Development of a novel in-water vaccination protocol for DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine in adult sheep

Vaccine 30 (2012) 1481–1491

Contents lists available at SciVerse ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

Development of a novel in-water vaccination protocol for DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine in adult sheep V.L. Mohler a , D.M. Heithoff b , M.J. Mahan b , M.A. Hornitzky c , P.C. Thomson a , J.K. House a,∗ a

University of Sydney, Faculty of Veterinary Science, Camden, NSW 2570, Australia Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106, USA c Elizabeth Macarthur Agricultural Institute, Industry & Investment NSW, Menangle, NSW 2570, Australia b

a r t i c l e

i n f o

Article history: Received 15 July 2011 Received in revised form 11 November 2011 Accepted 17 December 2011 Available online 31 December 2011 Keywords: Salmonella ovine vaccine DNA adenine methylation Dam

a b s t r a c t Intensive livestock production is associated with an increased incidence of salmonellosis. The risk of infection and the subsequent public health concern is attributed to increased pathogen exposure and disease susceptibility due to multiple stressors experienced by livestock from farm to feedlot. Traditional parenteral vaccine methods can further stress susceptible populations and cause carcass damage, adverse reactions, and resultant increased production costs. As a potential means to address these issues, in-water delivery of live attenuated vaccines affords a low cost, low-stress method for immunization of livestock populations that is not associated with the adverse handling stressors and injection reactions associated with parenteral administration. We have previously established that in-water administration of a Salmonella enterica serovar Typhimurium dam vaccine conferred significant protection in livestock. While these experimental trials hold significant promise, the ultimate measure of the vaccine will not be established until it has undergone clinical testing in the field wherein environmental and sanitary conditions are variable. Here we show that in-water administration of a S. Typhimurium dam attenuated vaccine was safe, stable, and well-tolerated in adult sheep. The dam vaccine did not alter water consumption or vaccine dosing; remained viable under a wide range of temperatures (21–37 ◦ C); did not proliferate within fecal-contaminated trough water; and was associated with minimal fecal shedding and clinical disease as a consequence of vaccination. The capacity of Salmonella dam attenuated vaccines to be delivered in drinking water to protect livestock from virulent Salmonella challenge offers an effective, economical, stressor-free Salmonella prophylaxis for intensive livestock production systems. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Despite good husbandry practices, salmonellosis continues to be a significant problem in intensive production systems. Much of this is caused by increased pathogen exposure and disease susceptibility attributed to cumulative stressors experienced by stock on farm (mustering, yarding, food and water deprivation prior to transport), during transport (food and water deprivation, environmental stress), and in sale yards (co-mingling, pathogen exposure). Good animal husbandry at the feedlot is directed at minimizing stress following feedlot entry by minimizing handling to allow animals to settle, eat, drink and establish social groups. Parenteral administration of vaccines at feedlot entry require livestock to be

∗ Corresponding author. Tel.: +61 2 4655 0777; fax: +61 2 4655 1212. E-mail addresses: [email protected], [email protected] (J.K. House). 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.12.079

handled by personnel which can further stress susceptible populations and cause carcass damage, adverse reactions, and resultant increased production costs [1–5]. In-water delivery of live attenuated vaccines affords a potential low cost, low-stress approach for immunization of livestock populations that is not associated with the adverse handling stressors and injection reactions associated with parenteral administration. The use of in-water medication and vaccination to control and prevent disease is a common practice in intensively managed poultry [6] and swine [7–9]. Additionally, use of impregnated baits and sachets have been employed to successfully vaccinate feral dogs and wildlife against rabies [10–12] and wild deer populations against tuberculosis [13]. Conversely, very little information has been reported on the use of in-water or oral vaccination in livestock species such as cattle, goats and sheep, wherein the use of such practices may improve animal health and well-being as well as conferring significant reductions in pre- and post-harvest bacterial loads.

1482

V.L. Mohler et al. / Vaccine 30 (2012) 1481–1491

Recent innovations have resulted in the development of modified live Salmonella vaccines that elicit cross-protective immunity to homologous and heterologous salmonellae serovars [14–20]. These vaccine candidates are attenuated live Salmonella that contain mutations in global regulatory networks that are thought to increase antigen exposure and/or modulate the immune response to confer protection. Salmonella enterica serovar Typhimurium, containing loss of function mutations in the gene encoding the DNA adenine methylase (dam), are capable of providing protection against a diversity of salmonellae and are well tolerated when applied as modified live vaccines in mice [16,17,21,22], poultry [23,24], sheep [25] and calves [26–28]. Moreover, administration of the Salmonella dam vaccine via drinking water conferred protection in livestock, providing a potential low cost and low-stress immunization method for livestock populations [25]. While these experimental trials are encouraging, commercial vaccine utility requires testing under conditions that simulate that found in the field, whereby adverse environmental and sanitary conditions may affect vaccine stability and safety. Here we show that a protective S. Typhimurium dam attenuated vaccine was readily consumed in drinking water by adult sheep; remained viable under a wide range of temperatures; did not proliferate in drinking water contaminated with animal feces; and has a tenfold safety margin when administered per os to adult sheep.

to enrollment on study for qualitative detection of Salmonella. Approximately 1 g of feces was placed into 10 mL mannitol selenite (MSB) broths, incubated for 24 h at 37 ◦ C and then plated on to plain XLD. Suspect Salmonella colonies were sub-cultured for pure growth, tested for agglutination with Salmonella Test Kit [for detection of Salmonella Antigen (DR1108A) (Oxoid Ltd., UK)] and then assessed using standard biochemical tests (urea, TSI, ONPG). Salmonella isolates were sent to the Institute of Medical and Veterinary Science, Salmonella Reference Laboratory (SA, Australia) for serotyping. No Salmonella were detected in the sheep during the acclimation period. Sheep were transported to the Medium Security Animal Housing Facility at Elizabeth MacArthur Agricultural Institute (EMAI), Department of Industry & Investment (NSW, AU), and randomly assigned to experimental groups. Sheep were individually housed in wire mesh pens on raised mesh flooring. Each pen was equipped with three plastic all-purpose buckets for administration of water and feed. Biosafety protocols approved by the Australian Office of the Gene Technology Regulator (OGTR) for containment of PC2 level organisms were followed to prevent cross contamination between groups and prevent release of the vaccine into the environment. Sheep received a diet of oaten chaff (8 megajoules of metabolisable energy per kg of dry matter [MJ ME/kg DM]) and commercially prepared grain pellet (12 MJ ME/kg DM). Sheep were offered 600 g of oaten chaff and 300 g of grain pellets during morning, roughly 8–9 MJ ME/d.

