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Passive Immunity of Progeny from Broiler Breeders Vaccinated with Oil-EmulsionBacterin Against 
Salmonella 
Enteritidis
 A. Y. Inoue,
 AC
 A. Berchieri Jr.,
B
 A. Bernardino,
 A 
 J. B. Paiva,
B
and E. V. Sterzo
B
 A 
Fort Dodge Animal Health, Rua Luiz Fernando Rodriguez, 1701, CEP 13064-798, Campinas, Sa ˜o Paulo, Brazil
B
Department of Veterinary Pathology, Avian Pathology Laboratory, Sa ˜o Paulo State University, Jaboticabal, Sa ˜o Paulo, BrazilReceived 4 March 2008; Accepted and published ahead of print 6 June 2008SUMMARY. Young poultry are very susceptible to
Salmonella 
Enteritidis (SE) infections because of the absence of completeintestinal flora colonization and an immature immune system. This study evaluated the role of passive immunity on the resistanceof young birds against early infections caused by SE. The progeny of broiler breeders vaccinated with an oil-emulsion bacterin wascompared to the progeny of unvaccinated birds. Efficacy was determined by challenging birds at 1 and 14 days of age with SE NalSpc strain, phage type 4. After challenge at 1 day of age, the progeny of vaccinated birds presented a significantly lower number(log 
10
) of SE Nal Spc reisolation (
P 
,
0.05) in liver (2.21), spleen (2.31), and cecal contents (2.85) compared with control groups(2.76, 3.02, and 6.03, respectively). The examination of the internal organs, 3 days after infection, revealed that 28% of the birds(7/25) from vaccinated breeders were positive, whereas 100% (25/25) of the chicks derived from unvaccinated birds were positive.Birds challenged at 14 days of age presented a lower number of positive samples compared with those challenged at 1 day of age,and the progeny of vaccinated birds presented statistically lower numbers (log 
10
) of colony-forming units/ml of SE Nal Spc only inthe cecal contents compared with nonvaccinated breeder progeny (2.11
vs 
. 2.94). Age seems to influence the susceptibility of birdsto SE infections: in control groups, the number of positive birds at 14 days of age (9/25) was lower when compared with the groupinfected at 1 day of age (25/25). The number of positive fecal samples of the progeny of vaccinated birds was significantly lower(36) than those of the control group (108) after challenge at 1 day of age. Unchallenged progeny of vaccinated birds presentedpassive antibodies detectable by enzyme-linked immunosorbent assay (ELISA) up to 21 days of age. On the other hand, antibodiesof the control group were detected by ELISA 14 days after challenge. These results show a significant contribution of breedervaccination by increasing the resistance of the progeny against early SE infections. However, the bacteria were not completely eliminated, suggesting that additional procedures are needed to effectively control SE infections.RESUMEN. Inmunidad pasiva de la progenie de reproductoras pesadas vacunadas con una bacterina emulsionada en aceitecontra 
Salmonella 
Enteritidis.Las aves jo´venes son muy susceptibles a las infecciones con
Salmonella 
Enteritidis debido a la ausencia de una colonizacio´n completa por parte de la flora intestinal y a la inmadurez de su sistema immune. Este estudio evaluo´el papel de la inmunidad pasiva en la resistencia de aves jo´venes contra las infecciones tempranas causadas por
Salmonella 
Enteritidis. La progenie de reproductoras pesadasvacunadas con una bacterina emulsionada en aceite fue comparada con la progenie de aves no vacunadas. La eficacia fue determinada por el desafı´o con la cepa de
Salmonella 
Enteritidis Nal Spec, fagotipo 4. Despue´s del desafı´o al dı´a de edad, la progenie de avesvacunadas presento´un nu´mero significantemente (
P 
,
0.05) menor (log 
10
) de reaislamientos de
Salmonella 
