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AVIAN DISEASES 52:567–571, 2008

Passive Immunity of Progeny from Broiler Breeders Vaccinated with Oil-Emulsion


Bacterin Against Salmonella Enteritidis
A. Y. Inoue,AC A. Berchieri Jr.,B A. Bernardino,A J. B. Paiva,B and E. V. SterzoB
A
Fort Dodge Animal Health, Rua Luiz Fernando Rodriguez, 1701, CEP 13064-798, Campinas, São Paulo, Brazil
B
Department of Veterinary Pathology, Avian Pathology Laboratory, São Paulo State University, Jaboticabal, São Paulo, Brazil
Received 4 March 2008; Accepted and published ahead of print 6 June 2008

SUMMARY. Young poultry are very susceptible to Salmonella Enteritidis (SE) infections because of the absence of complete
intestinal flora colonization and an immature immune system. This study evaluated the role of passive immunity on the resistance
of young birds against early infections caused by SE. The progeny of broiler breeders vaccinated with an oil-emulsion bacterin was
compared to the progeny of unvaccinated birds. Efficacy was determined by challenging birds at 1 and 14 days of age with SE Nal
Spc strain, phage type 4. After challenge at 1 day of age, the progeny of vaccinated birds presented a significantly lower number
(log10) 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 (log10) of colony-forming units/ml of SE Nal Spc only in
the cecal contents compared with nonvaccinated breeder progeny (2.11 vs. 2.94). Age seems to influence the susceptibility of birds
to SE infections: in control groups, the number of positive birds at 14 days of age (9/25) was lower when compared with the group
infected 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 presented
passive antibodies detectable by enzyme-linked immunosorbent assay (ELISA) up to 21 days of age. On the other hand, antibodies
of the control group were detected by ELISA 14 days after challenge. These results show a significant contribution of breeder
vaccination 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 aceite
contra Salmonella Enteritidis.
Las aves jóvenes son muy susceptibles a las infecciones con Salmonella Enteritidis debido a la ausencia de una colonización completa
por parte de la flora intestinal y a la inmadurez de su sistema immune. Este estudio evaluó el papel de la inmunidad pasiva en la
resistencia de aves jóvenes contra las infecciones tempranas causadas por Salmonella Enteritidis. La progenie de reproductoras pesadas
vacunadas con una bacterina emulsionada en aceite fue comparada con la progenie de aves no vacunadas. La eficacia fue determinada
por el desafı́o con la cepa de Salmonella Enteritidis Nal Spec, fagotipo 4. Después del desafı́o al dı́a de edad, la progenie de aves
vacunadas presentó un número significantemente (P , 0.05) menor (log10) de reaislamientos de Salmonella Enteritidis Nal Spec en
hı́gado (2.21), bazo (2.31) y contenido cecal (2.85) cuando se comparó con los resultados obtenidos para el grupo control (2.76, 3.02 y
6.03, respectivamente). La evaluación de los órganos internos 3 dı́as después de la infección, reveló 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 no
vacunadas fueron positivas. Las aves desafiadas a los 14 dı́as de edad presentaron un número menor de muestras positivas comparado
con aquellas desafiadas al dı́a de edad, y la progenie de aves vacunadas presentó valores estadı́sticamente menores (log10) de Unidades
Formadoras de Colonia/ml de Salmonella Enteritidis Nal Spec, únicamente en el contenido cecal, comparados con los obtenidos en la
progenie de reproductoras no vacunadas (2.11 Vs. 2.94). Aparentemente la edad influyó en la susceptibilidad de las aves a la infección
con Salmonella Enteritidis: en los grupos control, el número de aves positivas a los 14 dı́as de edad (9/25) fue menor cuando se
comparó con el grupo infectado al dı́a de edad (25/25). El número de muestras provenientes de heces positivas de la progenie de aves
vacunadas fue significativamente menor (36) que el originado en las del grupo control (108) después del desafı́o al dı́a de edad. La
progenie no desafiada de aves vacunadas presentó anticuerpos maternos detectables por medio del inmunoensayo asociado a enzimas
(ELISA) hasta los 21 dı́as de edad. De otra parte, los anticuerpos del grupo control fueron detectados por la prueba ELISA 14 dı́as
después del desafı́o. Estos resultados muestran una contribución significante de la vacunación 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 infección.
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 important (29). SE is associated with illness in young birds and with foodborne
bacterial diseases in domestic chickens and turkeys. Among many diseases in humans (11).
serotypes of Salmonella, Salmonella Enteritidis (SE) has been Vertical transmission and early exposure to SE are the main causes
described as the most prevalent in commercial broilers and layers of clinical diseases because of the high susceptibility of newly
hatched birds to infections. Intestinal microflora colonization and
mature immune systems provide older birds resistance against the
C
Corresponding author. E-mail: inouea@fdah.com effects of SE, lowering the morbidity and mortality (13). Birds