2. Materials and methods 2.1. Bacterial strains and growth conditions Experiments were carried out with S. Typhimurium dam vaccine MT2313, which contains an insertion element dam102::Mud-Cm (encoding chloramphenicol resistance) in Typhimurium strain UK1 (obtained from Roy Curtiss III [15]). The wild-type Typhimurium strain MT2315 is a virulent derivative of strain UK-1, containing a Lac+ MudJ transcriptional fusion which is used to discern it from other Salmonella which are inherently Lac− [23,29]. Strains used in these stability studies were grown overnight in Luria broth at 37 ◦ C. Cells were re-suspended in phosphate buffered saline (PBS), 137 mM 8.0 g/L of NaCl, (8.45 mM) 1.2 g/L Na2 HPO4 , (1.5 mM) 0.2 g/L KH2 PO4 , and (2.7 mM) 0.2 g/L of KCl pH 7.2–7.4 for these studies unless otherwise described. Dose titers were confirmed via serial dilutions plated on Luria agar plates; colony serotype was confirmed via standard biochemical tests (urea, TSI, ONPG) and agglutination with serogroup B specific antisera [somatic O antisera group B, factors 1, 4, 5, 12, and 27 (BD Diagnostics, USA)]. 2.2. Salmonella isolation from test solutions Samples were serially diluted into PBS 1:10, plated onto xylose lysine deoxycholate (XLD) and XLD containing chloramphenicol at 20 ␮g/mL, to select for the Typhimurium dam vaccine (XLDCHL) and incubated for at least 24 h at 37 ◦ C. Several colonies from each experiment were evaluated using standard biochemical tests (urea, TSI, ONPG) and agglutinated with serogroup B specific antisera [somatic O antisera group B, factors 1, 4, 5, 12, and 27 (BD Diagnostics, USA)]. 2.3. Safety dose range of S. Typhimurium dam vaccine when administered orally to adult Merino wethers 2.3.1. Sheep and husbandry Merino wethers, approximately 2–4 years in age, were sourced from a single flock in New South Wales, Australia. The flock was routinely monitored for and determined to be free of Johne’s and virulent foot rot. Fecal samples were collected from all sheep prior

2.3.2. Clinical assessment Clinical parameters evaluated twice daily included attitude, water consumption, and fecal characteristics. Rectal temperatures were recorded in the mornings prior to feeding twice a week during the week prior to vaccination and daily following vaccination. The normal range of rectal temperatures for the sheep used in these experiments based on the pre-vaccination mean ± two standard deviations was 38.3–39.5 ◦ C. Clinical pyrexia was defined as a sheep with a rectal temperature above 40 ◦ C [30,31]. Attitude and fecal composition were scored on an ordinal scale. Attitude was scored on a scale of 0 to 4 as follows: 0 = standing to be fed (bright, alert and responsive [BAR]), 1 = standing, quiet and alert (QAR), 2 = stands with head low, 3 = stands with stimulus or stands but leaning on pen gates, and 4 = unable to stand or lateral recumbency. Fecal scores were assessed on a scale from 0 to 4 as follows: 0 = normal (pellets), 1 = soft (clumped pellets), 2 = very soft (formed but no pellets), 3 = soft pat, or 4 = diarrhea (liquid, watery or containing blood or casts). Appetite was recorded in grams of chaff and pellets not consumed over each 24 h feeding period. Percent refusal of chaff and pellets was determined for each animal following vaccination. An attitude score of 4 at any time of the day or failure to consume chaff or pellets (100% refusal of feed) for three consecutive feedings constituted grounds for euthanasia. The study protocol was approved by the Animal Ethics Committee at EMAI. 2.3.3. Experimental design A total of 21 sheep were used in the conduct of this study. The sheep were randomly assigned to three dose groups on day −10 during the acclimation period: unvaccinated controls (n = 7; [Control]); 107 CFU oral bolus vaccinates (n = 7; [107 OB]); and 108 CFU oral bolus vaccinates (n = 7; [108 OB]). Doses of vaccine selected for this safety study were based on S. Typhimurium dam vaccine dose levels used to provide protective efficacy in neonate calves [26–28]. Prior to vaccination, all sheep were food- and water-fasted overnight. The sheep in the 107 OB and 108 OB dose groups received the S. Typhimurium dam vaccine per os reconstituted into 10 mL of PBS at t = 0 h. The unvaccinated controls received 10 mL of PBS per os at t = 0 h.

V.L. Mohler et al. / Vaccine 30 (2012) 1481–1491

2.3.4. Fecal sampling Prior to initiating the study fecal samples collected from the sheep were cultured for Salmonella as described in Section 2.3.1, Salmonella was not detected prior to vaccination of the sheep used in this trial. Fecal samples were assessed for shedding of the dam vaccine at 3, 7, 10 and 14 post-vaccination. Between 2 and 5 g of feces were collected per rectum of each sheep using individual disposable latex gloves. The fecal material was homogenized as a 1:4 dilution in sterile PBS, serially diluted, plated on XLD-CHL to select for the vaccine strain, and incubated for 24 h at 37 ◦ C. Additionally, 1 mL of the homogenized sample was placed into 9 mL of MSB, incubated for 24 h at 37 ◦ C, and streaked for single colonies onto XLD-CHL. The number of Salmonella organisms present in the fecal sample was calculated based on the sample weight, dilution factor, and number of colonies counted. Two colonies from each Salmonella positive fecal culture were sub-cultured and tested with O-antigen specific antisera to verify isolate identification. 2.3.5. Isolation of S. Typhimurium dam vaccine from ovine tissues Sheep were euthanized and immediately necropsied using standard techniques 12 d following vaccination. Approximately 1–2 g of tissue was obtained from the mesenteric lymph node (MLN), liver, and spleen of each animal. Tissue sections were homogenized as a 1:4 dilution in sterile PBS, serially diluted, plated onto XLD-CHL, and incubated for 24 h at 37 ◦ C. Additionally, 1 mL of the homogenized sample was placed into 9 mL of MSB enrichment media, and incubated for 24 h at 37 ◦ C. Colony counts were enumerated and MSB enrichments were streaked onto XLD-CHL. Samples positive by selective enrichment in MSB were recorded as 40 CFU/g and negative samples were recorded as 1 CFU/g. 2.4. Stability of S. Typhimurium dam vaccine strain in water Twenty 50 mL polypropylene tubes were filled with 9 mL of sterile Milli-Q® water (DIH2 O). Three tubes each were inoculated with 1 mL of either S. Typhimurium dam vaccine (dam− ) or S. Typhimurium dam+ (dam+ ) strains prepared following methods in Section 2.1 and reconstituted in PBS to dose concentrations of 104 , 106 and 108 CFU/mL. The remaining two tubes served as negative controls. Samples were incubated at room temperature with loose caps. Viability of the bacteria strains was assessed 24, 48, 72, and 120 h post inoculation. Fixed effects used to analyze viability and stability of the organisms were time, strain and inoculation dose. 2.5. Stability of the S. Typhimurium dam vaccine in trough water with and without fecal contamination Ten liters of trough water were collected from the Mayfarm Sheep Yards at the University of Sydney (Camden, NSW, Australia) for use in this experiment. Feces used in this trial were obtained from sheep and determined to be Salmonella free via inoculation into enrichment cultures. At time zero (t = 0), 3 mL of S. Typhimurium dam vaccine was prepared following methods in Section 2.1 and reconstituted in PBS then inoculated into 500 mL polypropylene jars containing solutions of either 297 mL of sterile DIH2 O (n = 6), 297 mL trough water (TRW; n = 6) or 294 mL of trough water with 3 g sheep feces (pellets) (TRW + F; n = 6). The final concentration of the S. Typhimurium dam vaccine in each jar was approximately 104 CFU/mL. Samples were incubated at room temperature with loose caps. To determine the homogeneity of the S. Typhimurium dam vaccine in drinking water, 3 jars from each group were vortexed prior to sampling (vortex) and the remaining 3 jars from each group were sampled from the upper layer of the sample (still) at each time point. Viability of the vaccine was assessed at 6, 24 and 48 h post inoculation. Fixed effects used to analyze

