Salmonella Incidence in Broilers from Breeders Vaccinated with Live and Killed Salmonella

©2007 Poultry Science Association, Inc.

Salmonella Incidence in Broilers from Breeders Vaccinated with Live and Killed Salmonella S. D. Young, O. Olusanya, K. H. Jones, T. Liu, K. A. Liljebjelke, and C. L. Hofacre1 Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens 30602

Primary Audience: Veterinarians, Production Managers, Quality Assurance Management

Salmonella reduction in broilers from commercial broiler breeders vaccinated with live and killed Salmonella vaccines was evaluated. Broiler breeders were vaccinated with Poulvac ST live Salmonella Typhimurium vaccine at 1 d of age, and this was repeated at 2 and 6 wk of age. The breeders were then administered a killed autogenous oil emulsion adjuvanted vaccine containing Salmonella Kentucky, Salmonella Heidelberg, and Salmonella Hadar, at 10 and 18 wk of age. From the ages of 36 to 52 wk, eggs from the breeder flock were hatched, and progeny were challenged in 4 separate experiments at 1 d of age by oral gavage with 1 × 106 cfu/chick by either Salmonella Kentucky, Salmonella Heidelberg, Salmonella Hadar, or Salmonella Enteritidis, each containing resistance to naladixic acid at 32 ␮g/mL. At 17 to 21 d of age, the broilers were euthanized, and 1 side of the cecum was cultured for Salmonella, and the other side of the cecum was used for enumeration on positive samples. Recovered Salmonella was confirmed to be the challenge strain by O-antisera grouping. This study indicated a difference in Salmonella incidence and enumeration between the vaccinated and nonvaccinated breeder groups for certain species. When challenged with serotypes Salmonella Kentucky, Salmonella Hadar, and Salmonella Heidelberg, protection was noted with a reduction of 28, 17, and 11%, respectively, when compared with the control groups. However, protection was not seen when challenged with S. enteritidis. Under the conditions of this study, live and killed vaccination of commercial broiler breeders with Salmonella contributes protection to progeny when challenged at 1 d of age. Key words: Salmonella, maternal antibody, broiler chicken, vaccine, protection 2007 J. Appl. Poult. Res. 16:521–528 doi:10.3382/japr.2007-00009

DESCRIPTION OF PROBLEM With an estimated 800,000 to 4 million human cases of Salmonella infections each year in the United States, reducing Salmonella incidence has become monitored and regulated by the USDA Food Safety and Inspection Service [1]. Because poultry is one of the leading sources of Salmonella illnesses found in tracebacks, the industry has taken measures to reduce the 1

Corresponding author: [email protected]

amount of exposure from poultry foods. Currently, programs such as Hazard Analysis and Critical Control Points have focused on reducing Salmonella within the processing plant. As a result, Salmonella-positive samples in small broiler processing plants have decreased from 37.3% in 2001 to 8.4% in 2002 [2]. Salmonella can be reduced in all stages of poultry production, but in the past, the processing plant has had the most active control

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SUMMARY

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only have they been shown to reduce the number of positive colonization in the chicks from vaccinated breeding hens but have also been shown to reduce the colonization numbers dependent on the strain of vaccine used [16]. However, vaccination before onset of laying is important, because infection usually occurs within the first part of lay from the farm environment [17]. Even though 1 study demonstrated that turkey breeder hens vaccinated with a single species bacterin did not protect against Salmonella Hadar in progeny [18], by vaccinating with more than 1 serotype, there may be more widespread protection. Some studies have shown that progeny from broiler breeders vaccinated with a live Salmonella Typhimurium and a killed Salmonella Enteritidis have fewer Salmonella organisms colonized in the intestine [19]. As a result, some poultry companies have begun vaccinating broiler breeders with live and bacterin-derived vaccines. Some of these companies have noted there has been a reduction in overall Salmonella incidence in the broiler processing plant, approximately 1 yr after implementing this vaccination plan. However, there have been few studies to see if the broiler offspring from these vaccinated flocks have a reduced Salmonella colonization when challenged at 1 d of age. The current study evaluates Salmonella colonization and organism load of progeny from broiler breeders administered commercially available live and inactivated Salmonella vaccines.

