Control of Salmonella enterica serovar Enteritidis in laying hens by inactivated Salmonella Enteritidis vaccines.

Salmonella Enteritidis is one of the agents that is responsible for outbreaks of human foodborne salmonellosis caused by Salmonella Enteritidis and is generally associated with the consumption of poultry products. Inactivated Salmonella Enteritidis cell vaccine is one of the available methods to control Salmonella Enteritidis in breeders and laying hens, however results in terms of efficacy vary. This vaccine has never been tested in Brazil, therefore, the present work was carried out to assess three commercial inactivated Salmonella Enteritidis vaccines allowed in Brazil. Four hundred white light variety commercial laying hens were obtained at one-day-of age. At eight weeks old, the birds were divided into four groups with one hundred animals each. Birds from three groups (V1, V2 and V3) received different intramuscular vaccines, followed by a booster dose at 16 weeks of age. Birds from another group (CG) were not vaccinated. When the laying hens were 20, 25 and 31 weeks old, 13 from each group were transferred to another room and were challenged by inoculating 2 mL neat culture of Salmonella Enteritidis. On the second day after each challenge, the caecal contents, spleen, liver and ovary of three birds from each group were analyzed for the presence of Salmonella Enteritidis. Twice a week a cloacal swab of each bird was taken and all eggs laid were examined for the presence of Salmonella Enteritidis. After four consecutive negative cloacal swabs in all the groups, the birds were sacrificed so as to examine the liver, caecal contents and ovaries. Overall, the inactivated vaccine used in group V3 reduced Salmonella Enteritidis in the feces and eggs. A very small amount of Salmonella was found in the spleen, liver, ovary and caeca of the birds in the four groups during the whole experiment. In general, inactivated Salmonella Enteritidis vaccines was able to decrease the presence of Salmonella Enteritidis in the birds and in the eggs as well. Nevertheless, they must be associated with general hygiene and disinfection practices in poultry husbandry.

INTRODUCTION

In many countries, outbreaks of human salmonellosis have been occurring since the 1980s. These events are mostly related to the consumption of poultry products, specially eggs and food containing raw eggs contaminated by Salmonella enterica serovar Enteritidis (3,26,28,35).

Salmonella Enteritidis was introduced in poultry flocks mainly by vertical transmission. Once Salmonella Enteritidis reaches a flock of birds it is easily disseminated through the feces (11,34) and remains in the environment (16,40). Therefore, commercial birds may be contaminated throughout their lives. The Salmonella control program should pay attention to the vertical via in addition to other measures taken during the birds’ life. Inactivated Salmonella Enteritidis vaccines have been used in several countries (9,21,22,37,39) and in some of them they have been used in breeder flocks (13,30,39).

The inconvenience of using inactivated Salmonella Enteritidis vaccines is the need for individual application and the reaction caused by the lipopolysaccharide bacterium plus the adjuvant vaccine. Nevertheless, there is a chance of including these vaccine antigens in other polyvalent inactivated preparations that have already been adopted. In addition, since they do not have live Salmonella Enteritidis cells there is no harm to public health.

The efficacy of vaccine preparation is judged by the level of intestinal and systemic colonization, morbidity and mortality rates after vaccination and experimental infection using the oral or parenteral routes of administration. However, the level of protection depends on the challenge strain, the route of administration, infection dose, bird age and species/line/breed. Consequently, it is difficult to compare the efficacy of the currently available vaccine preparations precisely (37).

Inactivated vaccines have been used to control non-specific host Salmonella infections in poultry with varying success (9,17,21,22,30,33,36). Thus, several publications are favorable to their application due to the reduction of fecal shedding and decrease in organ colonization and contamination of eggs. Single oral or intramuscular immunization with formalin-inactivated S. Enteritidis at 2 weeks of age decreases fecal shedding and organ colonization of Salmonella Enteritidis after oral infection with 109 colony forming units (CFU) at 6 weeks of age (29). The vaccination of hens with oil-emulsion inactivated S. Enteritidis vaccine reduced fecal shedding of Salmonella Enteritidis after the challenge. In vaccinated hens, 58% of fecal samples were positive, while in unvaccinated hens, 81% were positive (22). Laying hens vaccinated intramuscularly with a commercial inactivated Salmonella Enteritidis vaccine and challenged intravenously with Salmonella Enteritidis culture, produced less Salmonella Enteritidis positive eggs (54/439 batches of eggs) than the unvaccinated ones (99/252 batches) (39).

