Effect of egg powder containing anti- salmonella enteritidis igy on the excretion of the se micro-organism in broiler chickens
Published: October 20, 2011
By: PA Chacana1*, ML Cigoy2, E Bisesti3, G Sigal3, V Parreño1, HR Terzolo2, A Wigdorovitz1 - 1Institute of Virology, CICVyA, INTA, Argentina; 2EEA Balcarce, INTA, Argentina; 3Vetanco S.A., Argentina
Summary
Paratyphoid caused by Salmonella Enteritidis (SE) is an important problem in both poultry industry and public health. Control of the pathogen includes several strategies: implementation of management and biosecurity measures, vaccination, restoration of normal competitive flora, etc. A supplementary alternative for the control of paratyphoid is supplementation of balanced food with antibodies against SE. In this work, 1-day-old broiler chickens were fed with egg powder enriched with IgY anti-SE. Thereafter, they were challenged with SE. Excretion levels of SE were significantly lower on the 6th day post-challenge in chicks fed hyper-immune egg powder. These results suggest that supplementation with hyper-immune egg powder should be considered as an effective supplementary strategy to control SE in poultry.
Key words: Salmonella Enteritidis, Broilers, Egg powder.
Introduction
Paratyphoid in farm animals is a major problem worldwide, not only because of the direct economic losses caused by mortality of young birds, which varies according to serotype variety involved, and the health state and maternal immunity of birds, but also, primarily by causing a decrease in weight gain and increased health and prevention costs. On the other hand, feces of infected animals cause a wide spread of the bacteria due to pollution of soil, water, plants, tools and livestock facilities, etc (Suárez & Mantilla, 2000). Some birds may have the disease in asymptomatic form, which contributes to transmission to humans through the processing of apparently healthy carrier birds in food processing of poultry origin that are thus contaminated, either directly or indirectly, in slaughter plants (Sandoval et al., 1989; Guard-Petter, 2001; Cogan & Humphrey, 2003; Terzolo, 2006). Globalization, economic openness and growth in the poultry industry have increased consumption and distribution of the chicken, eggs and their byproducts and, therefore, have also increased the risk salmonellosis transmission caused by S. Enteritidis (SE), so that the control of this zoonosis is becoming increasingly important (Suárez & Mantilla, 2000; SAGPyA, 2009). In recent years, the use of antimicrobials and inactivated and attenuated vaccines to limit or prevent Salmonella spp. Infection in veterinary medicine has been questioned, due to the development of bacterial resistance to antibiotics, as well to the potential risks posed by residues of antibiotics, and vaccines adjuvants in food products derived from animals for human consumption (Kramer et al., 2001, Hasenstein et al., 2006). For these reasons, the use of these agents has been gradually restricted and other tools for the control of this disease, such as competitive exclusion through administration of prebiotics have been explored (Edens et al., 1997; Rahimi et al., 2007). Thus, the IgY technology is a supplementary tool for the prevention of poultry diseases and the generation of products and food by-products with higher microbiological safety. This biotechnology is based on the use of egg yolk immunoglobulin (IgY), which can be produced by immunization of chickens with different antigens (Chacana et al., 2004). Various trials have linked IgY with Salmonella. For example, it was demonstrated that IgY could be used for classification of the different serotypes of this micro-organism (Terzolo et al., 1998), and the potential of specific IgY for the control of salmonella in poultry has also been evaluated. Lee et al. (2002) conducted a cross reaction study between IgY produced against SE and Salmonella Typhimurium (ST). After hyper-immunizing different chickens with both inactivated bacteria, these researchers developed hyper-immune egg powder through liophilization of the water-soluble portion of the egg yolk. Through specific ELISA tests for both serotypes, it was demonstrated that the antibodies produced against SE showed cross-reaction against ST in 55.3% and, conversely, the IgY produced against ST reacted against SE in 42.4%. In addition to this, these authors demonstrated that the incubation of SE and ST together with antibodies significantly reduced the development of these organisms in liquid culture media. These results indicate the need to evaluate the protective ability of specific IgY in laboratory tests and in experimental infection in birds, using products that contain specific immunoglobulin and may be easily scalable to the industrial development of additives and nutritional supplements. For this reason, the objective of this work was the evaluation of the effect of administering egg powder of hyper-immunized chickens against SE on the excretion of bacteria in an experimental infection trial performed on broiler chickens.