1483

CFU/mL of the S. Typhimurium dam vaccine were time, solution and sampling method. 2.6. Stability and viability of the S. Typhimurium dam vaccine in buffered water In this experiment, the S. Typhimurium dam vaccine was prepared following methods in Section 2.1 and reconstituted in sterile de-ionized water then inoculated into 50 mL polypropylene tubes containing 19 mL of either DIH2 O, PBS or PBS serial diluted 1:1 with DIH2 O to give solutions of 0.5× PBS, 0.25× PBS, 0.125× PBS, 0.062× PBS or 0.031× PBS. The final concentration of the S. Typhimurium dam vaccine in each tube was approximately 105 CFU/mL at t = 0. Viability of the S. Typhimurium dam vaccine was assessed at 24 and 48 h post inoculation. Fixed effects used to analyze CFU/mL of the S. Typhimurium dam vaccine were time and PBS concentration (solution). 2.7. Effect of temperature on the viability of the S. Typhimurium dam vaccine in buffered water The S. Typhimurium dam vaccine was prepared following methods in Section 2.1 and reconstituted in PBS then inoculated into 500 mL polypropylene tubes containing 199 mL of either PBS (n = 6), 0.2× PBS [PBS dilute 1:5 in DIH2 O] (n = 6) or DIH2 O (n = 6) test solutions. The final concentration of the S. Typhimurium dam vaccine in each jar was approximately 104 CFU/mL. Three jars from each test solution were maintained at 37 ◦ C in an incubator and 3 jars were kept at room temperature (range: 22–26 ◦ C). Viability of the S. Typhimurium dam vaccine and the pH of each the test solutions (CyberScan pH 510, Eutech Instruments, AU) were assessed at 6, 12 and 24 h post inoculation. One 200 mL jar of each solution without vaccine served as the controls. The controls were maintained at room temperature throughout the experiment and pH measurements were performed in quadruplet and sterility was monitored at each time point. Fixed effects used to analyze CFU/mL of the S. Typhimurium dam vaccine were time, test solution, temperature and pH. 2.8. Delivery of S. Typhimurium dam vaccine to adult Merino wethers via drinking water 2.8.1. Experimental design A total of 22 sheep were used in the conduct of this study. Sheep husbandry and clinical assessments were conducted following procedures outlined in Sections 2.3.1 and 2.3.2. Two vaccination trials were conducted using 15 and 7 sheep, respectively. In experiment A, 15 sheep were randomly assigned to two dose groups on day −8: 107 CFU oral bolus vaccinates (n = 7; [107 OB]) and 107 CFU in-water vaccinates (n = 8; [107 IW]). In experiment B, seven sheep were assigned to a 106 CFU in-water vaccinate group (n = 7; [106 IW]). On day 6 during the acclimation period, all sheep received drinking water containing 20% phosphate buffered saline (0.2× PBS) to determine if the buffering agent would affect consumption prior to use during the vaccine trial. Prior to vaccination, all sheep were food and water fasted overnight. The S. Typhimurium dam vaccine was delivered in the drinking water of the 106 IW and 107 IW vaccinates ad libitum over a 24 h period (t = −24 h to 0 h) via plastic drinking buckets maintained within each pen. The vaccine was inoculated into fresh water containing 20% PBS to a final volume of 6 L at the morning and evening observation periods. The target concentration of the vaccine was 106 and 107 CFU/L for the 106 IW and 107 IW vaccinate groups, respectively. Consumption of water containing the S. Typhimurium dam vaccine was recorded to the nearest 0.1 L on vaccination day.

1484

V.L. Mohler et al. / Vaccine 30 (2012) 1481–1491

The sheep in 107 OB vaccination group received the vaccine per os reconstituted into 10 mL of PBS at t = 0 h. 2.8.2. Fecal sampling and isolation of S. Typhimurium dam vaccine from ovine tissues Prior to initiating the study, fecal samples collected from the sheep were cultured for Salmonella as described in Section 2.3.4. Salmonella was not detected prior to vaccination of the sheep used in this trial. Fecal samples were assessed for shedding of the dam vaccine at 72 h post-vaccination following procedures outlined in Section 2.3.4. Sheep were euthanized and immediately necropsied using standard techniques 96 h following vaccination. Tissue samples were assessed for colonization with the dam vaccine at 72 h post-vaccination following procedures outlined in Section 2.3.5. 2.9. Statistical analysis The statistical program Genstat (12th edition, VSN International, UK) was utilized to perform residual (or restricted) maximum likelihood (REML) analysis. Data from the stability and viability studies were analyzed using a single variate repeated measures model where fixed effects of the model were time, treatment or temperature and their interactions, and the random effect was sample tube/jar, outcome variables included CFU/mL and pH. Results of the sheep vaccination trial were analyzed using a single variate, repeated measures model where the fixed effects of the model were time, treatment and their interaction, and random effects were animal and experiment (A vs. B), outcome variables included fecal shedding (CFU/g), rectal temperature, chaff and pellet refusal, and water intake. Prior to analysis, CFU data was converted to log base 10. The Wald chi-square test was used to determine significant individual effects and or significant interactions between factors. Any non-significant terms were dropped from the model and analysis repeated. Following analysis, data are presented as predicted model based means. Predicted means are those obtained from the fitted model rather than the raw sample means. This is important as predicted means represent means adjusted to a common set of variables, thus allowing valid comparison between means. A P value less than 0.05 was considered to be statistically significant. Differences between the individual means were determined by calculating an approximate least significant difference (LSD). A difference of means that exceeded the calculated LSD was considered significant. The statistical program StatsDirect (version 2.7.8, http:// www.statsdirect.com, England: StatsDirect Ltd., 2008 [32]) was utilized to perform analysis of variance and unpaired t-tests. Tissue colonization was analyzed using analysis of variance. An unpaired t-test was employed to analyze the following: the effect of vaccine dose on pyrexia in sheep following vaccination; the effect of 0.2× PBS on the water consumption in sheep; and the effect of in-water vaccination on the water consumption in sheep. 3. Results 3.1. Safety of Typhimurium dam vaccine delivered as an oral bolus to adult Merino wethers Administration of the Salmonella dam vaccine via drinking water conferred protection in livestock, providing a potential low cost and low-stress alternative for livestock immunization [25]. While these experimental trials show considerable promise, an in-water S. Typhimurium dam vaccine for commercial application requires that the vaccine is safe and well-tolerated in immunized stock. Here we established that the vaccine has a dosing safety margin by evaluating the response to the previously established protective

