Salmonella DNA Adenine Methylase Mutants Elicit Protective Immune Responses to Homologous and Heterologous Serovars in Chickens

Dam⁻Salmonella is attenuated for virulence in day-of-hatch chicks. Salmonella DNA adenine methylase (Dam) mutants are attenuated for virulence in mice and elicit protective (10, 16) and cross-protective (15) immune responses against murine typhoid fever. To examine whether Dam⁻Salmonella cells were attenuated for virulence, we challenged day-of-hatch chicks with either Dam⁻ or Dam⁺Salmonella entericaserovar Typhimurium UK-1 (Table1). All chicks (15 out of 15) survived that were infected on the day of hatch with 10¹⁰Dam⁻ UK-1 (MT2313) cells (Table2). In contrast, 8 of 15 chicks survived after challenge with 10⁵ Dam⁺ UK-1 (MT2315) cells. These data indicate that a mutation in damattenuated the virulence of serovar Typhimurium UK-1 in day-of-hatch chickens by ≥100,000-fold.

Immunization with Dam⁻Salmonella elicits protective immunity.To determine whether Dam⁻serovar Typhimurium elicited protective immune responses, chicks were orally vaccinated with 10⁷ CFU of Dam⁻Typhimurium UK-1 (MT2313) within 10 h of hatching and again at 1 week of age. Chicks were challenged at 5 weeks of age with 10⁸ CFU of serovar Typhimurium F98 (MT2318). Vaccine efficacy was determined by comparison of vaccinate (n = 62) and control (n = 62) quantitative cultures of the spleen, bursa of Fabricius, ileum, cecum, and feces; cultures were performed at 2, 3, 5, 7, 9, 12, and 14 days postchallenge. The mean log₁₀ CFU of homologous challenge with Typhimurium F98 by organ and day of termination for vaccinated birds relative to controls are presented in Fig. 1A. Vaccination with Dam⁻ UK-1 (MT2313) resulted in significantly lower CFU (P < 0.05) in the spleen and feces of vaccinates on all 7 postchallenge days examined. Significantly lower CFU in vaccinates relative to controls were observed in the bursa on 4 out of 7 termination days, in the ileum on 5 of 7 days, and in the cecum on 3 of 7 days.

• Open in new tab
• Download powerpoint

Fig. 1.

Dam⁻Salmonella elicits protective responses in chicken tissue sites. The protective capacity of Dam⁻ serovar Typhimurium was assessed by orally immunizing 62 chicks with 10⁷ CFU of Dam⁻ UK-1 (MT2313) within 10 h of hatch and boosted with the same dose at 1 week of age (hatched bars); 62 additional chicks remained as nonvaccinated controls (filled bars). All chicks were challenged at 5 weeks of age with 10⁸ CFU of serovar Typhimurium F98 (MT2318). Data are depicted as mean log₁₀ CFU by organ. Nine control and nine vaccinated chickens were terminated at 2, 3, 5, 7, 9, and 12 days postchallenge; 8 birds per group were terminated 14 days postchallenge. Cross-protective immunity was assessed as follows. Sixty chicks were orally vaccinated with 10⁷ Dam⁻ UK-1 (MT2313) cells within 10 h of hatching and again at 1 week of age; 60 additional chicks remained as nonvaccinated controls. All chicks were challenged at 5 weeks of age with either 10⁸ CFU of serovar Enteritidis 4973 (MT2314) or 10⁹ CFU of S. enterica serovar O6,14,24:e,h-monophasic (MT2339). Ten control and 10 vaccinated chickens were terminated at 4, 5, and 6 days postchallenge for the serovar Enteritidis challenge or 5, 6, or 7 days postchallenge for theS. enterica serovar O6,14,24:e,h-monophasic challenge. For each experiment, a separate cohort of 8 to 14 nonvaccinated, nonchallenged negative control birds were maintained and necropsied as described below. Approximately 1 g of each organ was obtained in the following order: spleen, bursa of Fabricius, ileum and ileal contents, cecum and cecal contents, and rectum and feces. Organs were weighed, homogenized, and serially diluted, and 100 μl of each dilution was plated on 1% lactose MacConkey agar plates containing kanamycin. For detection of salmonellae at concentrations below 40 CFU/g, the 1:4 dilution homogenate sample was incubated for 24 h in 9 ml of tetrathionate solution, streaked for single colonies on lactose MacConkey agar, and incubated for 24 h at 37°C. Samples positive by selective enrichment in tetrathionate broth were recorded as 10 CFU, and negative samples were recorded as 0 CFU. For tissue experiments, bacterial titers were confirmed via serial dilutions plated on 1% lactose MacConkey agar; colony serotype was confirmed via standard biochemical tests (urea, triple sugar iron, and ONPG [O-nitrophenyl-β-d-galactopyranoside]) and agglutination with appropriate antisera on two randomly selected colonies from each organ of each bird. ∗, significant difference (P < 0.05) between vaccinates and controls as assessed by the Mann-Whitney test.