The immunization of 38 week old laying hens with an inactivated S. Enteritidis vaccine, followed by a booster four weeks later, reduced colonization of ovary, spleen and fecal shedding of Salmonella Enteritidis after intravaginal challenge. After the challenge, 19% of the eggs laid by vaccinated hens were positive, resulting in a significantly lower frequency than in unvaccinated hens (37%) (33). On the other hand, in a field trial conducted in 10 laying hen flocks, there was no difference in the recovering of Salmonella Enteritidis from bird organs and the environment, despite the administration of inactivated Salmonella Enteritidis vaccine (17). In another field study, in which inactivated Salmonella Enteritidis vaccine was administered to laying hens, Salmonella Enteritidis although not completely eliminated, was reduced in the flocks (15). According to Inoue (27) broiler chicks from vaccinated breeder flock shedded less Salmonella Enteritidis than those from unvaccinated breeder flock after experimental challenge on the first day of life.

Publications on Salmonella control by vaccines present approaches which are much more suitable to experimental conditions than to the real situation in the field. In this study, three commercial vaccines containing inactivated Salmonella Enteritidis cells in oil-emulsion were assessed, trying to simulate field conditions.

MATERIAL AND METHODS

Bacteria

The challenge was carried out with a mutant strain of Salmonella Enteritidis PT4 resistant to nalidixic acid and spectinomycin (SE Nalr/Specr). Bacterial cultures were set in 10 mL LB broth (Difico-244620) incubated in a shaking incubator (100 rev/min) at 37°C overnight. This culture contained approximately 2.13 x 109 CFU/mL.

Experimental animals

The experiment was carried out with a white light variety of commercial laying hens (Hyline W-36). Four-hundred birds were obtained at one-day-of age. They were reared and fed according to producer recommendations. At 8-weeks they were divided into four groups (V1, V2, V3 and CG) with 100 birds each.

On arrival, the birds were inspected for Salmonella sp according to Zancan et al. (40).

Vaccines

Three commercial vaccines (V1, V2 and V3) were used which are produced by different companies allowed for use in breeder and commercial laying hen flocks. They contained inactivated Salmonella Enteritidis cells in oil-emulsion. At 8 and 16 weeks of age, the birds in each group were vaccinated intramuscularly as recommended by the manufacturer.

Experimental design

Groups V1, V2 and V3 were vaccinated with different vaccines and CG group received no vaccine.

When birds were 20, 25 and 31 weeks old, 13 from each group were transferred to another room and were challenged by being inoculating with 2 mL neat culture of Salmonella Enteritidis Nalr/Specr.

On the second day after each challenge, the caecal contents, spleen, liver and ovary of three birds from each group were analyzed for Salmonella Enteritidis Nalr/Specr. Twice a week a cloacal swab was taken from each bird and all eggs laid were examined for the presence of Salmonella Enteritidis Nalr/Specr. Birds were sacrificed for the examination of liver, caecal contents and ovaries after four consecutive negative results of cloacal swab examination in all groups.

Bacteriological analysis

The bacteriological analysis was carried out as described by Barrow & Lovell (10) with some modification. The Salmonella Enteritidis Nalr/Specr fecal shedding was inspected by cloacal swabs, which were placed in selenite broth (CM 395, Oxoid) containing novobiocin (40 μg/mL) (SN) incubated overnight at 37°C before being plated on Brilliant Green agar (CM 263, Oxoid) containing sodium nalidixate (100 μg/mL) and spectinomycin (100 μg/mL) (BGA NalSpc). In the absence of growth, new plating was performed from the incubated swab. Eggs collected during the experiment were dropped into sterile glass jars, the shell broken and contents mixed by agitation. Yolk from the ovaries was collected in a jar with SN broth. The jars were incubated at 37°C overnight, and their contents were plated on BGA NalSpc and incubated at 37°C. Samples from spleen and liver were homogenized in a pestle and mortar. The tissue homogenates and the caecal contents were mixed and diluted in phosphate-buffered saline, pH 7.4. The viable count of Salmonella Enteritidis NalrSpcr in the samples was estimated by plating aliquots of decimal dilutions on BGA NalSpc incubated overnight at 37°C. When no Salmonella Enteritidis was found, the first dilution of the sample was added to an equal volume of double-strength SN broth, which was incubated at 37°C overnight and plated on BG NalSpc agar.