567
568 A. Y. Inoue et al.

challenged at 1 day of age can remain infected and shed SE until Table 1. Number of positive samples of liver, spleen, and cecal
maturity (12). contents for SE Nal Spc in birds challenged at one or 14 days of age, and
The benefits of passive immunity in reducing the damages caused examined 3 days after infection.
by early exposure to infectious agents have been studied for many Challenged at Challenged at
poultry diseases (8,10,26). Maternal antibodies from breeder’s blood 1 day of ageA 14 days of ageB
can be transferred through the egg yolk as immunoglobulin G (IgG; Group 1 Group 2 Group 3 Group 4
15,24). It is also reported that other immunoglobulins, like IgA and
IgM, are also secreted in reproductive tract of vaccinated birds (25). Breeders Vaccinated Unvaccinated Vaccinated Unvaccinated
These immunoglobulins can be transferred to the eggs during their Liver 4/25 15/25 0/25 2/25
Spleen 3/25 18/25 0/25 2/25
passage through the oviduct. After absorption, the antibodies are
Cecal contents 4/25 17/25 2/25 8/25
diffused in amniotic liquid and are ingested by the embryo (7). In Total positive
commercial poultry industry, the importance of passive immunity birds 7/25 25/25 2/25 9/25
against SE is also related to the ability of the bacteria to infect large A
Challenged with 2.12 3 105 CFU/ml.
number of birds at the hatchery, where the temperature and B
Challenged with 2.17 3 109 CFU/ml.
moisture are favorable to proliferation (5,20).
In the present study, we investigated the protective effects of
Antibody detection. Sixty chicks were divided into three groups: 20
maternal antibodies on the progeny of breeders vaccinated with an birds from group 1 unchallenged, 20 birds from group 1 challenged, and
SE bacterin in oil emulsion. In addition to the effects on bacterial 20 birds from group 2 challenged. Both groups were challenged dir-
counts in the organs of challenged birds, we assessed other aspects, ectly into the crop with 2.12 3 105 CFU of SE Nal Spc at 1 day old.
such as fecal shedding and serology using an enzyme-linked Serum samples were collected at 1, 7, 14, 21, and 28 days of age.
immunosorbent assay (ELISA). Serum was diluted at 1:2 in phosphate-buffered saline solution and
tested for the presence of specific antibodies by an ELISA (FlockChek
SE, IDEXX Laboratories, Inc., Westbrook, ME). The results were
MATERIALS AND METHODS measured in a Visible Plate Reader (650 nm, Biotrak version 1.3;
Amersham Pharmacia Biotech, Uppsala, Sweden).
Birds. Day-old broilers chicks (Cobb-Vantress, Siloam Springs, AR) Statistical analysis. Chi-square contingency tables were used to
from two different commercial hatcheries (São José Farm, and Pena statistically analyze fecal-shedding results. ANOVA was used to
Branca Company, São Paulo, Brazil) were used in the study. The chicks statistically analyze viable SE log10 count in the organs (liver, spleen,
were divided into four groups: groups 1 and 3 were progeny of broiler and cecal contents).
breeders vaccinated with an inactivated SE vaccine (POULVAC SE; Fort
Dodge Animal Health, Overland Park, KS) at 12 and 20 wk old.
Groups 2 and 4 were chicks from unvaccinated breeders. The chicks RESULTS
used in this study were from breeders between 38 and 40 wk old.
The birds were weighed (40 6 1 g), then reared in isolation units All the samples from day-old chicks, examined at arrival, had
maintained with filtered air under negative pressure (Fort Dodge negative bacteriologic exams.
Research Center, Paulinia, São Paulo, Brazil). All birds were fed and Organs samples. Results of bacteriologic exams (Table 1) for all
watered ad libitum. At arrival, 20 birds from each group were assessed organs showed a higher number of positive birds in the progeny
according to Waltman et al. (30), to ensure that the newly hatched
from unvaccinated breeders (groups 2 and 4) compared with the