MATERIALS AND METHODS Experimental Design Laying Hens. Broiler breeder meat-type hens from a Georgia broiler integrator were vaccinated with live and inactivated water in the oil emulsion autogenous vaccination program, Poulvac ST [20], followed by 2 killed autogenous vaccines containing Salmonella Kentucky, Salmonella Heidelberg, and Salmonella Hadar [21]. The first live Salmonella Typhimurium vaccination was administered by coarse spray at 1 d of age. The second and third live Salmonella Typhimurium vaccines were administered through the drinking water at 2 and 6 wk of age. At 10 and 18 wk of age, the autogenous killed vaccine was administered by i.m. injection. Eggs

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program [3]. It is known that one of the major sources of Salmonella to broilers is by vertical transmission from the broiler breeder and horizontal transmission in the broiler house [3]. It has been shown that the same Salmonella species found in the broiler processing plant can often be traced back to the hatchery and to the parent broiler breeder flock [4]. As a result, the poultry industry has been focused on reducing the Salmonella incidence in live production, to further cause reduction of Salmonella numbers in the processing plant. By reducing incidence of Salmonella in the grow-out phase, there will be less contamination in the processing plant. The overall goal is not only to reduce the number of positive samples but also reduce the load of organisms brought to the processing plant. It is known that both humoral and cell-mediated immunity play a role in prevention and the clearance of Salmonella, although the mechanism is not completely understood [5]. Because the foodborne Salmonella pathogens rarely cause systemic disease in adult poultry, it can be difficult to produce a humoral immune response by natural exposure. However, exposing young chicks (<2 d old) to Salmonella can cause a systemic response resulting in immunity [6]. Vaccination of chickens with killed bacterins has been shown to decrease intestinal colonization and mortality [7, 8]. However, Salmonella Enteritidis bacterins have had more effect on reducing colonization of organs in addition to the intestine and have only reduced the numbers of intestinal shedding, not eliminating Salmonella alltogether [9, 10]. Live vaccination procedures have been shown to be more effective against fecal shed of Salmonella than killed bacterins alone [11]. Certain invasive strains of Salmonella Typhimurium have been shown to have IgG titers at detectable levels by enzyme-linked immunosorbent assay up to 9 mo after exposure [12, 13]. It has been shown that a live Salmonella Typhiumurium vaccine can protect against Salmonella Enteritidis [14]. Certain killed Salmonella Enteritidis vaccines can induce high levels of circulating specific IgG to Salmonella protein antigens of unidentified species [15]. In the past, studies have demonstrated that there can be maternal antibody transmission for Salmonella from vaccinated or exposed breeders to the progeny with some protection [14]. Not

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Table 1. Comparison of Salmonella ceca total culture results from broilers challenged at d 1 by serovar in each column Group1 Vaccinated challenged Nonvaccinated challenged

Salmonella Kentucky

Salmonella Hadar

Salmonella Heidelberg

Salmonella Enteritidis

29/50 (58%)2 43/50 (86%)

36/48 (75%) 45/49 (92%)

40/48 (83%) 46/49 (94%)

49/50 (98%) 45/49 (92%)

1

No statistically significant differences between vaccinated or control. Culture positive number/total number cultured (percentage positive).

2

Kentucky. Salmonella Enteritidis was the fourth challenge, and even though it was not included in the breeder vaccine protocol, previous studies have shown that live Salmonella Typhimurium exposure can protect against colonization when

Figure 1. Mean Salmonella ceca enumeration (log10) from broilers from either Salmonella-vaccinated or nonvaccinated breeders when challenged at 1 d of age by individual Salmonella serovar.