Materials & Methods
Egg Powder
Complete egg powder (egg yolk and white), made from eggs of hyper-immunized hens (hyper-immune egg powder) with a bacterium against the regional SE INTA 86/360 strain, fagotype 4 (Sandoval et al., 1989) . Furthermore, egg powder made from eggs of non-hyper-immunized hens (control egg powder) was used. The title of anti-SE agglutinative antibodies in egg powder was determined through on-plate micro-agglutination of 96 U-bottom bowls. Serial dilutions in log2 base were made of both the hyper-immune egg powder and the control egg powder.
Egg Powder
Complete egg powder (egg yolk and white), made from eggs of hyper-immunized hens (hyper-immune egg powder) with a bacterium against the regional SE INTA 86/360 strain, fagotype 4 (Sandoval et al., 1989) . Furthermore, egg powder made from eggs of non-hyper-immunized hens (control egg powder) was used. The title of anti-SE agglutinative antibodies in egg powder was determined through on-plate micro-agglutination of 96 U-bottom bowls. Serial dilutions in log2 base were made of both the hyper-immune egg powder and the control egg powder.
Preparation of food with the addition of egg powder
Food supplemented with 10% (p/p) of hyper-immune egg powder or 10% (p/p) of control egg powder was prepared. The food was mixed with the egg powder to ensure proper homogenization.
Food supplemented with 10% (p/p) of hyper-immune egg powder or 10% (p/p) of control egg powder was prepared. The food was mixed with the egg powder to ensure proper homogenization.
Experimental Challenge of Broiler Chicks
Strain used. The SE INTA 1222 strain, isolated from a case of avian paratyphosis, was used to implement the experimental infection model.
Birds. 56 one-day-old broiler chicks (508 Cobb-Vantress line) provided by the company Toledo S.A., Mar del Plata, province of Buenos Aires were used. These birds came from 28-week-old heavy breeders. The chicks were housed in the challenge room of the EEA Balcarce, INTA bird vivarium. Upon arrival, a sampling of all birds´ meconium was performed to verify the absence of Salmonella spp. During the entire experimental period, animals access to water and food ad libitum. The birds were fed a Salmonella-free commercial feed, made without animal products and free of aflatoxins (Tapia, 1985). The absence of Salmonella spp. in drinking water and food was verified by means of selective enrichment cultures for Salmonella spp in tetrathionate broth additioned with bright green 0.1% v/v, and lactose broth, respectively. For subcultures, xylose-lysine-deoxycholate agar plates, with the addition of 4 tergitol at 0.46% v/v (XLDT) were used.
Strain used. The SE INTA 1222 strain, isolated from a case of avian paratyphosis, was used to implement the experimental infection model.
Birds. 56 one-day-old broiler chicks (508 Cobb-Vantress line) provided by the company Toledo S.A., Mar del Plata, province of Buenos Aires were used. These birds came from 28-week-old heavy breeders. The chicks were housed in the challenge room of the EEA Balcarce, INTA bird vivarium. Upon arrival, a sampling of all birds´ meconium was performed to verify the absence of Salmonella spp. During the entire experimental period, animals access to water and food ad libitum. The birds were fed a Salmonella-free commercial feed, made without animal products and free of aflatoxins (Tapia, 1985). The absence of Salmonella spp. in drinking water and food was verified by means of selective enrichment cultures for Salmonella spp in tetrathionate broth additioned with bright green 0.1% v/v, and lactose broth, respectively. For subcultures, xylose-lysine-deoxycholate agar plates, with the addition of 4 tergitol at 0.46% v/v (XLDT) were used.
Treatments
The broiler chicks were divided into three groups of 20 chicks each. Form the first day of life and for 14 days the chicks were fed: 1) conventional feed; ((2) feed supplemented with 10% w/w of hyper-immune egg powder; 3) feed supplemented with control egg powder. All birds were slaughtered by cervical dislocation of the occipitoatloid joint after 15 days of life.
The broiler chicks were divided into three groups of 20 chicks each. Form the first day of life and for 14 days the chicks were fed: 1) conventional feed; ((2) feed supplemented with 10% w/w of hyper-immune egg powder; 3) feed supplemented with control egg powder. All birds were slaughtered by cervical dislocation of the occipitoatloid joint after 15 days of life.
Experimental infection
On the 5th day of life the chicks from each group were individually challenged with 105 CFU/bird at SE INTA 1222, fagotype 4, by ingluvial catheter.