dose of 107 CFU [25] and a dose tenfold the protective dose in sheep. Clinical manifestations were assessed after two immunizing doses (108 OB; 107 OB) were delivered via oral bolus to adult sheep. Pyrexia (rectal temperature > 40.0 ◦ C) [30,31] was not observed in sheep from the 107 OB vaccinate group and the unvaccinated controls (Fig. 1A). These groups remained within the normal temperature range throughout the study and had significantly lower predicted mean rectal temperatures relative to those exhibited by the 108 OB vaccinates from 3 to 6 d post vaccination (P < 0.05). Pyrexia was observed in 2 of 7 sheep in the 108 OB group (P < 0.06). Mean rectal temperatures of the 108 OB vaccinates had returned to normal ranges by day 6 post vaccination. There was no significant effect of vaccination treatment on chaff (P = 0.786) and pellet appetite (P = 0.663) of the sheep over the course of the experiment and remained within the normal ranges throughout the study period. However, chaff refusal of the 108 OB vaccinates were significantly increased when compared to the unvaccinated control group on days 6 and 7 post vaccination (P < 0.05; Fig. 1B). Pellet refusal of the 108 OB vaccinates were significantly increased when compared to the unvaccinated controls on day 7 post-vaccination (P < 0.05; Fig. 1C). No abnormal attitude or fecal scores were observed in any of the vaccinated or nonvaccinated sheep. Vaccination of sheep via an oral bolus had a significant effect on sheep water consumption (P < 0.001; Fig. 1D). Vaccinated sheep had significantly increased water consumption when compared to the unvaccinated controls on day 1 post vaccination (P < 0.05). The water consumption of the vaccinates was 5.7 ± 0.79 L and 5.4 ± 0.47 L for the 107 OB and 108 OB groups, respectively, which was 219.9 and 200.7% of the mean daily water consumption during the 5 d preceding vaccination for each group. The water consumption of the unvaccinated controls was 3.8 ± 0.47 L which was 102.4% of mean water consumption. And significantly lower water consumption was observed in 107 OB vaccinates on day 3 post vaccination and in the 108 OB vaccinates on day 6 post vaccination. However, sheep in this study consumed between 5 and 16% of their body weight in water per day which was within the reported normal daily range of 5–20% [33,34]. Taken together, these data demonstrate that 107 OB immunization dose had minimal effects on chaff appetite, pellet appetite, water consumption, attitude and fecal composition of adult sheep. Sheep administered tenfold the protective vaccine dose (108 OB immunization dose) developed transient fevers and a mild brief reduction in chaff consumption 3–6 d following vaccination. The 108 OB immunization dose did not affect pellet consumption and attitude or fecal scores. Colonization of lymphoid tissue (e.g., intestine [Peyer’s patches]; mesenteric lymph nodes [MLN]; spleen) and visceral organs (e.g., liver) leads to the initiation of protective innate and adaptive immune responses. Here we assessed the ability of the S. Typhimurium dam vaccine to colonize such host tissue sites via enumeration of CFU recovered from feces, MLN, spleen, and liver at 12 d post-vaccination. Fecal shedding: No differences in fecal shedding of the dam vaccine were observed between 107 OB and 108 OB vaccinates over the entire study period (P = 0.198; Fig. 1E). However, significantly lower numbers of dam vaccine shedding (1 log less CFU/g feces) was observed in the 107 OB vaccinates when compared to the 108 OB vaccinates on day 12 post vaccination (P < 0.05). Over the 12 d study period the dam vaccine was recovered from the feces of 7 of 7 sheep in the 107 OB vaccinate group on one or more occasions and from 6 of the 7 sheep in the 108 OB vaccinate group (Fig. 1E). MLN colonization: Significantly lower numbers of the dam vaccine were observed in the MLN of the 107 OB vaccinates (1 log less CFU/g) when compared to the 108 OB vaccinates (P < 0.001; Fig. 2). Additionally, the S. Typhimurium dam vaccine was recovered from the MLN of 5 of 7 and 7 of 7 sheep from the

V.L. Mohler et al. / Vaccine 30 (2012) 1481–1491

1485

Fig. 1. Clinical assessment of adult sheep following oral immunization with S. Typhimurium dam vaccine. Seven sheep were immunized with a 10 mL oral bolus (OB) of S. Typhimurium dam vaccine strain (MT2313) in PBS at dose levels of 107 CFU (107 OB, grey triangles) or 108 CFU (108 OB, grey circles); and 7 unvaccinated sheep received a 10 mL oral bolus of PBS (white squares, control). Sheep were vaccinated at t = 0. (A) Rectal temperatures: Rectal temperatures were determined using a digital thermometer twice weekly prior to vaccination and daily following vaccination. Data is presented as predicted mean rectal temperature ± SE. The normal range (38.3–39.5 ◦ C) of rectal temperatures for sheep used in this experiment was based on the pre-vaccination mean ± two standard deviations (dashed grey lines). The threshold for pyrexia (dashed black line) reflects the published normal range for sheep [30,31]. *Rectal temperatures of the non-vaccinated controls were significantly lower than the 108 OB vaccinates from days 3 to 6 post vaccination (P < 0.05). There was no difference in the rectal temperatures of the 107 OB and 108 OB vaccinates. (B) Chaff refusal: Percent of chaff refused was determined for each animal during the vaccination period based on amount of offered chaff refused. Data are predicted mean chaff refusal ± SE. *Chaff refusal was significantly higher in the 108 OB vaccinates when compared to the non-vaccinated controls on days 6 and 7 post vaccination (P < 0.05). There was no difference in the chaff refusals of the 107 OB and 108 OB vaccinates. (C) Pellet refusal: Percent of pellet refused was determined for each animal during the vaccination period based on amount of pellet refused over offered. Data are depicted as predicted mean pellet refusal ± SE. Pellet refusal was significantly higher in the 108 OB vaccinates when compared to the non-vaccinated controls on day 6 post vaccination (P < 0.05). There was no difference in the pellet refusals of the 107 OB and 108 OB vaccinates. (D) Water consumption: Data are depicted as predicted mean water consumption in L ± SE. *The water consumption of the 107 OB and 108 OB vaccinates was significantly higher than unvaccinated controls 24 h post-vaccination (day 1) (P < 0.05). On day 3 and day 7 post vaccination the water consumption of the 107 OB vaccinates and 108 OB vaccinates, respectively, was significantly decreased when compared to unvaccinated controls (P < 0.05). (E) Fecal shedding of vaccine: Bacterial CFU in the feces was enumerated by direct and enriched colony counts as described in Section 2. Data are depicted as a predicted mean log10 CFU of dam vaccine per g of feces on days 3, 7, 10 and 12 post vaccination. *On day 12 post vaccination fecal shedding of the vaccine in the 108 OB vaccinates was significantly increased compared to 107 OB vaccinates (P < 0.05).

107 OB and 108 OB vaccination groups, respectively. Spleen and liver colonization: dam vaccine was not recovered from the spleen and liver of the 107 OB and 108 OB vaccinates. Collectively, these data indicate that 107 OB is safe, well tolerated, and capable of bypassing the rumen of adult sheep. Transient fevers and a mild transient

reduction in chaff consumption were observed in sheep when the vaccine was administered at a dose ten times the protective dose indicating that the vaccine has a good dosing safety margin to cater for potential fluctuation in the dose consumed when it is delivered in drinking water.

1486

V.L. Mohler et al. / Vaccine 30 (2012) 1481–1491

viability over time at lower inoculation doses over the 120 h study period with 82.2% and 70.4% of the original inoculums remaining viable, respectively (P < 0.05). (3) Significant CFU/mL differences were observed between dam− and dam+ strains at 24 h and from 24 to 72 h post inoculation at the 106 and 108 dose levels, respectively (P < 0.05); however, no differences were observed in the predicted mean CFU/mL between the dose levels at 120 h post inoculation, and strain alone did not have a significant effect on predicted mean CFU/mL (P = 0.728). These data demonstrate that the S. Typhimurium dam vaccine has similar viability to the dam+ strain when inoculated into DIH2 O, and inoculation dose has a significant effect on stability over the 5-d study period.