chicks were free of Salmonella. The samples (drag swabs, liver, spleen,
heart, and ceca) were examined using selenite broth (CM0395 and progeny of vaccinated birds (groups 1 and 3). The chicks that were
LP0121A, Oxoid, Ogdensburg, NY) for 24 hr at 37 C and plated onto challenged at 14 days of age (groups 3 and 4) had fewer positive
brilliant green agar (CM0263, Oxoid). samples than the groups challenged at 1 day old (groups 1 and 2).
Salmonella Enteritidis strain. A mutant strain of Salmonella Comparing different organs, cecal contents were the site of greater
Enteritidis (SE), phage type 4, resistant to nalidixic acid (Nal) and positive samples at 14 days of age.
spectinomycin, (Spc) from the Laboratory of Avian Pathology (São Data in Table 2 show a statistically higher number of viable SE
Paulo State University, Jaboticabal, São Paulo, Brazil) was used as Nal Spc (P , 0.05) in all samples (liver, spleen, and cecal contents)
challenge. Bacterial cultures were prepared in 10 ml of nutrient broth at of birds from unvaccinated breeders (group 2) challenged at 1 day
37 C for 24 hr under stirring. This mutant was named SE Nal Spc. old compared with chicks from group 1. In chicks challenged at 14
Experimental design. Organ samples. One hundred chicks were days old, the number of viable SE Nal Spc of the control group
divided in four groups: birds from groups 1 and 2 were challenged at 1
(group 4) was statistically higher (P , 0.05) than group 3 only in the
day of age (25 birds per group) and birds from groups 3 and 4
challenged at 14 days of age (25 birds per group). Both groups were cecal contents.
challenged directly into the crop with different doses of SE Nal Fecal samples. Table 3 shows that the birds from vaccinated
Spc: groups 1 and 2, with 2.12 3 105 colony-forming units (CFU) per breeders (group 1) presented a statistically lower number of positive
bird; groups 3 and 4, with 2.17 3 109 CFU per bird. The birds from cloacal swab samples (P , 0.05) than the control group (group 2).
each group were sacrificed and were examined 3 days after challenge. Group 1 had fewer positive results than group 2 in direct evaluation
Samples of liver, spleen and ceca contents were analyzed individually to of the samples and after culture in selenite broth. After 13 dpi, the
recover viable SE Nal Spc. The number of cells was estimated by plating number of positive samples was similar in both groups, and no
0.1 ml of the decimal dilutions on brilliant green agar containing shedding was observed after 20 dpi. In broilers derived from
sodium nalidixate (20 mg/ml) and novobiocin (1 mg/ml) (BGNalNov). vaccinated breeders, the peak of shedding occurred at 9 dpi, whereas
Fecal samples. Eighty chicks were divided into two different groups:
the peak was at 1 dpi in the control group (Fig. 1).
birds from groups 1 and 2. Both groups were challenged directly into the
crop with 2.12 3 105 CFU of SE Nal Spc. Cloacal swabs were taken at Antibody detection. Table 4 shows the differences in antibody
1, 3, 6, 9, 13, 15, 20, 23, and 26 days postinfection (dpi). Cloacal swabs detection in the three groups of birds. Birds from unvaccinated
were cultured in selenite broth (CM0395 and LP0121A, Oxoid) breeders (group 2) challenged at 1 day old had detectable
containing novobiocin (40 mg/liter) or were directly plated on seroconversion in the ELISA at 14 days old. More than 70% of
BGNalNov. The samples were incubated at 37 C for 24 hr. the birds from vaccinated breeders (group 1) challenged at 1 day old
Passive immunity in vaccinated broiler breeder progenies 569
Table 2. Log10 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 that
were 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 age
Group 1 Group 2 Group 3 Group 4
Breeders Vaccinated Unvaccinated Vaccinated Unvaccinated
Liver 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).