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from the vaccinated broiler breeder flock were collected from 36 wk of age through 52 wk of age. The serovars used to challenge the progeny at hatch in 3 experiments were Salmonella Hadar, Salmonella Heidelberg, and Salmonella

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Salmonella serovar. Each dose contained approximately 106 viable Salmonella sp. cells. The gavaged birds served as seeders for the remaining birds in each group [24]. Cecal Samples. Birds were euthanized by cervical dislocation. Gloves were changed between birds, and disinfected instruments were used for each bird and sample to prevent crosscontamination between birds and groups. Ceca were separated by their 2 sides and each placed in an individual sterile Whirl Pak bag [25]. Therefore, there were 2 sample bags for each bird. Salmonella Culture and Identification. One side of the cecum was sampled for enrichment culture from each bird. Each individual sample was enriched with 25 mL of tetrathionate brilliant green broth, stomachered with the Stomacher Lab Blender 80 [26] for approximately 10 s, and incubated for 18 to 24 h at 42°C. Loopfuls (10 ␮L) of the sample broth were streaked for colony isolation on brilliant green agar [27] supplemented with 0.02 mg/mL of novobiocin [28] and xylose-lysine-tergitol 4 with 32 ␮g/mL of naladixic acid. Typical cherry red colonies on brilliant green agar or individual black colonies on xylose-lysine-tergitol 4 were considered positive for Salmonella. Verification was done by taking 20 colonies from 20 different samples per trial and growing on blood agar. Blood agar plates were grown at 37°C with 5 to 10% CO2 for 18 to 24 h. Colonies from blood agar plates were tested with Salmonella 0 antiserum [29] for serogrouping to verify that the Salmonella was the same serogroup as our challenge strain. Polymerase chain reaction was then performed to verify the species [30]. Negative samples on primary culture were then held for delayed secondary enrichment and culture [31]. Salmonella-Nested Polymerase Chain Reaction. Samples taken for Salmonella sampling using the nested polymerase chain reaction method were taken from the same bag of tetrathionate broth (TTB)-enriched media as the culture samples 24 h after incubation at 37°C [32]. This method detects whether Salmonella is present. The nested polymerase chain reactions were used to know which samples were negative. Note that all positive results reported were based on primary and secondary culturing techniques

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exposed to Salmonella Enteritidis [14]. The same Salmonella-vaccinated broiler breeder hen flock [22] was used for the entire experiment. The breeders were 36 wk when eggs were collected for the Salmonella Hadar progeny challenge, 39 wk for the Salmonella Kentucky challenge, 45 wk for the Salmonella Heidelberg challenge, and 52 wk for the Salmonella Enteritidis challenge. The nonvaccinated broilers were from hen flocks approximately the same age as the vaccinated flock when eggs were collected for the challenges; however, these 3 nonvaccinated hen flocks were from a different broiler integrator in North Georgia. The eggs from the vaccinated and nonvaccinated hens were brought to the University of Georgia, where they were incubated and hatched in separate hatcher cabinets. The chicks were divided into 4 groups with 50 chicks per group. The 50 chicks were placed into 2 pens of 25 each. Group 1 was from vaccinated hens and challenged, group 2 was from vaccinated hens and not challenged, group 3 was from nonvaccinated hens and challenged, and group 4 was from nonvaccinated hens and not challenged. The broiler chickens were housed in floor pens with softwood shavings for bedding material, and strict biosecurity was practiced to avoid cross-contamination between groups. The broiler chicks challenged with Salmonella Hadar and Salmonella Heidelberg were euthanized at 18 d of age, Salmonella Kentucky at 21 d of age, and Salmonella Enteritidis at 25 d of age. Broilers. At day of hatch, 10 chicks from each group were sampled for Salmonella, to be sure the flock was not contaminated with Salmonella transovarilly or from the eggshell. Livers, yolk sacs, and cecum were cultured for Salmonella (as described below). On the day of hatch, chicks were separated into groups of 25 and orally challenged with Salmonella as described below. All birds were provided with ad libitum antibiotic-free feed and water. Salmonella Challenge. An overnight brain heart infusion broth [23] culture of 32 ␮g/mL of naladixic acid-resistant mutant of Salmonella Hadar, Salmonella Heidelberg, Salmonella Enteritidis, or Salmonella Kentucky was diluted with brain heart infusion. Ten out of 25 of the chicks in the challenge groups were orally gavaged with a 0.10-mL dose of the designated