Tissue Colonization of the Vaccine (CFU/g Log10)

4.0

107 OB

*

3.0

108 OB

2.0

1.0

5/7

0.0

7/7

MLN

SPLEEN

3.3. Viability of S. Typhimurium dam vaccine in trough water with and without fecal contamination

LIVER

Fig. 2. Colonization of lymphoid tissue and visceral organs in adult sheep following oral immunization of a S. Typhimurium dam vaccine. Seven sheep were immunized with a 10 mL oral bolus (OB) of S. Typhimurium dam vaccine strain (MT2313) in PBS at dose levels of 107 CFU (light grey bars) or 108 CFU (dark grey bars); and 7 unvaccinated sheep received a 10 mL oral bolus of PBS. Sheep were vaccinated at t = 0. At 12 d post-vaccination, sheep were euthanized and immediately necropsied. Bacterial CFU in tissues [mesenteric lymph nodes (MLN), spleen, and liver] was enumerated by direct and enriched colony counts as described in Section 2. Data are depicted as mean log10 CFU/g by tissue ± SE. Significantly higher numbers of the dam vaccine strain were isolated from the MLN of the 108 OB vaccinates when compared to the 107 OB vaccinates (*P < 0.001). The dam vaccine strain was not isolated from the liver or spleen of the vaccinated sheep. Limit of detection is 4 CFU/g tissue.

3.2. Viability and stability of S. Typhimurium dam vaccine strain in water In water delivery of a modified live vaccine requires vaccine viability and stability in drinking water for effective livestock immunization. Here, vaccine viability and stability were initially examined in DIH2 O to eliminate confounders such as pH, water hardness and bacterial contaminants. Note that since the bacterial strains were reconstituted in 1× PBS, the final concentration was equivalent to 0.1× PBS in these experiments. Three salient observations were made (Fig. 3): (1) The CFU/mL of both the S. Typhimurium dam vaccine (MT2313) and isogenic dam+ (MT2315) strains was markedly stable at a 108 inoculation dose over the 120 h study period with 100.2% and 99.0% of the original inoculums remaining viable, respectively. (2) The dam vaccine and dam+ strains exhibited relatively mild but significant reductions in

Predicted Mean CFU/mL Log10

8.5

7.5

§

§

§

6.5 Δ

*β

4.5

dam- 106 dam+ 106

dam- 104 dam+ 104

3.4. Viability of the S. Typhimurium dam vaccine in buffered water

Δ

5.5

dam- 108 dam+ 108

γ

3.5

α α

2.5 0

24

γ

48

γ

72

Salmonellae are capable of proliferating in moist fecal material [35,36] and, thus, fecal contamination may alter dose delivery for livestock immunization. Here, we assessed S. Typhimurium dam vaccine CFU/mL in DIH2 O; trough water (TRW); and TRW + feces (TRW + F) as a function of time at a dose concentration of 104 CFU/mL. Note that the final concentration of PBS was equivalent to 0.01× PBS following inoculation with the S. Typhimurium dam vaccine reconstituted in PBS. The CFU/mL of the S. Typhimurium dam vaccine in solutions of TRW + F was significantly higher than that observed in TRW at 24–48 h post inoculation (P < 0.05; Fig. 4A). The predicted mean CFU/mL of the S. Typhimurium dam vaccine in TRW declined over the course of the experiment with 98%, 72.3% and 53.1% of the original inoculum viable at 6, 24 and 48 h post inoculation, respectively; the predicted mean CFU/mL of the S. Typhimurium dam vaccine in TRW + F was relatively stable over the course of the 48 h experiment with 102.2%, 105.9% and 93.8% of the original inoculum viable at 6, 24 and 48 h, respectively. Further, sampling of the S. Typhimurium dam vaccine from the surface of the trough vs. vortexed samples did not have an effect on predicted mean CFU/mL over the course of the study (P = 0.94; Fig. 4B). These data indicate that the vaccine was uniformly distributed within the trough water following inoculation and, thus, water consumption from the surface of the trough will not limit delivery of an effective vaccination dose. Taken together, these data establish the viability and stability of the S. Typhimurium dam vaccine in trough water with and without fecal contamination, suggesting that such vaccine persistence should be sufficient for oral dosing of livestock. Moreover, the observed decline in vaccine viability in clean trough water over time provides an inherent mechanism for elimination of the vaccine following trough inoculation.

α γ

120

Hours Post Inoculation Fig. 3. Viability of a S. Typhimurium dam vaccine following inoculation into deionized water. Three tubes each containing de-ionized water were inoculated with S. Typhimurium dam vaccine strain (MT2313) at dose levels of 104 (grey triangles), 106 (grey squares) or 108 (grey circles) CFU/mL or with dam+ (MT2315) at dose levels of 104 (white triangles), 106 (white squares) or 108 (white circles) CFU/mL at t = 0. Bacterial CFU/mL in samples was enumerated by direct colony count as described in Section 2. Data are depicted as a predicted mean log10 CFU/mL ± SE. Significant difference between strains at same dose level are denoted by * for 106 and § for 108 CFU/mL (P < 0.05). A significant difference from t = 0 inoculation dose is denoted by ␣ for dam− 104 , ␤ for dam− 106 , ␥ for dam+ 104 and  for dam+ 106 CFU/mL.

Vaccine persistence may be prolonged by the addition of buffers to enhance vaccine stability [8,37]. Thus, we examined the minimum concentration of PBS required for stability of the S. Typhimurium dam vaccine when inoculated into drinking water. The concentration of PBS had a significant effect on the viability of the vaccine over the 48 h study period (P = 0.005; Fig. 5). Solutions containing greater than or equal to 0.062× PBS had significantly increased predicted mean CFU/mL when compared to the DIH2 O (P < 0.05), with solutions of PBS, 0.5× PBS and 0.25× PBS containing 103.2%, 103.0 and 90.5% of the initially seeded 105 dose at 48 h post inoculation. The minimum concentration of PBS required for improved viability of the S. Typhimurium dam vaccine in drinking water over a 24 h period was greater than or equal to 0.125× PBS. Note that PBS concentrations of 0.2× PBS in combination with the S. Typhimurium dam vaccine does not compromise water intake in sheep [25]. These data suggest that inclusion of a buffering agent

V.L. Mohler et al. / Vaccine 30 (2012) 1481–1491

Predicted Mean CFU/mL Log 10

5.0

a

in vaccine formulation facilitates S. Typhimurium dam vaccine persistence without compromising livestock water consumption.

* α

*

* α

*

4.0

3.5. Effect of temperature and buffering agents on the viability of the S. Typhimurium dam vaccine

*

3.0 DIH2O

2.0

TRW TRW+F

1.0 0

6

24

48

Hours Post Inoculation Predicted Mean CFU/mL Log10

1487

5.0

b

4.0

3.0

Vortex Still

2.0 0

24

6

48

Hours Post Inoculation Fig. 4. (A) Viability of a S. Typhimurium dam vaccine when inoculated into trough water. Six jars each containing de-ionized water (DIH2 O; black square), trough water (TRW; white circle) and trough water with feces (TRW + F; grey triangle) were inoculated with S. Typhimurium dam vaccine strain (MT2313) at a dose of 104 CFU/mL at t = 0. Bacterial CFU/mL in samples was enumerated by direct colony count as described in Section 2. Data are depicted as a predicted mean log10 CFU/mL ± SE. * Denotes a significant difference from DIH2 O control (P < 0.05) and ␣ denotes a significant difference between TRW and TRW + F (P < 0.05). (B) Effect of sampling technique on a S. Typhimurium dam vaccine following inoculation into trough water. Six jars each containing either de-ionized water, trough water and trough water with feces were inoculated with S. Typhimurium dam vaccine strain (MT2313) at a dose of 104 CFU/mL at t = 0. Three jars from each solution were vortexed prior to sample collection (vortexed; white circle) while samples were collected from the surface of the remaining three jars (still; grey triangle). Bacterial CFU/mL in samples was enumerated by direct colony count as described in Section 2. Data are depicted as a predicted mean log10 CFU/mL ± SE.