RESULTS

Table 1 shows the results concerning the presence of Salmonella Enteritidis in the spleen, liver and caecal contents two days after each challenge. The presence of Salmonella Enteritidis was similar in the liver and spleen among groups. It was only in the third challenge that Salmonella Enteritidis counting in the caecal contents differed between group V1 and the Control Group (p < 0.05). Salmonella Enteritidis was not recovered from the ovaries. In the second trial, Salmonella Enteritidis was isolated from the caecal contents of two birds, one from the V1 group and the other from the V2 group. In the third trial, Salmonella Enteritidis NalrSpcr was found in the liver of one bird from the V2 group, in the caecal contents of two birds from the V2 group and in caecal contents of one bird from the CG group.

Table 1:

Means of viable count (Log10) of Salmonella Enteritidis Nalr/Specr of three birds in spleen, liver and caecal contents two days after each challenge.

Tissue	Challenge	Treatments
		V1	V2	V3	CG
Spleen	1st	2.33 A	< 2.00 A	< 2.00 A	< 2.00 A
	2nd	< 2.00 A	< 2.00 A	< 2.00 A	< 2.00 A
	3rd	< 2.00 A	< 2.00 A	< 2.00 A	2.67 A
Liver	1st	2.33 A	< 2.00 A	< 2.00 A	2.30 A
	2nd	< 2.00 A	< 2.00 A	< 2.00 A	< 2.00 A
	3rd	< 2.00 A	< 2.00 A	< 2.00 A	2.33 A
Cecal contents	1st	3.36 A	2.33 A	< 2.00 A	3.i9 A
	2nd	2.53 A	2.59 A	3.07 A	3.67 A
	3rd	2.67 B	3.57 AB	3.43 AB	4.50 A

Salmonella Enteritidis fecal shedding data are in Table 2. Only in the first trial was the difference between V3 group and the control group significant (p <0.05).

Table 2:

Recovery of Salmonella Enteritidis Nalr/Specr from cloacal samples after each challenge of vaccinated (V1, V2 and V3) and unvaccinated (CG) birds.

Dpi	1st Challenge												2nd Challenge												3rd Challenge
	V1			V2			V3			CG			V1			V2			V3			CG			V1			V2			V3			CG
	D	E	T	D	E	T	D	E	T	D	E	T	D	E	T	D	E	T	D	E	T	D	E	T	D	E	T	D	E	T	D	E	T	D	E	T
2	0	1	1	0	1	1	0	0	0	1	2	3	0	0	0	0	2	2	0	1	1	0	2	2	0	0	0	0	1	1	0	0	0	0	0	0
7	0	0	0	0	3	3	0	0	0	0	2	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2	2	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
13	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
16	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
20	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
∑	1ab/60			4a/60			0b/60			5a/60			0a/60			2b/60			1a/60			2a/60			0a/60			3a/60			0a/60			0a/60

The detection of Salmonella Enteritidis in eggs is shown in Table 3. In general, birds from the control group produced more contaminated eggs than birds from other groups, but the recovery means were variable. In the first challenge, the vaccine used in the laying hens from the V3 group reduced the presence of Salmonella Enteritidis (p < 0.05) and in the last trial all vaccines reduced the presence of Salmonella Enteritidis in eggs (p < 0.05).

Table 3:

Recovery of Salmonella Enteritidis Nalr/Specr from egg samples after challenge of vaccinated (V1, V2 and V3) and unvaccinated (CG) birds.

Dpi	1st Challenge				2nd Challenge				3rd Challenge
	V1	V2	V3	CG	V1	V2	V3	CG	V1	V2	V3	CG
1	1	5	1	5	3	3	0	3	2	1	1	4
2	1	0	0	0	0	0	0	1	0	0	0	0
3	0	0	1	2	0	0	1	1	0	0	0	1
4	0	1	0	0	2	1	3	3	0	0	0	1
5	0	0	0	0	0	0	0	0	0	0	0	1
6	1	0	0	0	0	0	0	0	0	0	0	0
7	0	0	0	0	0	0	0	0	0	0	0	1
8	0	1	0	1	0	0	0	0	0	0	0	0
9	0	0	0	0	0	0	0	0	0	1	0	0
10	0	0	0	0	0	0	0	0	0	0	0	0
11	0	0	0	0	0	0	0	0	0	0	0	0
12	0	0	0	0	0	0	0	0	0	0	0	0
13	0	0	0	0	0	0	0	0	0	0	0	0
14	0	0	0	0	0	0	0	0	0	0	0	0
15	0	0	0	0	0	0	0	0	0	0	0	0
16	0	0	0	0	0	1	0	0	0	0	0	0
Pos.*	3ab/ 123	7ab/136	2b/126	8a/122	5a/125	5a/126	4a/121	8a/124	2b/145	2b/149	1b/145	8a/133

Dpi= days after infection;

Pos. * = Number of positive samples/ total eggs examined; ab = Means followed by different letters in the line for each challenge indicate significant differences by Chi-Square’s test (p < 0.05).