had positive simples at 14 days of age, and 23.5% were positive at 28 infections (6,9,13). In the present study, the groups of chicks
days. Nevertheless, the unchallenged group 1 had a faster decrease in infected at 1 day of age had a higher number of positive samples and
positive findings, with 47% of the birds positive at 14 days of age, higher SE Nal Spc counts than the groups challenged at 14 days of
but no positive samples at 28 days of age. age in progeny from vaccinated or unvaccinated breeders.
It was also demonstrated that horizontal transmission and
environmental contamination is higher in birds from unvaccinated
DISCUSSION breeders because of SE shedding after challenge. Vaccinated groups
had lower shedding rates throughout the study. Another important
In groups of birds challenged at 1 day old, the results comparing
result is related to the age of fecal shedding after challenge: the
the number of positive samples and the number of SE viable cells
progeny of vaccinated breeders presented the highest numbers of
showed a significant reduction in both parameters for the group
positive samples on day 9 dpi, whereas, in progeny of unvaccinated
from vaccinated breeders. These results are interesting, once the
breeders, the shedding peak was at 1 dpi. Birds infected at 1 day of
number of SE cells is related directly to the potential for damage
age are important sources of infection because of intermittent SE
caused by SE infection (4).
shedding until they reach adulthood (12). In addition, chicken meat
The ceca is the main site of SE bacterial multiplication in birds;
cross-contamination in poultry processing plants, caused by contact,
spleen and liver are the most important nonintestinal organs where
can be increased by contaminated feces in carcasses (23). The control
this bacterium is found (13). SE can be recovered in internal organs
of SE fecal shedding is equally important in layers because
1 hr after experimental inoculation (18), and the highest levels of
contaminated feces can soil the egg shell surface, and penetrate
liver and spleen contamination are observed 3 days after infection
through the egg (2). Delay in fecal shedding may be related to the
(28). In the present study, in addition to the lower SE Nal Spc count
presence of maternal IgA in the intestinal tract. IgA in the intestinal
in the cecal contents of progeny from immunized breeders, as
tract has a significant role in immunity against Salmonella because it
previously described by Methner and Steinbach (24), there was less
inhibits bacterial adherence and colonization in the intestinal
SE reisolation in the liver and the spleen after challenge at 1 day of
mucosa, which is the primary site of infection (22). Higher IgA
age. The passive immunity could be important in reducing the
titers in the progeny of vaccinated birds have been reported during
transient depletion of lymphocytes in the lymphoid organs, which
the first week of age (15).
promote the development of carrier states in broilers that are exposed
The period of shedding lasted until 16 days of age for both
early to Salmonella, as described by Hassan and Curtis (14).
groups, which is similar to that described by Bailey (1), although
Comparing the groups challenged at different ages (1 and 14
Berchieri et al. (3) demonstrated that passive immunity in chicks
days), it is clearly demonstrated that the absence of complete
from infected breeders presented a longer shedding period after SE
intestinal flora and immature immune systems play an important
infection than birds without maternal immunity. The shedding
role in the higher susceptibility of young birds to Salmonella
period and the numbers of infected bird can be influenced by the
infectious dose and by the strain used in the challenge (27).
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
Progeny from Progeny from
vaccinated birds unvaccinated birds
Days postinfection 0 hr 24 hr Total 0 hr 24 hr Total
1 dpi 0 1 1 12 26 38
3 dpi 1 5 6 5 9 14
6 dpi 0 7 7 2 17 19
9 dpi 0 13 13 4 25 29
13 dpi 0 8 8 0 7 7
16 dpi 0 1 1 0 1 1
20 dpi 0 0 0 0 0 0
23 dpi 0 0 0 0 0 0
Total 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. Fig. 1. Number of SE positive samples in cloacal swabs from chicks
*Statistically significant (P , 0.05). challenged at 1 day of age.
570 A. Y. Inoue et al.

Table 4. Number of positive serum samples in ELISA SE in progeny from vaccinated and unvaccinated breeders, challenged or not at a day of
age with SE Nal Spc.
% Positive on days postinfection
A
Groups Results 1 7 14 21 28
Group 2 Positive 0.00 0.00 76.47 94.12 88.24
Suspect 0.00 5.88 11.76 5.88 5.88
Negative 100.00 94.12 11.76 0.00 5.88
Group 1 Positive 88.89 82.35 70.59 41.18 23.53
Suspect 0.00 5.88 29.41 23.53 29.41
Negative 11.11 11.76 0.00 35.29 47.06
Control Positive 88.89 100.0 47.06 17.65 0.00
Suspect 11.11 0.00 11.76 11.76 11.76
Negative 0.00 0.00 41.18 70.59 88.24
A
Group 2 5 progeny from unvaccinated breeders, challenged at 1 day of age (n 5 20 birds); group 1 5 progeny from vaccinated breeders,
challenged at 1 day of age (n 5 20 birds); Control 5 progeny from vaccinated breeders, not challenged (n 5 20 birds).

Maternal IgG was detected in the serum of the unchallenged Salmonella enterica serovar Enteritidis phage type 4 in chickens. Avian
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