Vaccinated birds also had significantly lower CFU than controls following homologous challenge with serovar Typhimurium F98 (MT2318) with maximum CFU/tissue/bird as the unit of analysis (Fig.2). All control birds had at least 40 CFU of challenge organism in at least one organ following challenge. However, no salmonellae were isolated in any organ from 7 out of 62 (11%) vaccinates; an additional 7 out of 62 (11%) vaccinates had only 10 CFU isolated from at least one organ. In vaccinates, 32 out of 62 (52%) birds had ≤10³ CFU in at least one organ, compared to 2 (3%) out of 62 controls in this category. Birds with ≤10⁴ CFU in at least one organ included 46 (74%) out of 62 vaccinates compared to only 7 (11%) out of 62 controls. Taken together, these data indicate that immunization of chicks with Dam⁻ serovar Typhimurium confers protection against homologous challenge with the organ or maximum CFU/tissue/bird as the unit of analysis.

• Open in new tab
• Download powerpoint

Fig. 2.

Salmonella Dam⁻ vaccine elicits protective responses as determined by the maximum CFU/tissue/bird analysis. Vaccinated (open boxes) and nonvaccinated (closed boxes) chicks were challenged as described in the Fig. 1 legend. The maximum CFU from the five tissues examined for each bird determined the category into which the bird was included. For example, if no CFU were found in the spleen, bursa, ileum, or cecum but 10⁶ CFU were found in feces, the bird was included in the ≤10⁶ CFU category.

Immunization of chicks with Dam⁻Salmonella elicits cross-protective immunity.Next, we examined the cross-protective capacity of chicks immunized with Dam⁻Salmonella. Sixty chicks were orally vaccinated with 10⁷ Dam⁻ UK-1 (MT2313) cells within 10 h of hatching and again at 1 week of age; 60 additional chicks remained as nonvaccinated controls. All chicks were challenged at 5 weeks of age with either 10⁸ CFU of serovar Enteritidis 4973 (serogroup D; MT2314) or 10⁹ CFU of serovar S. enterica O6,14,24:e,h-monophasic (serogroup H; MT2339). Figure 1B and C show the mean log₁₀ CFU of serovars Enteritidis 4973 and S. entericaO6,14,24:e,h-monophasic challenge organisms by organ and day of termination for vaccinated birds relative to nonvaccinated controls. Six days postchallenge with serovar Enteritidis 4973 (MT2314), vaccinates had significantly lower CFU in the spleen, bursa, cecum, and feces: no challenge organisms were recovered from any vaccinated bird organs, whereas 50 to 60% of control organs were positive forSalmonella. No challenge organisms were recovered from the spleens of vaccinates on day 6 or 7 postchallenge withS. enterica serovar O6,14,24:e,h-monophasic (MT2339); in contrast, salmonellae were recovered from 19 out of 20 control spleens on these 2 days.

Vaccinated birds had significantly lower CFU than controls following heterologous challenge when the maximum CFU/tissue/bird was used as the unit of analysis (Fig. 2). No serovar Enteritidis 4973 was recovered from 18 out of 30 (60%) challenged vaccinates compared to only 7 out of 30 (23%) noninfected control birds. Vaccinated birds also showed protection against challenge with serovar S. entericaO6,14,24:e,h-monophasic, as 7 out of 30 (23%) vaccinates had ≤100 CFU in at least one organ; no control birds were in this category. Taken together, these data indicate that chicks vaccinated with Dam⁻ serovar Typhimurium UK-1 elicited cross-protective immune responses to challenge with serovars Enteritidis 4973 andS. enterica O6,14,24:e,h-monophasic for the organ or maximum CFU/tissue/bird as the unit of analysis.

