Cross agglutination of egg yolk anti-salmonella enteritidis antibodies with other serovars of salmonella enterica

The egg yolk antibodies (IgY) have been successfully applied in the design of diagnostic systems and products for the prevention and treatment of different diseases, both human and veterinary (Chacana et al., 2004, Schade & Chacana, 2007). For example, the IgY technology has been successfully employed in studies for the control of neonatal diarrhea, human dental caries, gastric ulcer caused by Helicobacter pylori, fish diseases, inhibition of the development of Salmonella enterica, etc. (Schade et al., 2005).

Salmonella enterica encompasses a wide variety of serovars (over 2,000), that according to the Kauffmann-White scheme are classified by their somatic and flagellar antigens. Under this scheme, different serovars are classified into groups and subgroups, based on the occurrence of common antigens (Grimont & Weill, 2007). For example, Lee et al. (2002) conducted a cross-reactivity study between IgY produced against Salmonella Enteritidis (SE) (antigenic formula: 9,12: g: m) and S. Typhimurium (ST) (antigenic formula 4,12: i: 1,2) . After different laying hens were hyperimmunized with both inactivated bacteria, these researchers produced hyperimmune egg powder by freeze-drying the water soluble fraction of the egg yolk. By specific ELISA tests for both serovars, it was shown that antibodies produced against SE cross-reacted against ST by 55.3% and, conversely, IgY produced against ST reacted against SE by 42.4%. In addition, the authors showed that incubation of SE and ST together with antibodies significantly decreased the development of these microorganisms in liquid culture media. This work demonstrates that, although ST and SE do not have high antigenic identity according to the Kauffmann and White scheme, they share other antigens and epitopes characteristic of the species and that antibodies directed against them have the ability to cross-neutralize the different serovars. On the other hand, Terzolo et al. (1998) demonstrated the ability of egg yolk antibodies to bind to Salmonella and its potential to be used as an alternative to mammalian antibodies for serotyping. Based on this, the aim of this study was to evaluate the binding capacity of egg powder produced against SE to bind different serovars of S. enterica.

We used whole egg powder (yolk and white), prepared from eggs of hyperimmunized laying hens (hyperimmune egg powder) with a bacterin against Salmonella Enteritidis regional strain INTA 86/360 phage type 4 (Sandoval et al., 1989) . In addition, egg powder produced from not hyperimmunized hen eggs (control egg powder) was used.

Different strains of Salmonella enterica isolated from different veterinary cases were used. The serovars included were: S. Braenderup, S. Typhimurium, S. Gallinarum, S. Infantis, S. Agona, S. Newport and S. Muenster.

The different strains of salmonella were sown on agar XLDT₄ and after incubation at 37°C for 24 h, growth was used to inoculate brain-heart infusion broth, which was incubated under the same conditions above. Broth was inactivated with 0.05% formalin (v/v) and finally the bacterial concentration was adjusted to 10⁸ bacteria/mL as per the turbidity of McFarland nephelometric scale. The antigens were stained using 0.01% (v/v) crystal violet

The binding antibody titer of egg powder against each of the antigens was determined by microagglutination in 96-well "U" bottom plate. Serial dilutions were made ​​based on log₂, both for the hyperimmune egg powder an the control egg powder. The titer was determined as the inverse of the highest dilution with binding ability.

Table 1 shows the binding antibody titers in control and hyperimmune egg powder against SE. In all cases, the control egg powder binding titers were not higher than 40. The presence of agglutinating antibodies in eggs derived from not hyperimmunized hens can be due to both nonspecific reactions or antibodies directed against other microorganisms with epitopes in common  with Salmonella spp. Agglutinating antibody titers obtained from anti-SE egg powder varied according to different serovars. Higher titers were obtained when egg powder was faced with different strains of S. Enteritidis tested. Egg powder had the ability to bind even serovars that do not show antigens in common with SE, according to the Kauffmann-White scheme (Grimont & Weill, 2007). For example, titer against S. Muenster, S. Branderup and S. Newport was of 640.

Table 1. Agglutination titres of anti-Salmonella Enteritidis control and hyperimmune egg powder against different serovars of Salmonella. The titer was determined as the reciprocal of the highest dilution with binding ability.

The powdered eggs produced from hyperimmunized hens with Salmonella Enteritidis shows binding capacity against both the Salmonella strains included in the vaccine and against other serovars of Salmonella who do not share common antigens.

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