times, intra vena) respectively.One week after the last vaccination, the serum was collected.

Preparation of Dissolved Antigen for Cross-Reaction Test
The bacteria used for the cross-reaction test are the antigen vaccine S. enteritidis strain Sm24/Rif12/Ssq, S. enteritidis ATCC 13076, S. enteritidis BCC B2691 (collection of The Indonesian Research Centre for Veterinary Science), S. enteritidis, S. typhimurium, S. pullorum (collection of laboratory of Microbiology, Faculty of Veterinary Medicine, Bogor Agricultural University), S. typhi ATCC 35250, and E. coli ATCC 25250.

Wyanda Arnafia, et al
Jurnal Veteriner complex, thus it can be seen by the eye.The presence of precipitation lines between serum well with antigen wells indicates that the serum contains immunoglobulins which are specific to the antigen tested.Table 1 presents the results of cross-reaction test of serum in chickens vaccinated with the commercial vaccine S. enteritidis strain Sm24/Rif12/SSQ to some bacteria.
Vaccination is likely to have a central role in the reduction of Salmonella in poultry, because research studies indicate that it may reduce both the horizontal and vertical transmission of Salmonella.Vaccination works by reducing the prevalence of Salmonella or by increasing the passive immunity and blocking the horizontal transmission of Salmonella in poultry (Dórea et al., 2010).The isolates used as a master seed vaccine must have the ability to induce the formation of antibodies against the disease agent contained on field.This study uses some antigens S. enteritidis to test the ability of antibodies produced by vaccinated chickens to recognize such types of S. enteritidis isolates.S. enteritidis BCC and S. enteritidis collection of laboratory of Microbiology, Faculty of Veterinary Medicine, Bogor Agricultural University are the on-field isolates of S. enteritidis in Indonesia.The AGID test results showed positive results on all isolates of S. enteritidis used in this study.This indicates that the serum produced by chickens vaccinated with S. enteritidis strain Sm24/Rif12/Ssq contains antibodies capable of recognizing all S. enteritidis isolates used in the study.The serum contains the antibodies specific to the on-The soluble antigen was produced by an extraction technique using HCl.Extraction using HCl used for extract surface antigens.Enteribactericeae have complex surface antigens.The main surface antigens are somatic antigens (O antigen), flagellar antigens (H antigen), and envelope or capsule antigens (Vi antigen).The antigenic structure is used to differentiate organism within a genus, species, or serotype.Technique HCl extraction based on that of Abrar et al. (2012) with minor modifications.All isolates were inoculated in 50 mL brain heart infussion broth and incubated at 37°C for 18-24 hours.Bacteria was harvested with centrifuge 10.000×g for 15 minutes.The supernatant was disposed, the pellet was added with 0.35 mL of 0.2 N HCl, one drop of phenol red, and one drop of 1 N NaOH.This suspension was heated on waterbath at 56°C for two hours.Suspension will centrifuge and the supernatant will use as a dissolved antigen.

Agar Gel Immunodiffusion Test
Cross-reaction tests done by using the agar gel immunodiffusion test (AGID).Soluble antigens of all selected bacterial isolates were used in this test.A total of 0.5 g of agarose and 0.05 g of sodium azide were dissolved into 25 mL of phosphate-buffered saline (PBS) and 25 aqua bidest.The mixture was heated and poured on a glass object up to 4 mL, then cooled, and hexagonal holes were made with a diameter of 4 mm, depth of 3-4 mm, and the distance between holes of 2 mm.A total of 25 µL of serum resulted from vaccination was put into in the hole located in the center, and the other holes were filled with soluble antigen of each bacteria in the same volume.Gel was incubated at 25°C in a moist state for 18-24 hours.Cross-reactions were observed for the visible precipitation lines between the serum hole and the isolate hole.