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RESULTS AND DISCUSSION All 10 chicks from each broiler group from each of the 4 experiments tested before challenge were negative for Salmonella. All challenge studies consisted of 2 pens of vaccinated challenge and 2 pens of nonvaccinated challenge, each consisting of 25 birds. However, results will be reported by combining the results of both groups; therefore, it will be out of 50 birds. During all experiments and challenges with different Salmonella serovars, no clinical symp-

toms of systemic infection were observed. Mortality was less than 1% in all groups, including the controls. The control chickens remained free of Salmonella, with the exception of 1 group. One of the 2 nonchallenged, vaccinated control groups in the Salmonella Hadar challenge experiment became contaminated with Salmonella. However, the Salmonella cultured from the cecum in 22 out of the 25 birds in the group was not the challenge strain. This was confirmed by the sensitivity of the Salmonella to naladixic acid and when sampled by polymerase chain reaction, and it was not the challenge Salmonella Hadar serogroup (C1). Therefore, that control group was eliminated from the study results. There was no statistically significant difference between the vaccinated and nonvaccinated groups in any of the 4 challenge studies. Colonization by the Salmonella Kentucky Challenge Strain Between the Progeny of Nonvaccinated and Vaccinated Broiler Breeder Hens On primary enrichment culture from the chicks from vaccinated hens, 29 out of 50 (58%) were positive for Salmonella Hadar (Table 1). There were 43 out of 50 (86%) of the nonvaccinated birds positive. After delayed secondary enrichment, there were no additional samples positive. Enumeration of the positive samples from the vaccinated group had a mean of 0.90. Also, the progeny from the nonvaccinated group had a mean of 1.13 (Figure 1). When the enumeration numbers were averaged and the groups compared, there was a numerically lower number of Salmonella isolation in the vaccinated group but no statistical difference by t-test. Colonization by the Salmonella Hadar Challenge Strain Between the Progeny of Nonvaccinated and Vaccinated Broiler Breeder Hens On primary culture, the group from the vaccinated breeder flocks had 36 out of 48, or 75%, positive samples for Salmonella (Table 1). The nonvaccinated challenge group had 45 out of 49, or 92% positive. No other samples were positive after delayed secondary enrichment. Enumeration showed that the vaccinated challenge mean for the number of Salmonella

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and species confirmation by polymerase chain reaction. Salmonella Enumeration. The other side of the cecum was refrigerated overnight at 4°C. TTB-enriched samples that were positive for Salmonella by nested polymerase chain reaction were chosen to direct plate for enumeration. Refrigerated samples were then diluted with 10 mL of sterile 0.85% physiologic buffered saline. Bags were stomachered for approximately 10 s. Three serial dilutions were made and evenly spread on 3 individual plates of xylose-lysinetergitol 4 plates with 32 ␮g/mL of naladixic acid and were incubated at 37°C for 18 to 24 h. Therefore, the dilutions were 102, 103, and 104. Black colonies were counted and recorded based on the dilution plate. Positive samples for the challenge groups were determined based on positive culture methods as described above and then confirmed by polymerase chain reaction. Results are compared based on the number of cultures positive after primary enrichment with TTB, delayed secondary enrichment with TTB, and by enumeration. The numbers of Salmonella shed in each treatment were transformed to log10, and then the means were calculated. Samples that had no growth on primary culture were considered as containing no Salmonella and assigned to 0 for statistical analysis. Samples that had no growth on the primary culture plates but were culturepositive following TTB-delayed enrichment were presumed to contain 9 cfu/mL. This number was assigned due to the number of organisms that can possibly be detectable by enrichment but not direct plating [33]. Statistical Analysis. The t-test was used for statistical evaluation for the enumeration comparison.

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526 colonies was 2.43 and the nonvaccinated group was 2.69. Again, there was no significant difference on the t-test (Figure 1). Colonization by the Salmonella Heidelberg Challenge Strain Between the Progeny of Nonvaccinated and Vaccinated Broiler Breeder Hens