Enteritidis Nal Spec enhı´gado(2.21),bazo(2.31)ycontenidocecal(2.85)cuandose comparo´con losresultados obtenidosparaelgrupocontrol(2.76,3.02 y 6.03, respectivamente). La evaluacio´n de los o´rganos internos 3 dı´as despue´s de la infeccio´n, revelo´que el 28% de las aves (7/25)provenientes de reproductoras vacunadas fueron positivas, mientras que el 100% (25/25) de las provenientes de reproductoras novacunadas fueron positivas. Las aves desafiadas a los 14 dı´as de edad presentaron un nu´mero menor de muestras positivas comparadocon aquellas desafiadas al dı´a de edad, y la progenie de aves vacunadas presento´valores estadı´sticamente menores (log 
10
) de UnidadesFormadoras de Colonia/ml de
Salmonella 
Enteritidis Nal Spec, u´nicamente en el contenido cecal, comparados con los obtenidos en la progenie de reproductoras no vacunadas (2.11 V 
s 
. 2.94). Aparentemente la edad influyo´en la susceptibilidad de las aves a la infeccio´ncon
Salmonella 
Enteritidis: en los grupos control, el nu´mero de aves positivas a los 14 dı´as de edad (9/25) fue menor cuando secomparo´con el grupo infectado al dı´a de edad (25/25). El nu´mero de muestras provenientes de heces positivas de la progenie de avesvacunadas fue significativamente menor (36) que el originado en las del grupo control (108) despue´s del desafı´o al dı´a de edad. La progenie no desafiada de aves vacunadas presento´anticuerpos maternos detectables por medio del inmunoensayo asociado a enzimas(ELISA) hasta los 21 dı´as de edad. De otra parte, los anticuerpos del grupo control fueron detectados por la prueba ELISA 14 dı´asdespue´s del desafı´o. Estos resultados muestran una contribucio´n significante de la vacunacio´n al incrementar la resistencia de la progenie contra infecciones tempranas con
Salmonella 
Enteritidis. Sin embargo, la bacteria no fue completamente eliminada,sugiriendo que se requieren procedimientos adicionales para el control de la infeccio´n.Key words: passive immunity,
Salmonella 
Enteritidis, vaccination, broiler breeder Abbreviations: CFU
5
colony-forming units; dpi
5
days postinfection; ELISA 
5
enzyme linked immunosorbent assay;Ig 
5
immunoglobulin; Nal
5
nalidixic acid; SE
5
Salmonella 
Enteritidis; Spc
5
spectinomycin
Salmonella 
infection is considered one of the most importantbacterial diseases in domestic chickens and turkeys. Among many serotypes of 
Salmonella 
,
Salmonella 
Enteritidis (SE) has beendescribed as the most prevalent in commercial broilers and layers(29). SE is associated with illness in young birds and with foodbornediseases in humans (11).Vertical transmission and early exposure to SE are the main causesof clinical diseases because of the high susceptibility of newly hatched birds to infections. Intestinal microflora colonization andmature immune systems provide older birds resistance against theeffects of SE, lowering the morbidity and mortality (13). Birds
C
Corresponding author. E-mail: inouea@fdah.com
 AVIAN DISEASES 52:567–571, 2008
567
 
challenged at 1 day of age can remain infected and shed SE untilmaturity (12).The benefits of passive immunity in reducing the damages causedby early exposure to infectious agents have been studied for many poultry diseases (8,10,26). Maternal antibodies from breeder’s bloodcan be transferred through the egg yolk as immunoglobulin G (IgG;15,24). It is also reported that other immunoglobulins, like IgA andIgM, are also secreted in reproductive tract of vaccinated birds (25).These immunoglobulins can be transferred to the eggs during theirpassage through the oviduct. After absorption, the antibodies arediffused in amniotic liquid and are ingested by the embryo (7). Incommercial poultry industry, the importance of passive immunity against SE is also related to the ability of the bacteria to infect largenumber of birds at the hatchery, where the temperature andmoisture are favorable to proliferation (5,20).In the present study, we investigated the protective effects of maternal antibodies on the progeny of breeders vaccinated with anSE bacterin in oil emulsion. In addition to the effects on bacterialcounts in the organs of challenged birds, we assessed other aspects,such as fecal shedding and serology using an enzyme-linkedimmunosorbent assay (ELISA).