RESULTS AND DISCUSSION
Cross-reaction tests were performed on the serum produced by the chickens vaccinated with the commercial vaccine S. enteritidis.A total of nine bacterial antigens were used in the crossreaction tests.These tests were carried out using the agar gel immunodiffusion (AGID) technique.Positive results were characterized by the formation of precipitation lines between serum well with antigen wells (Figure 1).The precipitation line is a visualized antigen-antibody It has been demonstrated that Salmonella live vaccines can elicit cross immunity against members of the same Kauffmann-White scheme serogroup (Chacana and Terzolo, 2006).The S. enteritidis, S. pullorum, and S. typhi belong to the serogroup of D1 in Kauffmann scheme (Grimont and Weill, 2007).The D1 serogroup is composed of those serotypes having the somatic antigen O9.This study found the presence of precipitation lines between the serum in S. enteritidis strain Sm24/Rif12/Ssq-vaccinated chickens with the antigens of S. pullorum and S. typhi.This is because the serum contains specific immunoglobulins that recognize the antigen section similar to that of the vaccine isolates.This antigen is likely to be the somatic antigen O9.The S. pullorum has similarities in all somatic antigen O with S. enteritidis, namely, somatic antigens O1, O9, and O12.This indicates that the formation of precipitation line between the serum and the S. pullorum antigen is influenced by not only the similarity of somatic antigen O9 but also the similarity of somatic antigen O1 and O12.The same thing happened between the serum with the S. typhi antigen.The S. typhi and S. enteritidis had similarity not only in the somatic antigen O9 but also in the somatic antigen O12.The field S. enteritidis isolates of Indonesia.The presence of specific antibodies against the onfield isolates can hamper commercial poultry infections on field.This implies that the vaccine is expected for an effective use in poultry farms in Indonesia.
Live Salmonella vaccines have been reported to induce cross-immunity against related serovars (Matulova et al., 2013;Nandre et al., 2015;Lee, 2015).Cross-reactions test of the serum in S. enteritidis strain Sm24/Rif12/Ssqvaccinated chickens showed positive results when they were reacted with the antigens of S. typhimurium.This is consistent with the study by Nandre et al. (2015).Nandre et al. (2015) reported that vaccination with an S. enteritidis strain induced significantly higher levels of systemic IgG and mucosal sIgA antibodies specific for S. typhimurium.
Based on Kauffmann-White scheme, S. enteritidis and S. typhimurium have similar somatic antigens of O1 and O12 (Grimont and Weill, 2007).The formation of precipitation lines between the serum in S. enteritidis strain Sm24/Rif12/Ssq-vaccinated chickens with the S. typhimurium antigen showed compatibility between immunoglobulin contained in serum and its specific antigen.This is because the serum recognizes the specific somatic antigens of O1 and O12 on S. typhimurium antigen that  The test results showed that chickens vaccination using a live vaccine S. enteritidis could induce humoral immunity that recognize not only S. enteritidis, but also some other serotypes of S. enterica, namely S. typhimurium, S. pullorum and S. typhi.Systemic antibodies are essential to kill Salmonella bacteria, which escape from infected cells to reach distant tissue sites and form new foci of infection (Dougan et al., 2011).The ability of systemic antibodies to recognize some strains of Salmonella is expected to inhibit the infection by these strains.This can increase the efficient use of the vaccine.The potential cross-reaction of the serum from chickens vaccinated with S. enteritidis to some serotypes of S. enterica is expected to play a role in reducing the infection by the serotypes.
The serum of S. enteritidis-vaccinated chickens could not react with the E. coli antigen.Cross-reaction testing for E. coli was done to see the presence of immunoglobulin specific to the bacteria included in the Enterobactericeae family.The negative result of the cross reaction test for E. coli indicates that the serum does not contain the immunoglobulin specific to this bacteria.Enterobactericeae have complex surface antigens.The main surface antigens are somatic antigens (O antigen), flagellar antigens (H antigen), and envelope or capsule antigens (Vi antigen).The antigenic structure is used to differentiate organism within a genus, species, or serotype.The difference of surface antigens of Salmonella and E. coli resulted in specific antibodies against Salmonella do not recognize surface antigens of E. coli.This implies that the use of S. enteritidis vaccines could not induce the formation of antibodies specific to other Enterobactericeae, particularly E. coli, so it could not inhibit the infection by this bacteria.

CONCLUSION
The serum in S. enteritidis strain Sm24/ Rif12/Ssq-vaccinated chickens can react with some on-field S. enteritidis isolates of Indonesia.It can recognize some serotypes of S. enterica such as S. pullorum, S. typhimurium, and S. typhi, but cannot react with other Enterobactericeae bacteria, especially E. coli.S. enteritidis vaccine is potential to be used against several serotypes of S. enterica.

SUGGESTION
The serum in S. enteritidis strain Sm24/ Rif12/Ssq-vaccinated chickens can react with some serotypes of S. enterica such as S. enteritidis, S. pullorum, S. typhimurium, and S. typhi.This vaccine can be used to against that bacteria in poultry farm.