3.6. Delivery of S. Typhimurium dam vaccine to adult Merino wethers via drinking water

Predicted Mean CFU/mLLog10

5.5 5 4.5 *

4 3.5 3

Under field conditions, trough water temperatures are likely to vary and may affect viability or favor proliferation of the vaccine strain. Here, we evaluated the effect of water temperature on viability and/or proliferation of the S. Typhimurium dam vaccine in drinking water with and without buffering agents. Although the viability of the S. Typhimurium dam vaccine declined post inoculation, no significant differences in the predicted mean number of CFU/mL were observed across all solutions incubated at either room temperature (20–25 ◦ C) or 37 ◦ C over the 24 h time course (P = 0.22; Fig. 6A). The capacity for the addition of PBS to significantly improved S. Typhimurium dam vaccine viability over the 24 h time course when compared to DIH2 O was independent of incubation temperature with a greater predicted mean CFU/mL in PBS and 0.2× PBS solutions when compared to the DIH2 O control from 6 to 24 h post inoculation (P < 0.05; Fig. 6B). The improved S. Typhimurium dam vaccine viability conferred by PBS was correlated, in part, to stabilization of pH, which was significantly lower in DIH2 O relative to that observed in PBS and 0.2× PBS solutions from 6 to 24 h post inoculation (P < 0.05; Fig. 6C). Control solutions of DIH2 O and PBS without inoculation of the S. Typhimurium dam vaccine exhibited only a mild decrease in pH over the experimental time course (data not shown). Thus, the buffering capacity of PBS likely influences vaccine viability by preventing vaccine-mediated acidification of the solution over the 24 h study period. Note that the mild (half log) decrease in S. Typhimurium dam vaccine viability at the 6 h time point in buffered solutions (Fig. 4B) was not attributed to pH as no measurable change in pH was observed over the entire time 24 h time course. Taken together, these data demonstrate that the viability of S. Typhimurium dam vaccine is similar during the first 24 h in solutions maintained at room temperature or 37 ◦ C and that the vaccine does not proliferate when inoculated into warm water. Moreover, the buffering capacity of PBS in solution likely influences vaccine viability by preventing acidification and, thus, vaccine persistence can be controlled via inclusion/exclusion of buffering agents and stabilizers, providing an effective means for vaccine delivery in drinking water for ad libitum oral delivery under on-farm conditions.

DIH2O 0.031X PBS 0.062X PBS 0.125X PBS 0.25X PBS 0.5X PBS PBS

* * * * *

2.5 0

24

48

Hours Post Inoculation Fig. 5. Effect of PBS concentration on the viability of a S. Typhimurium dam vaccine. S. Typhimurium dam vaccine strain (MT2313) was inoculated into tubes containing either de-ionized water (DIH2 O, black square), phosphate buffered saline (PBS, white triangle) and 0.031× PBS (grey square), 0.062× PBS (grey triangle), 0.125× PBS (grey diamond), 0.25× PBS (grey circle), 0.5× PBS (white circle) at a concentration of 105 CFU/mL at t = 0. Bacterial CFU/mL in samples was enumerated by direct colony count as described in Section 2. Data are depicted as a predicted mean log10 CFU/mL ± SE. * Denotes significantly different from PBS control (P < 0.05).

Delivery of an effective S. Typhimurium dam vaccine in drinking water to adult sheep requires that water consumption is not compromised; no adverse clinical reactions are manifested; effective colonization occurs in host lymphoid tissues; and the vaccine is subsequently cleared, with minimal vaccine shedding from the immunized animals. The cost of vaccine production is influenced by the vaccine dose, and by growth characteristics of the organism which will influence how much media is required to produce the vaccine. Previously we demonstrated that an in-water vaccine dose of 107 organisms induces protective immunity in adult sheep [25]. Prior to conducting a vaccine challenge study with a lower vaccine dose that would reduce the costs associated with vaccine production and potentially increase vaccine safety, we wished to determine if a lower vaccine dose administered in drinking water would be sufficient to achieve transient colonization of host tissues. The influence of vaccine dose on tissue colonization was evaluated by in water vaccination of sheep at 107 and 106 CFU. To address the potential issue of buffering agents on water

Predicted Mean CFU/mL Log10

1488

V.L. Mohler et al. / Vaccine 30 (2012) 1481–1491

4.5

a 37oC Room Temperature

4.0

3.5 0

6

12

24

PredictedMeanCFU/mL Log10

Hours Post Inoculation 4.5

b * *

*, β

4.0 * *

*

PBS

3.5

0.2X PBS DIH2O

3.0 0

6

12

24

Hours Post Inoculation

Predicted Mean pH

7.5

c *

7.0

*

*

*

*

*

*

12

24

*

6.5 PBS

6.0

0.2X 0 PBS DIH2O

5.5 0

6

Hours Post Inoculation Fig. 6. (A) Effect of incubation temperature on viability of a S. Typhimurium dam vaccine. S. Typhimurium dam vaccine strain (MT2313) was inoculated into six jars each containing either PBS; 0.2× PPS or DIH2 O to concentration of 2.5 × 104 CFU/mL at t = 0. Three jars from each solution were incubated at room temperature (20–26 ◦ C) (black square, dashed lines) or 37 ◦ C (grey circle; solid lines). Bacterial CFU/mL in samples was enumerated by direct colony count as described in Section 2. Data are depicted as a predicted mean log10 CFU/mL ± SE. No differences between incubation temperatures were observed (P = 0.22). (B) Effect of PBS on viability of a S. Typhimurium dam vaccine following incubation at room temperature and 37 ◦ C. Six jars each containing either PBS (white circle); 0.2× PBS (grey triangle) or DIH2 O (black square) were inoculated with S. Typhimurium dam strain (MT2313) to concentration of 2.5 × 104 CFU/mL at t = 0. Three jars from each solution were incubated at room temperature or 37 ◦ C. Bacterial CFU/mL in samples was enumerated by direct colony count as described in Section 2. Incubation temperature (RT vs. 37 ◦ C) did not have a significant effect on viability (P = 0.22) hence the predicted mean CFU calculated by the model for each solution includes the data from both incubation temperatures. Data are depicted as a predicted mean log10 CFU/mL ± SE. * Denotes significant difference from DIH2 O and ␤ denotes significant difference between 0.2× PBS and PBS (P < 0.05). (C) Effect of a S. Typhimurium dam vaccine on solution pH. Six jars each containing either PBS (white circle); 0.2× PBS (grey triangle) or DIH2 O (black square) were inoculated with S. Typhimurium dam vaccine strain (MT2313) to a concentration of 2.5 × 104 CFU/mL at t = 0. Three jars from each solution were incubated at room temperature or 37 ◦ C. Bacterial CFU/mL in samples was enumerated by direct colony count as described in Section 2. Incubation temperature (RT vs. 37 ◦ C) did not have a significant effect on pH (P = 0.13) hence the predicted mean CFU calculated by the model for each solution includes the data from both incubation temperatures. Data are depicted as a predicted mean pH ± SE. * Denotes significant difference from DIH2 O (P < 0.05).

