Salmonella is a ubiquitous pathogen which, in addition to causing poultry diseases, has a growing zoonotic impact. It has demanded the implementation of diverse control strategies, in which vaccines play a major role. The understanding of the immune pathways elicited by the different vaccines is important, contributing for the establishment of strong immune correlates of protection, for instance. With the purpose of determining the dynamics of the humoral and cellular immune responses to vaccination, broiler breeders (Cobb Slow) were immunized with live or inactivated vaccines against Salmonella Enteritidis. Lymphocyte and macrophage subsets were analyzed in the peripheral blood by flow cytometry and antigen-specific circulating IgY and mucosal IgA were quantified. The markers analyzed by flow cytometry were CD8/CD28, CD4/TCRVβ1, Kul/ MHC II and Bu-1. Both live and inactivated vaccines induced an increase in the proportion of circulating monocytes (Kul +MHCII +) in some time points compared to non-vaccinated controls. However, whereas the live vaccine leads to an increase in CD8 -CD28 + and Bu-1 + lymphocytescompared to the control group, the inactivated vaccine prompteda reduction in the percentage of severalleucocyte subsets (Kul -MHCII +, Bu-1 +, CD8 +CD28 +, CD8 -CD28 +, CD4 +TCRVβ1 -, CD4 +TCRVβ1 +, CD4 -TCRVβ1 +) after the boost dose. Both vaccines induced specific serum IgY and mucosal IgA production; however, the inactivated vaccine stimulated higher titers in a shorter period. These results contribute to the understanding of mechanisms of action of live and inactivated Salmonella vaccines in chickens.
Salmonella spp. remains as one of the main causes of alimentary infection in humans. Poultry meat and eggs are constantly linked to clinical cases. The success of control strategies for salmonellosis in chickens is associated with several factors, among which is the control of bacterial contamination at its sources [
Among the most relevant serovars is S. Enteritidis, with its prevalence reaching more than 1% (up to 8%) of breeding flocks in several European countries [
The immunization of parent flocks with live and inactivated vaccines is a common practice inanimal biosecurity control programs of S. Enteritidis and S. Typhimurium, aiming to reduce the excretion and the contamination of the carcass and eggs [
This study had the objective of testing the immune effects of live and inactivated vaccines against Salmonella in broiler breeders. Several cellular subsets and antibody titers were analyzed serially in the peripheral blood and mucosae with the intent of clarifying the immune responses underlying the protection conferred by vaccination.
One hundred and fifty birds of the Cobb broiler breeder lineage were divided equally and randomly into 3 groups. The “negative control” group did not receive any treatment throughout the experiment. The “live vaccine” group received three doses of said compound on days 1, 42 and 112 of age. The “inactivated vaccine” group was inoculated on days 84 and 126 of age. The experimental protocol is illustrated in
Procedures with animals followed ethical standards and were approved by the Bioethics Committee from Universidade Positivo, protocol 142/2013.
The inactivated vaccine (AviPro® 109 SE4, Lohmann Animal Health) was injected subcutaneously (0.25 ml/bird), in the dorsal region of the neck, using disposable needles. The vaccine was used according to the manufacturer’s instructions. AviPro® 109 SE4 contains Salmonella Enteritidis of the phagotypes 8, 24, 14B e 23 in oil emulsion. The live vaccine (AviPro® Salmonella VAC E, Lohmann Animal Health) is composed of Salmonella Enteritidis (Sm24/Rif12/Ssq), which is a mutant strain derived from metabolic alterations (Metabolic Drift Mutants) (EFSA, 2004). The first dose was administered via large particle aspersion; the second and third doses were administered via drinking water.
Presence of live Salmonella was assessed in the environment and in birds throughout the experiment. Previous to housing, presence of Salmonella was verified on the surfaces of the cages, in the water and on the air filters. In the birds, swabs of cloaca were analyzed for Salmonella on day 1 and at 6, 12 and 16 weeks of age. Microbiological isolation and bacterial analyses were made by Laboratório Porto Belo (Porto Alegre, Brazil).