Colonization by the Salmonella Enteritidis Challenge Strain Between the Progeny of Nonvaccinated and Vaccinated Broiler Breeder Hens For the progeny that were from vaccinated hens, primary culture revealed 49 out of 50, or 98%, positive samples. The nonvaccinated group was 42 out of 49. After delayed enrichment, the vaccinated group had not changed, but the nonvaccinated group had 45 out of 49, or 92%, positive samples (Table 1). On enumeration, the vaccinated challenge mean for Salmonella colonies was 2.90 and the nonvaccinated group was 2.39. Again, there was no significant difference on the t-test (Figure 1). Three out of the 4 challenge strains used, Salmonella Kentucky, Salmonella Heidelberg, and Salmonella Hadar, showed a numerical decrease in Salmonella-positive samples as well as a numerical decrease in Salmonella colonization numbers. However, the Salmonella Enteritidis study revealed no reduction between the 2 groups (Table 1). It was later found that the nonvaccinated group of breeders used for the Salmonella Ken-

tucky challenge were culture positive for Salmonella Kentucky. As a result, some of the hens within the flock could have seroconverted by natural exposure and passed maternal antibody to the embryo to offer some mild protection. This may account for the lack of statistical difference between the Salmonella Kentucky vaccinated vs. nonvaccinated. Salmonella Enteritidis was used as a challenge strain to determine if there was any crossprotection from the live Salmonella Typhimurium vaccine given at 1 d, 2 wk, and again at 6 wk. Immunity from a live Salmonella Typhimurium vaccine is known to be short-lived, and most of the immunity is cell-mediated, not humoral. The breeder flock used for this challenge study was over 50 wk at the time of egg collection for this study. Because the birds were 44 wk when they received the live Salmonella Typhimurium vaccine, any cross-protective antibodies to the Salmonella Enteritidis may have gone by the time of the challenge. Our results are consistent with what is known about shortlived cross-immunity, and the progeny exposed with Salmonella Enteritidis would not have been expected to have much maternal protection. In our study, the challenge dose for the infected seeder birds was large, 106 cfu/mL, to allow for lateral spread of infection. However, such a large dose may not reflect the natural exposure load in most field challenges. Also, the seeder birds were not sampled independently from the horizontally challenged birds. Even with the large challenge dose, it appears that in 3 of the 4 different Salmonella challenges, not only were there numerically fewer number of intestinally positive birds from the groups of progeny from vaccinated breeders, but the overall number of organisms able to colonize was numerically reduced. This is not only evident in the numerical counts of the cecum but also in the number of positive samples from primary culture vs. the secondary enrichment culture. The fewer the numbers colonized in the intestine, the more difficult it is to recover Salmonella on the primary enrichment culture. Although there was no statistical difference between positive samples and enumeration samples, this information and reduction is useful for the broiler industry. A small amount of decrease in Salmonella in the field has the potential to

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On primary culture, the group from the vaccinated breeder flocks had 34 out of 48, or 71%, positive samples for Salmonella. The nonvaccinated challenge group had 41 out of 49, or 84%, positive. After secondary delayed enrichment, the total number of positive samples for Salmonella Heidelberg in the progeny from vaccainted breeders was 40 out of 48, or 83% (Table 1). For the progeny from nonvaccinated breeders, the total was 46 out of 49, or 94% positive samples. The enumeration plates revealed that the vaccinated challenge mean for Salmonella colonies was 1.55 and the nonvaccianted group was 1.95. Again, there was no significant difference on the t-test (Figure 1).

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YOUNG ET AL.: SALMONELLA IN BROILERS decrease the amount of Salmonella brought in the processing plant and may have an effect on the numbers of cross-contaminated Salmonella carcasses. Under the conditions of this study, it appears that when commercial broiler breeders

527 are given 3 live Salmonella Typhimurium and 2 killed autogenous Salmonella vaccines, there is some maternal protection passed to the progeny when chicks are exposed to Salmonella at 1 d of age.

CONCLUSIONS AND APPLICATIONS

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1. Vaccination of broiler breeder hens with 3 live Salmonella Typhimurium and 2 autogenous bacterins results in reduced Salmonella colonization of their broiler offspring when challenged with the same serovars contained in the bacterin. 2. Vaccination of broiler breeder hens with 3 live Salmonella Typhimurium and 2 autogenous bacterins resulted in reduced Salmonella in the offspring for those Salmonella serovars used as antigens in the vaccine. 3. Because the results of the study were numerically but not significantly reduced by the use of this vaccination program, additional studies using larger numbers would be needed to confirm that Salmonella vaccination of breeder hens will reduce Salmonella colonization of their offspring.

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