consumption, sheep were offered drinking water containing 0.2× PBS during the acclimation period. Water consumption was compared to the mean water consumption from the previous 5 d. No differences in consumption of PBS treated water was observed when compared to mean untreated drinking water consumption (P = 0.86). These data establish that 0.2× PBS does not affect palatability or inhibit consumption of water by adult sheep. Further, there was no evidence of refusal of the S. Typhimurium dam vaccine when delivered in-water containing 0.2× PBS (P = 0.81). On the day of vaccination, water consumption of the in-water vaccinates was 1.9 ± 0.24 L and 2.4 ± 0.22 L for the 107 IW and 106 IW groups, respectively, which was 85.4 and 105.6% of the mean daily water consumption during the 5 d preceding vaccination for each group. Thus, sheep consumed 4–8% of their body weight in water per day which was within the reported normal daily range of 5–20% [33,34]. Moreover, the mean in-water vaccination dose consumed was: 1.1 × 106 CFU (range 8.7 × 105 to 1.5 × 106 CFU/sheep) in the 106 IW group and 1.5 × 107 CFU (range 1.2 × 107 to 1.8 × 108 CFU/sheep) in the 107 IW group. Such 107 IW group dosing was similar to that received by the OB107 group (9.4 × 106 CFU/sheep in a 10 mL oral bolus). These data indicate that the S. Typhimurium dam vaccine was effectively delivered in water to sheep under field conditions. Next, we assessed clinical manifestations as a consequence of vaccination. Pyrexia (rectal temperature > 40.0 ◦ C) [30,31] was not observed in sheep from the 106 IW vaccinate group (Fig. 7A). This group remained within the normal temperature range throughout the study and had significantly lower predicted mean rectal temperatures relative to that exhibited by the 107 OB and 107 IW vaccinates from 24 to 72 h post vaccination (P < 0.05). In contrast, pyrexia was observed in 4 of 7 sheep in the 107 OB group and 7 of 8 sheep in the 107 IW group at 48 and 72 h following vaccination (P < 0.001). However, no difference in the predicted mean rectal temperatures was observed between these two groups, even though the 107 IW vaccinates consumed a half log more vaccine that the 107 OB group (P < 0.001; Fig. 7A). Taken together, these data indicate that the 106 IW vaccinate group remained within the normal temperature range throughout the study period; moreover, no significant pyrexia differences were observed between the 107 IW and 107 OB vaccinate groups. There was no significant effect of vaccination treatment on chaff appetite, pellet appetite, and water consumption of the sheep over the course of the experiment (Fig. 7B–D). Moreover, the attitude and fecal scores of all sheep remained at baseline values over the course of the study period. However, chaff refusal of the 107 OB vaccinates was significantly increased when compared to the 106 IW vaccinates at 72 h post vaccination (P < 0.05; Fig. 7B); and the water consumption of the sheep in the 107 OB vaccinates was significantly higher than the 106 IW and 107 IW vaccinates during vaccination period, t 0–24 h (P < 0.05; Fig. 7D). Taken together, these data indicate that vaccine treatment had minimal effects on chaff appetite, pellet appetite, and water consumption. Colonization of lymphoid tissue (e.g., intestine [Peyer’s patches]; mesenteric lymph nodes [MLN]; spleen) and visceral organs (e.g., liver) leads to the initiation of protective innate and adaptive immune responses. Here we assessed the ability of the S. Typhimurium dam vaccine to colonize such host tissue sites via enumeration of CFU recovered from feces, MLN, spleen, and liver at 96 h post-vaccination. Fecal shedding: Significantly lower numbers of dam vaccine shedding (2 logs less CFU/g feces) was observed in the 106 IW vaccinates when compared to the 107 OB and 107 IW vaccinates (P < 0.01; Fig. 8); no differences were observed between 107 OB and 107 IW vaccinates (P = 0.9579). Moreover, the dam vaccine was recovered from the feces of 1 of 7 of the 106 IW vaccinates as compared to 8 of 8 and 7 of 7 sheep from the 107 IW and 107 OB vaccinates, respectively. MLN colonization: Significantly lower numbers of the dam vaccine were observed in the MLN of the

42.0

107OB 106IW 107IW

41.0

a

Baseline Normal Range Pyrexia

40.0 39.0 38.0 *

*

*

*

Predicted Mean Chaff Refusal (%)

Predicted Mean Rectal Temperature (O C)

V.L. Mohler et al. / Vaccine 30 (2012) 1481–1491

37.0 -24

0

24

48

100 90 80 70 60 50 40 30 20 10 0 -10

106IW 107IW

*

0

72

106IW 107IW

72

Predicted Mean Water Consumption (L)

Predicted Mean Pellet Refusal (%)

c

24 48 Hours Post Vaccination

24

48

72

Hours Post Vaccination

107OB

0

b

107OB

Hours Post Vaccination 100 90 80 70 60 50 40 30 20 10 0 -10

1489

4

d 3 *

2

* 7OB 107OB 10

1

106IW 107IW

0 0

24 48 Hours Post Vaccination

72

Fig. 7. Clinical assessment of adult sheep following in-water delivery of a S. Typhimurium dam vaccine. Seven sheep were immunized with 0.2× PBS drinking water containing target dose of 106 CFU of S. Typhimurium dam vaccine strain (MT2313) (white circles, solid white bars) (106 IW); 8 sheep were immunized in 0.2× PBS drinking water with target dose 107 CFU S. Typhimurium dam (grey triangles, grey bars) (107 IW); and 7 sheep were immunized in an oral bolus with 107 CFU of S. Typhimurium dam (black squares, black bars) (107 OB). In water vaccination groups had access to vaccine over a 24 h period (t = −24 to t = 0). Sheep vaccinated via oral bolus were vaccinated at t = 0. (A) Rectal temperatures: Rectal temperatures were determined using a digital thermometer twice weekly prior to vaccination, 24 h prior to vaccination (−24 h) and daily for 3 d following vaccination and data presented as predicted mean rectal temperature ± SE. The normal range (37.6–39.5 ◦ C) of rectal temperatures for sheep used in these experiments was based on the pre-vaccination mean ± two standard deviations (dashed grey lines). The threshold for pyrexia (dashed black line) reflects the published normal range for sheep [30,31]. *Rectal temperatures of the 106 IW vaccinates were significantly lower than the 107 IW and 107 OB vaccinates from time 0 to 72 h post vaccination (P < 0.05). There was no difference in the rectal temperatures of the 107 IW and 107 OB vaccinates. (B) Chaff refusal: Percent of chaff refused was determined for each animal during the vaccination period based on amount of offered chaff refused. Data are predicted mean chaff refusal ± SE. *Chaff refusal was significantly lower in the 106 IW when compared to the 107 OB vaccinates at 72 h post vaccination (P < 0.05). There was no difference in the chaff refusals of the 106 IW and 107 IW vaccinates. (C) Pellet refusal: Percent of pellet refused was determined for each animal during the vaccination period based on amount of pellet refused over offered. Data are depicted as predicted mean pellet refusal ± SE. There was no difference in the pellet refusals of the 107 OB, 106 IW and 107 IW vaccinates over the 72 h observation period. (D) Water consumption: Data are depicted as predicted mean water consumption in L ± SE. *The water consumption of the 106 IW and 107 IW vaccinates was significantly lower than the 107 OB vaccinates during 24 h post-vaccination period (t = −24 to t = 0) (P < 0.05).