Blood collection for flow cytometry immunophenotyping was performed by wing vein puncture with heparinized syringe. For the live vaccine, blood collections were made before the first vaccine dose, then 3, 7, 14 and 21 days after the first dose, 3 and 14 days after the second dose and 3 days after the third dose. After immunization with the inactivated vaccine, samples were collected on day 1, and then 3, 7, 14 and 21 days after both doses (
Flow cytometry was performed as previously described [
For the detection of local secretory IgA, ten animals per treatment were analyzed. 200 µl of intestinal content was aspirated from the cloaca of each bird using a nasogastric probe (n˚ 10). The sample was homogenized with 0.5 ml of PBS and 0.02% sodium azide. The suspension was centrifuged at 7000 × g, the supernatant was collected and stored at −18˚C until analysis. Quantification of intestinal antigen-specific IgA was carried out by antibody capture ELISA (enzyme-linked immunosorbent assay) [
For the detection of serum IgY, blood collection was performed by wing vein puncture. Serum was separated and kept at −18˚C until analysis in sterile tubes. Twenty three birds per treatment were sampled. From live vaccine-treated animals and its control group, samples were collected on days 21, 63 and 147 of age. From the inactivated vaccine group, samples were collected on days 105 and 147 (
Treatments were compared by a two-way Analysis of Variance (ANOVA) with Bonferroni post-hoc test (P < 0.05). Analyses and graphs were made using GraphPad Prism Software (GraphPad Software, Inc.).
No Salmonella was isolated from cages, water, air filter or from the cloaca of birds throughout the experiment. This is in accordance with a previous study, which did not detect the mutant drift variant bacteria after immunization with the live vaccine [
In chickens immunized with the live vaccine, proportions of monocytes (Kul+MHCII+, on days 3 and 116), B-lymphocytes (Bu-1+, on day 116) and CD8−CD28+ cells (on day 3) were raised following vaccination compared to the control group (
Anti-Salmonella-specific serum IgYtiterswere increased after immunization and are shown in
Anti-Salmonella-specific fecal IgA titers were also increased after immunization and are shown in
Immunization with the live Salmonella vaccine did not cause environmental shedding of the bacteria, probably due to low persistence of the mutant drift variants outside the host. The immunization led to an increase in the percentage of circulating monocytes (Kul+MHCII+) 6 days after the first dose (day 7) and 4 days after the third inoculation (day 116) in comparison to the control (
After a Salmonella infection, there is a rapid influx of macrophages into the intestinal lamina propria and these phagocytes can be found until 6 days post-infection in large amounts [
ings. The detection of the pathogen in the intestine by pattern recognition receptors, many of which are expressed abundantly in macrophages, is crucial for the initiation of the immune response against Salmonella and the subsequent activation of the cells of the adaptive immune system [
Tissue demand for B-lymphocytes is also increased following Salmonella challenge [
CD28+ T cells are T lymphocytes that have not yet undergone terminal differentiation, being still capable of replication in response to stimulus. This subset of cells includes activated lymphocytes, naïve and memory cells [
An increase in the circulating Salmonella-specific IgY could be observed even after the first dose of the live vaccine (
Group | 21 d | 63 d | 147 d |
---|---|---|---|
Control | 2.65b | 9.00b | 92.40b |
Live vaccine | 11.96a | 892.87a | 1468.70a |
Probability | 0.0007 | <0.0001 | <0.0001 |
Group | 105 d | 147 d |
---|---|---|
Control | 35.00b | 92.40b |
Bacterin | 3295.30a | 3279.20a |
Probability | 0.0007 | <0.0001 |
orally failed to affect serum IgY [
The live vaccine was also able to induce higher levels of intraluminal IgA antibodies in relation to control (
In summary, the mechanism of action of the live vaccine may rely on the production of antibodies, both systemically and secreted at mucosal sites. Cellular immune responses, especially those dependent on monocytes/ macrophages, are likely to be important for protection, especially at early time points after infection. Efficacy of vaccination was not assessed, but these data may assist in the understanding of the known differences between live and inactivated vaccines in conferring protection [
The cellular responses following immunization with the inactivated vaccine are considerably different from those seen after the live vaccine administration. Kul−MHCII+, B-lymphocytes (Bu-1+), CD8+CD28+, CD8−CD28+, CD4+TCRVβ1−, CD4+TCRVβ1+ and CD4−TCRVβ1+ were reduced in the peripheral blood of vaccinated animals in comparison to control animals following the administration of the second dose of the inactivated vaccine. CD4−TCRVβ1+ was briefly increased after the first dose, while Kul+MHCII+ were increased after both inactivated vaccine doses (
The importance of monocytes (Kul+MHCII+) in the initiation of the immune response has been discussed for the live vaccine, and the similar responses by these cells to both vaccines demonstrate the importance of this first step in the induction of protection. For the inactivated vaccine, the Kul+MHCII+ cells constituted up to 30% of all blood leucocytes following the second dose administration, which may be due also to the reduction in the percentage of almost all other leucocytes.