DISCUSSION

Human foodborne salmonellosis has been strongly associated with eggs and egg products contaminated with Salmonella Enteritidis (3,26,28,35,38), although many efforts have been made for the control of Salmonella Enteritidis in laying hens to prevent egg contamination. Following oral infection, Salmonella Enteritidis colonizes the intestinal tract and may invade organs such as the liver, spleen, ovary and heart (10,20). In this study, quite a few Salmonella Enteritidis organisms were found in the liver and spleen (Table 1) of birds from all the groups during the experiment, with no difference between vaccinated and unvaccinated birds (p > 0.05). These findings might have been expected since Berchieri Jr. et al. (12) demonstrated that Salmonella Enteritidis did not persist longer in mature birds. It was also showed that 20-40 weeks old laying hens are naturally more resistant to Salmonella Enteritidis infection (25). In contrast, the beneficial effect of Salmonella Enteritidis inactivated oil-emulsion vaccines in preventing organ colonization by Salmonella Enteritidis was demonstrated by Nakamura et al. (33) and Gast et al. (21).

In the present study, Salmonella Enteritidis fecal shedding (Table 2) did differ between the V3 group and the control group in the first challenge (p < 0.05). Depending on the composition, the inactivated vaccine can decrease of Salmonella Enteritidis fecal shedding (29). A study conducted by Barbour et al. (5), comparing six inactivated Salmonella Enteritidis vaccines, showed a variable decrease in the Salmonella Enteritidis fecal shedding. These authors suggested that several factors regarding the type and composition of the adjuvant, strain of Salmonella Enteritidis and inactivation method, could be responsible for this variation, and could explain the results depicted in Table 2. Unfortunately, not all information on the commercial vaccines used was available.

It is proposed that cell-mediated immunity is more important for tissue clearance of Salmonella Enteritidis, while humoral response seems to be responsible for the reduction of intestinal colonization (6,7,24,32,37). One of the criteria for an ideal vaccine is the promotion of bird protection against mucosal and systemic infection by effective stimulation of both immune responses (37). Some authors showed that the Salmonella Enteritidis inactivated vaccines induce only a good humoral immune response, which reduces the intestinal colonization by Salmonella Enteritidis (4,6,14,18,24,32). This might be in the reason for the decrease of Salmonella Enteritidis in bird feces (caecal Salmonella Enteritidis counting and cloacal swabs) of the V1 and V3 groups (Tables 1 and 2) in the present work.

There was a positive effect of the vaccination of birds in the V3 group in the first challenge and in all the groups of vaccinated birds in the third challenge (Table 3). These results are in agreement with previous works in which the presence of Salmonella Enteritidis in eggs laid was reduced by a vaccination program using an inactivated vaccine (21, 30 e 39). About 68.2% of the outbreaks of human foodborne salmonellosis caused by Salmonella Enteritidis is related to egg and food containing raw eggs (31), despite the fact that one out of 20,000 eggs laid is supposed to be contaminated by Salmonella Enteritidis (19). Therefore, any reduction is very welcome. In addition, inactivated vaccines may induce enough passive immunity to protect the progeny (27).

In the first and second challenges, it was possible to observe some correlation between Salmonella Enteritidis in feces and in eggs. Similar results were observed by Gast & Beard (20) and Woodward et al. (39). According to Barrow & Lovell (10) eggs are mainly contaminated with Salmonella Enteritidis by contact with fecal material in the cloacae, although transovarian contamination also occurs.

Intrinsic factors in the eggshell and in the albumen may interrupt the multiplication of Salmonella. At low temperatures, Salmonella can be kept viable in the yolk (2) and can multiply in 72hs, at 15°C (1), and in hot weather these factors become less active. Thus, the best way to prevent human foodborne salmonellosis is by taking precautions during the bird’s lifetime. It is known that Salmonella Enteritidis may persist in vaccinated flocks of laying hens (15). But in view of the results obtained in this work, a vaccination program to control the presence of Salmonella Enteritidis in laying hens can be adopted as an additional tool to minimize the presence of Salmonella Enteritidis in eggs, in association with general practices of hygiene and disinfection in poultry husbandry.