MATERIALS AND METHODSBirds.
Day-old broilers chicks (Cobb-Vantress, Siloam Springs, AR)from two different commercial hatcheries (Sa ˜o Jose´Farm, and Pena Branca Company, Sa ˜o Paulo, Brazil) were used in the study. The chickswere divided into four groups: groups 1 and 3 were progeny of broilerbreeders vaccinated with an inactivated SE vaccine (POULVAC SE; FortDodge Animal Health, Overland Park, KS) at 12 and 20 wk old.Groups 2 and 4 were chicks from unvaccinated breeders. The chicksused in this study were from breeders between 38 and 40 wk old.The birds were weighed (40
6
1 g), then reared in isolation unitsmaintained with filtered air under negative pressure (Fort DodgeResearch Center, Paulinia, Sa ˜o Paulo, Brazil). All birds were fed andwatered
ad libitum
. At arrival, 20 birds from each group were assessedaccording to Waltman
et al.
(30), to ensure that the newly hatchedchicks were free of 
Salmonella
. The samples (drag swabs, liver, spleen,heart, and ceca) were examined using selenite broth (CM0395 andLP0121A, Oxoid, Ogdensburg, NY) for 24 hr at 37 C and plated ontobrilliant green agar (CM0263, Oxoid).
Salmonella 
Enteritidis strain.
A mutant strain of 
Salmonella
Enteritidis (SE), phage type 4, resistant to nalidixic acid (Nal) andspectinomycin, (Spc) from the Laboratory of Avian Pathology (Sa ˜oPaulo State University, Jaboticabal, Sa ˜o Paulo, Brazil) was used aschallenge. Bacterial cultures were prepared in 10 ml of nutrient broth at37 C for 24 hr under stirring. This mutant was named SE Nal Spc.
Experimental design.
Organ samples 
. One hundred chicks weredivided in four groups: birds from groups 1 and 2 were challenged at 1day of age (25 birds per group) and birds from groups 3 and 4challenged at 14 days of age (25 birds per group). Both groups werechallenged directly into the crop with different doses of SE NalSpc: groups 1 and 2, with 2.12
3
10
5
colony-forming units (CFU) perbird; groups 3 and 4, with 2.17
3
10
9
CFU per bird. The birds fromeach group were sacrificed and were examined 3 days after challenge.Samples of liver, spleen and ceca contents were analyzed individually torecover viable SE Nal Spc. The number of cells was estimated by plating 0.1 ml of the decimal dilutions on brilliant green agar containing sodium nalidixate (20
m 
g/ml) and novobiocin (1
m 
g/ml) (BGNalNov).
Fecal samples 
. Eighty chicks were divided into two different groups:birds from groups 1 and 2. Both groups were challenged directly into thecrop with 2.12
3
10
5
CFU of SE Nal Spc. Cloacal swabs were taken at1, 3, 6, 9, 13, 15, 20, 23, and 26 days postinfection (dpi). Cloacal swabswere cultured in selenite broth (CM0395 and LP0121A, Oxoid)containing novobiocin (40 mg/liter) or were directly plated onBGNalNov. The samples were incubated at 37 C for 24 hr.
 Antibody detection
. Sixty chicks were divided into three groups: 20birds from group 1 unchallenged, 20 birds from group 1 challenged, and20 birds from group 2 challenged. Both groups were challenged dir-ectly into the crop with 2.12
3
10
5
CFU of SE Nal Spc at 1 day old.Serum samples were collected at 1, 7, 14, 21, and 28 days of age.Serum was diluted at 1:2 in phosphate-buffered saline solution andtested for the presence of specific antibodies by an ELISA (FlockChek SE, IDEXX Laboratories, Inc., Westbrook, ME). The results weremeasured in a Visible Plate Reader (650 nm, Biotrak version 1.3; Amersham Pharmacia Biotech, Uppsala, Sweden).
Statistical analysis.
Chi-square contingency tables were used tostatistically analyze fecal-shedding results. ANOVA was used tostatistically analyze viable SE log 
10
count in the organs (liver, spleen,and cecal contents).