The safety of the food supply can be compromised by large-scale animal husbandry, agricultural methods, and distribution practices that are prone to microbial contamination. This public health problem has been recently exacerbated by the emergence of pathogens that are resistant to multiple antibiotics and/or cause more debilitating forms of disease (e.g., Escherichia coli O157:H7 and Salmonellaserovars Enteritidis and Typhimurium DT104). Vaccination of chickens offers a practical and economically feasible approach to reducing contamination of poultry products. Here, we show that anS. enterica serovar Typhimurium Dam⁻ mutant was severely attenuated for virulence in day-of-hatch chicks. Additionally, chicks immunized with this Salmonella Dam⁻vaccine strain exhibit protective immune responses against homologous and heterologous Salmonella serotypes that are implicated inSalmonella infection of poultry. Vaccines based on altered levels of Dam activity may prove effective in controllingSalmonella contamination of poultry, meat, and dairy products derived from animals susceptible to Salmonellainfection and colonization.

Enumeration of salmonellae isolated from the visceral organs and intestinal tract of vaccinated and nonvaccinated chickens challenged with virulent serovars Typhimurium or Enteritidis or S. enterica O6,14,24:e,h-monophasic was used to determine the degree of protection associated with vaccination. Significantly lower mean log₁₀ CFU were observed in visceral organs and the gastrointestinal tract of vaccinates versus nonvaccinates. Comparison of these results with challenge studies for other vaccines is problematic as the outcome of infection varies greatly with challenge strain, inoculation and immunization dose, use of multiple (booster) immunizations, the age of the birds at vaccination and challenge, statistical analysis of the data, etc. (11, 17, 18). Previous studies using live attenuated Salmonella aroA(11, 17, 18) and Δcya Δcrpmutants (12) showed reduced visceral invasion and colonization of the gastrointestinal tract in chickens by homologous and, to a lesser extent, heterologous challenge strains. Oral vaccination with attenuated aroA mutants of serovar Typhimurium (3) or Enteritidis (6, 7) reduced fecal shedding following homologous challenge, but not heterologous challenge (8). Vaccination with serovar Typhimurium Δcya Δcrp conferred protection against intestinal and visceral invasion by both homologous and heterologous challenge serotypes (12). Moreover, this vaccine also provided protection against intestinal, visceral, reproductive tract and egg colonization by Salmonella for at least 11 months postvaccination, with no effect on egg production (13).

Results of this study are promising in that significant protection was observed following homologous and heterologous challenge at high challenge doses. It should be noted that a single challenge may not reflect the field situation wherein animals can be exposed to various doses of several virulent serovars alone and in combination. That said, multiple and/or continuous exposures to several serovars in the field situation do not necessarily result in susceptibility of immunized animals to disease: repeated exposures may contribute to the maintenance of heightened levels of protection in vaccinated hosts.

The data presented here suggest that vaccines based on altered DNA methylation may reduce preharvest Salmonella contamination in poultry, ultimately decreasing the potential for food-borne transmission of this pathogen to humans. DNA methylation plays a role in the virulence of a wide variety of pathogens of the gamma subdivision of proteobacteria, including Salmonella (murine typhoid; 10, 15), Yersinia pseudotuberculosis(murine bacteremia; 21), and Vibrio cholerae(cholera; 21). Additionally, DNA methylation is required for the virulence of Brucella abortus (fetal calf abortion;30) via CcrM, a cell-cycle regulated DNA adenine methyltransferase present in members of the alpha group of proteobacteria (28, 33). Since Dam and CcrM affect the virulence of such distantly related pathogens, the function of DNA methylation in virulence may emerge as a general theme in bacterial pathogenesis.

The role of DNA methylation in virulence and the elicitation of protective immune responses may rely on its capacity as a global regulator of gene expression (16, 22, 24, 25, 26, 28, 29). Dam regulates the production of a number of adhesins in E. coli (23, 32) and Salmonella(27), as well as several genes required forSalmonella infection (14, 16). Such ectopic gene expression may result in the production of an expanded repertoire of antigens that contribute to the heightened immunity seen in vaccinated animals (15, 16, 21). Thus, dysregulation of Dam activity may be a means to elicit protective immune responses directed against diverse pathogens that infect a wide variety of animal hosts (24, 25).