On the 5th day of life the chicks from each group were individually challenged with 105 CFU/bird at SE INTA 1222, fagotype 4, by ingluvial catheter.
Sample collection
Cloacal swab samples of all the chicks in the experimental groups were taken on days 3 and 6 post-infection (PI). Each of the samples was incubated in tetrathioned both for 48 hours at 37 ° C. Then, an aliquot of each of the broths was taken and planted on XLDT agar plates. The plates were incubated for 24 hours at 37 ° C. Finally, the presence of colonies with typical Salmonella features was observed; primary identification thereof was confirmed by means of the urea metabolism biochemical test to differentiate them from other potential microorganisms with macroscopic characteristics similar to Salmonella. The percentage difference in the proportion of chicks infected among the different experimental groups was statistically analyzed using the Chi-square contingency test. P values of less than 0.05 were considered significant.
Cloacal swab samples of all the chicks in the experimental groups were taken on days 3 and 6 post-infection (PI). Each of the samples was incubated in tetrathioned both for 48 hours at 37 ° C. Then, an aliquot of each of the broths was taken and planted on XLDT agar plates. The plates were incubated for 24 hours at 37 ° C. Finally, the presence of colonies with typical Salmonella features was observed; primary identification thereof was confirmed by means of the urea metabolism biochemical test to differentiate them from other potential microorganisms with macroscopic characteristics similar to Salmonella. The percentage difference in the proportion of chicks infected among the different experimental groups was statistically analyzed using the Chi-square contingency test. P values of less than 0.05 were considered significant.
Results and Discussion
Title of anti-SE antibodies in the egg powder. The title of anti-SE antibodies determined by micro-agglutination in the hyper-immune egg powder was 1: 640, while the control egg powder presented titels below 1: 10. Therefore, the birds fed with food supplemented with hyper-immune egg powder a level of specific antibodies 64 times higher that the birds that received control egg powder as feed supplement.
Excretion of SE. On day 3 IP there were no significant differences among the different groups of birds, and the percentages of isolation of SE from the cloacal swab cultures in the group supplemented with control egg powder and the group that received feed without the addition of egg powder, were both very low. The inoculated Salmonella could be re-isolated only from some of the birds examined in these groups. In the group of chicks that received food supplemented with hyper-immune egg powder, on the contrary, the bacterium could not be isolated in any of the birds (Table 1). The low rates of excretion of SE in the untreated groups during the 3rd day PI are as expected, according to the infection model used. On the 6th day PI administration of food supplemented with hyper-immune egg powder managed to decrease in 30% the excretion of SE, compared to birds that received only conventional food. This significant decrease was due to the presence of anti-SE IgY antibodies in the hyper-immune egg powder.
Table 1. Number of isolates (Positive/Total) of SE 1222 on days 3 and 6 post-infection from cloacal swab cultures of groups of chicks fed conventional food, food supplemented with 10% w/w of control egg powder or food supplemented with 10% w/w of hyper-immune egg powder.
|
Days post
infection
|
Excretion of Salmonella according to treatment
(No. of Positive / Total number) |
||
|
Conventional feed (without addition)
|
Feed with control egg powder
|
Feed with anti-SE egg powder
|
|
|
3
|
3/20 a
|
4/19 a
|
0/17 a
|
|
6
|
17/20 a
|
15/19 a,b
|
10/18 b
|
a, b Different letters in the same row indicate significant differences.
Excretion of SE in birds that received food added with control egg powder was only 6% lower, compared to excretion of SE in the group of birds fed only conventional food. The low title of anti-SE IgY present in the food supplemented with control egg powder (1: 10) would not be sufficient to achieve a significant reduction in the excretion of SE. For excretion of SE to decrease significantly, the feed consumed by the birds must be supplemented with egg powder with a high title of anti-SE IgY-, as confirmed by the use of hyper-immune egg powder.
Conclusions
Supplementation of bird feed with egg powder obtained from hens hyper-immunized with SE considerably decreased excretion of SE after the experimental challenge. This is due to the presence of specific anti-SE antibodies in the hyper-immune egg powder. The results are encouraging for the use of hyper-immune egg powder as a food additive, thus, reducing the excretion of ES and, consequently, environmental pollution by reducing the re-infection cycle on the farm and the chances of contamination of food products and by-products intended for human consumption. Therefore, based on these results, further trials will be performed on broiler chicken during the 7 days prior to slaughter, in order to determine if the addition of hyper-immune egg powder is effective to reduce the excretion of ES during this period.
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