RESULTS
 All the samples from day-old chicks, examined at arrival, hadnegative bacteriologic exams.
Organs samples.
Results of bacteriologic exams (Table 1) for allorgans showed a higher number of positive birds in the progeny from unvaccinated breeders (groups 2 and 4) compared with theprogeny of vaccinated birds (groups 1 and 3). The chicks that werechallenged at 14 days of age (groups 3 and 4) had fewer positivesamples than the groups challenged at 1 day old (groups 1 and 2).Comparing different organs, cecal contents were the site of greaterpositive samples at 14 days of age.Data in Table 2 show a statistically higher number of viable SENal Spc (
P 
,
0.05) in all samples (liver, spleen, and cecal contents)of birds from unvaccinated breeders (group 2) challenged at 1 day old compared with chicks from group 1. In chicks challenged at 14days old, the number of viable SE Nal Spc of the control group(group 4) was statistically higher (
P 
,
0.05) than group 3 only in thececal contents.
Fecal samples.
Table 3 shows that the birds from vaccinatedbreeders (group 1) presented a statistically lower number of positivecloacal swab samples (
P 
,
0.05) than the control group (group 2).Group 1 had fewer positive results than group 2 in direct evaluationof the samples and after culture in selenite broth. After 13 dpi, thenumber of positive samples was similar in both groups, and noshedding was observed after 20 dpi. In broilers derived fromvaccinated breeders, the peak of shedding occurred at 9 dpi, whereasthe peak was at 1 dpi in the control group (Fig. 1).
 Antibody detection.
Table 4 shows the differences in antibody detection in the three groups of birds. Birds from unvaccinatedbreeders (group 2) challenged at 1 day old had detectableseroconversion in the ELISA at 14 days old. More than 70% of the birds from vaccinated breeders (group 1) challenged at 1 day old
Table 1. Number of positive samples of liver, spleen, and cecalcontents for SE Nal Spc in birds challenged at one or 14 days of age, andexamined 3 days after infection.
Challenged at1 day of age
 A 
Challenged at14 days of age
B
Group 1 Group 2 Group 3 Group 4
Breeders Vaccinated Unvaccinated Vaccinated UnvaccinatedLiver 4/25 15/25 0/25 2/25Spleen 3/25 18/25 0/25 2/25Cecal contents 4/25 17/25 2/25 8/25Total positivebirds 7/25 25/25 2/25 9/25
 A 
Challenged with 2.12
3
10
5
CFU/ml.
B
Challenged with 2.17
3
10
9
CFU/ml.
568
A. Y. Inoue
et al.
 
had positive simples at 14 days of age, and 23.5% were positive at 28days. Nevertheless, the unchallenged group 1 had a faster decrease inpositive findings, with 47% of the birds positive at 14 days of age,but no positive samples at 28 days of age.