There was a concurrent decrease in the percentage of several immune cells after the second inactivated vaccine dose. Another group suggested that the reduced percentage of CD4+ and CD8+ cells in the peripheral blood following bacterin immunization and Salmonella challenge was due to reduced activation of cellular immunity because infection had been limited by vaccination [
Cellular trafficking between secondary immune organs, the injection site and blood/lymph vessels may explain some of the findings of the bacterin immunization. Following a Salmonella challenge, CD8+CD28+ and
Group | 3 d | 7 d | 14 d | 21 d | 49 d | 56 d | 119 d | 126 d |
---|---|---|---|---|---|---|---|---|
Control | 0.090 | 0.109 | 0.082 | 0.101 | 0.109b | 0.136b | 0.164b | 0.136b |
Live vaccine | 0.090 | 0.106 | 0.096 | 0.119 | 0.155a | 0.382a | 0.322a | 0.256a |
Probability | 0.9135 | 0.8001 | 0.1030 | 0.2989 | 0.0404 | 0.0006 | 0.0005 | <0.0001 |
Group | 87 d | 91 d | 98 d | 105 d | 133 d | 140 d |
---|---|---|---|---|---|---|
Control | 0.145 | 0.141b | 0.083b | 0.213b | 0.135b | 0.129b |
Bacterin | 0.216 | 0.539a | 0.816a | 0.528a | 0.462a | 0.440a |
Probability | 0.1912 | <0.0001 | <0.0001 | 0.0005 | <0.0001 | <0.0001 |
CD4+CD8+ and CD8+TCRVβ1+ cells are reduced in the peripheral blood, for example, and it is known that lymphocytopenia also occurs [
Subcutaneous bacterin immunization is not commonly a strong stimulus for mucosal IgA production [
In summary, bacterin immunization induced an evident reduction in the relative number of several circulating leucocytes, notably with the exception of macrophages, which was also increased following live vaccine immunization. Although these immune cells were reduced in the peripheral blood, they were likely to be involved with protection after being directed to lymphoid and injection sites. Humoral immune responses are likely to be relevant to the protection conferred by the inactivated vaccine, as it was also able to induce high mucosal IgA and seric IgY levels.
The analysis of the fluctuation of immune parameters in the same animals through a long experimental period contributes to the comprehension of how protection is developed within one individual and which cellular/ humoral subsets are relevant for protection.
Letícia DalBérto,Breno C. B.Beirão,Tobias FernandesFilho,11,MaxIngberman,11,CelsoFávaro Jr.,11,RosangelaTavella,Rebeca Buest de MesquitaSilva,Luiz FelipeCaron, (2015) Live and Inactivated Salmonella Enteritidis Vaccines: Immune Mechanisms in Broiler Breeders. World Journal of Vaccines,05,155-164. doi: 10.4236/wjv.2015.54018