DISCUSSION
In groups of birds challenged at 1 day old, the results comparing the number of positive samples and the number of SE viable cellsshowed a significant reduction in both parameters for the groupfrom vaccinated breeders. These results are interesting, once thenumber of SE cells is related directly to the potential for damagecaused by SE infection (4).The ceca is the main site of SE bacterial multiplication in birds;spleen and liver are the most important nonintestinal organs wherethis bacterium is found (13). SE can be recovered in internal organs1 hr after experimental inoculation (18), and the highest levels of liver and spleen contamination are observed 3 days after infection(28). In the present study, in addition to the lower SE Nal Spc countin the cecal contents of progeny from immunized breeders, aspreviously described by Methner and Steinbach (24), there was lessSE reisolation in the liver and the spleen after challenge at 1 day of age. The passive immunity could be important in reducing thetransient depletion of lymphocytes in the lymphoid organs, whichpromote the development of carrier states in broilers that are exposedearly to
Salmonella 
, as described by Hassan and Curtis (14).Comparing the groups challenged at different ages (1 and 14days), it is clearly demonstrated that the absence of completeintestinal flora and immature immune systems play an importantrole in the higher susceptibility of young birds to
Salmonella 
infections (6,9,13). In the present study, the groups of chicksinfected at 1 day of age had a higher number of positive samples andhigher SE Nal Spc counts than the groups challenged at 14 days of age in progeny from vaccinated or unvaccinated breeders.It was also demonstrated that horizontal transmission andenvironmental contamination is higher in birds from unvaccinatedbreeders because of SE shedding after challenge. Vaccinated groupshad lower shedding rates throughout the study. Another importantresult is related to the age of fecal shedding after challenge: theprogeny of vaccinated breeders presented the highest numbers of positive samples on day 9 dpi, whereas, in progeny of unvaccinatedbreeders, the shedding peak was at 1 dpi. Birds infected at 1 day of age are important sources of infection because of intermittent SEshedding until they reach adulthood (12). In addition, chicken meatcross-contamination in poultry processing plants, caused by contact,can be increased by contaminated feces in carcasses (23). The controlof SE fecal shedding is equally important in layers becausecontaminated feces can soil the egg shell surface, and penetratethrough the egg (2). Delay in fecal shedding may be related to thepresence of maternal IgA in the intestinal tract. IgA in the intestinaltract has a significant role in immunity against
Salmonella 
because itinhibits bacterial adherence and colonization in the intestinalmucosa, which is the primary site of infection (22). Higher IgA titers in the progeny of vaccinated birds have been reported during the first week of age (15).The period of shedding lasted until 16 days of age for bothgroups, which is similar to that described by Bailey (1), althoughBerchieri
et al.
(3) demonstrated that passive immunity in chicksfrom infected breeders presented a longer shedding period after SEinfection than birds without maternal immunity. The shedding period and the numbers of infected bird can be influenced by theinfectious dose and by the strain used in the challenge (27).
Table 2. Log 
10
viable counts of SE Nal Spc/ml in liver, spleen, and cecal contents in birds challenged at 1 or 14 days of age, from breeders thatwere vaccinated or not vaccinated against SE; birds were examined 3 days after experimental infection.
Challenge at 1 day of age Challenge at 14 days of ageGroup 1 Group 2 Group 3 Group 4
Breeders Vaccinated Unvaccinated Vaccinated UnvaccinatedLiver 2.21 (
,
2.0–3.5)* 2.76 (
,
2.0–4.7)
,
2.0 (
,
2.0–
,
2.0) 2.02 (
,
2.0–2.30)Spleen 2.31 (
,
2.0–3.4)* 3.02 (
,
2.0–4.7)
,
2.0 (
,
2.0–
,
2.0) 2.13 (
,
2.0–4.1)Cecal contents 2.85 (
,
2.0–7.8)* 6.03 (
,
2.0–9.0) 2.08 (
,
2.0–4.0)* 2.94 (
,
2.0–7.0)*Values in the same row followed by an asterisk (*) are statistically different (
P 
,
0.05). Comparison within the groups: challenged at 1 day old(between groups 1 and 2) or challenged at 14 days of age (between groups 3 and 4).Table 3. Shedding of SE Nal Spc measured by the number of positive cloacal swab samples in chicks challenged at 1 day of age.
 A 
Days postinfectionProgeny fromvaccinated birdsProgeny fromunvaccinated birds0 hr 24 hr Total 0 hr 24 hr Total
1 dpi 0 1 1 12 26 383 dpi 1 5 6 5 9 146 dpi 0 7 7 2 17 199 dpi 0 13 13 4 25 2913 dpi 0 8 8 0 7 716 dpi 0 1 1 0 1 120 dpi 0 0 0 0 0 023 dpi 0 0 0 0 0 0Total 1 35 36* 23 85 108
 A 
Each group (vaccinated or unvaccinated) contained 40 birds. 0 hr
5
SE-positive results in direct plating in brilliant green agar; 24 hr
5
SE-positive results after enrichment in selenite broth.*Statistically significant (
P 
,
0.05).Fig. 1. Number of SE positive samples in cloacal swabs from chickschallenged at 1 day of age.
Passive immunity in vaccinated broiler breeder progenies
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