106 IW vaccinates (2 logs less CFU/g) when compared to the 107 IW vaccinates (P < 0.005); no differences were observed between the 107 IW and 107 OB vaccinates (P = 0.0854) and between the 107 OB and 106 IW vaccinates (P = 0.2439). Moreover, the S. Typhimurium dam vaccine was recovered from the MLN of 2 of 7, 8 of 8 and 4 of 7 sheep from the 106 IW, 107 IW and 107 OB vaccination groups, respectively. Spleen and liver colonization: dam vaccine was not recovered from the spleen and liver of the 106 IW vaccinates, with only very low CFU recovered from one sheep from the 107 IW and 107 OB vaccinates groups. Consistent with the finding that in water delivery of 107 CFU of the S. Typhimurium dam induces protective immunity to Salmonella challenge [25], sheep vaccinated with this dose shed low numbers of vaccine organisms in the feces, and exhibited low level colonization of the MLN 96 h post vaccination. By contrast, the vaccine strain was not shed in the feces or recovered from the MLN of most sheep from the 106 IW vaccinate group, suggesting that this dose may be insufficient to consistently induce a protective immune response in adult ruminants. 4. Discussion Ideally, stock should be vaccinated on farm of origin to provide protective immunity prior to the stock experiencing the stressors

and pathogen exposure associated with sale, transport, and the high risk period following entry into the feedlot. A major challenge is to convince producers supplying stock to the feedlots to vaccinate prior to sale as the cost of disease is not incurred on the property of origin. Such timing, exposure, and compliance issues currently necessitate vaccination during the high risk period immediately following entry into the feedlot to provide protection. As a potential means to address these issues, in-water delivery of live attenuated vaccines affords a low cost, low-stress method for immunization of livestock populations that is not associated with the adverse handling stressors and injection reactions associated with parenteral administration. Here we established that in-water administration of the Salmonella dam vaccine was safe, stable and well-tolerated in adult ruminants. The vaccine delivery platform was amenable to field applications and an evaluation of vaccine dose effects further supported vaccine safety in livestock and the environment along with a low cost means of vaccine production. There are a number of promising features of Salmonella dam vaccines that suggest they are likely to offer an effective method of salmonellae prophylaxis in livestock industries. Experimental challenge studies have established that Salmonella dam vaccines confer protection against a diversity of clinically relevant salmonellae that are commonly associated with disease in cattle and humans;

1490

V.L. Mohler et al. / Vaccine 30 (2012) 1481–1491

Tissue Colonization of the Vaccine (CFU/g Log10)

4.0

3.0

2.0

1.0

0.0 FECES

MLN

SPLEEN

LIVER

Fig. 8. Colonization of lymphoid tissue and visceral organs in adult sheep following in-water delivery of a S. Typhimurium dam vaccine. Eight sheep were immunized with 107 CFU of S. Typhimurium dam vaccine strain (MT2313) in drinking water (solid grey bars; 107 IW); seven sheep were immunized with 106 CFU of S. Typhimurium dam in drinking water (solid white bars; 106 IW); and seven sheep were immunized with 107 CFU of S. Typhimurium dam vaccine strain as an oral bolus (solid black bars; 107 OB). At 96 h post-vaccination, sheep were euthanized and immediately necropsied. Bacterial CFU in infected tissues [feces, mesenteric lymph nodes (MLN), spleen and liver] was enumerated by direct and enriched colony counts as described in Section 2. Data are depicted as mean log10 CFU/g by tissue ± SE. Significantly lower numbers of the S. Typhimurium dam vaccine strain were shed into the feces of the 106 IW vaccinates when compared to the 107 OB and 107 IW vaccinates (a P < 0.01). Significantly higher numbers of the dam vaccine strain were isolated from the MLN of the 107 IW vaccinates when compared to the 106 IW vaccinates (b P < 0.005). Limit of detection is 4 CFU/g tissue.

and safety studies have demonstrated they are well tolerated by a diversity of species including mice, poultry, sheep and cattle [16,17,23–28]. Induction of immunity is rapid [24,26], which is requisite for the high-risk period following feedlot entry. Additionally, in-water administration of the dam vaccine conferred protection in livestock, establishing vaccine efficacy of the in-water protocol [25]. Here, the safety of the vaccine was evaluated in livestock and the environment. In water delivery of the vaccine was safe and well-tolerated in immunized sheep, as evidenced by minimal fecal shedding and clinical manifestations at a vaccine dose 10 times that which has been previously demonstrated to induce protective immunity to virulent Salmonella challenge. These data indicate that the vaccine has a considerable dosing safety margin to cater to potential fluctuation in the dose consumed when it is delivered in drinking water. Further, the dam vaccine was stable over a wide range of temperatures; salt concentrations; and in trough water with fecal contamination, suggesting vaccine persistence should be sufficient for oral dosing of livestock without significant environmental persistence. Environmental parameters such as temperature and fecal contamination may adversely affect vaccine dosing of the animals. For example, although our data show that the S. Typhimurium dam vaccine is stable under temperatures between 20 ◦ C and 37 ◦ C, and does not adversely affect water consumption [33,34], ambient temperatures may need to be considered when delivering the S. Typhimurium dam vaccine to sheep as higher environmental temperatures (40 ◦ C) encourage increased water consumption [33] and could affect vaccine viability/stability. Fecal contamination may affect dosing as it is not uncommon to find feces or contaminated bedding/feed material in troughs used to supply animals with water, and salmonellae are capable of proliferating in moist fecal material [35,36]. Our studies show that although exposure to fecal-contaminated trough water increased the stability of the S. Typhimurium dam vaccine, no increase in vaccine CFU was observed under these conditions. Since vaccine proliferation was not observed in fecal-contaminated trough water, the S.

Typhimurium dam vaccine would likely not excessively proliferate in polluted water supplies. A potential limitation to oral vaccine delivery in ruminants is the capacity of volatile fatty acids produced in the rumen to attenuate the survival of Salmonella [38,39]. The establishment of the Salmonella dam vaccine in the mesenteric lymph nodes of adult sheep demonstrated that the vaccine is capable of bypassing the rumen and surviving in the gastrointestinal tract, indicating the opportunity for successful in-water prophylaxis in adult ruminants as demonstrated in our previous in-water protection studies [25]. From a farm-management perspective, many of these features would have general utility in intensive livestock production systems and could be modified accordingly depending on the specific management/environmental strategies implemented. For example, if the planned application called for a more prolonged vaccination period, the S. Typhimurium dam vaccine maintained highest viability in solutions containing at least 0.2× PBS. Indeed, PBS has been used to enhance survival of live Mycoplasma gallisepticum vaccine in aerosol delivery systems to chickens [37,40] and did not negatively impact on water consumption by sheep in our study. If the planned application were to deliver the vaccine over a 24 h period, it would not be necessary to include a buffering agent; moreover, replenishing water troughs with unbuffered water would promote elimination of the vaccine following the delivery period. Safety and efficacy studies have established that the dam vaccine was well-tolerated and conferred protection in adult ruminants, remained viable under field conditions, showed minimal environmental persistence, and was widely adaptable to diverse dosing regimens. Taken together, these studies support that in-water delivery of modified live Salmonella dam vaccines provides a potential means for low-cost and low-stress immunization method with potential utility from farm to feedlots. Acknowledgements This work was supported by USDA grant 2004-04574 (to M.J.M. and J.K.H.), and the G. Harold & Leila Y. Mathers Foundation (to M.J.M.). Many thanks to all the staff of the Bacteriology Section of the State Veterinary Diagnostic Laboratory at the Elizabeth Macarthur Agricultural Institute, Department of Industry & Investment NSW, Australia for providing support with media and laboratory space. And a special thank you to Ms. Emily Funnell and Ann-Marie House for their assistance in the laboratory. References [1] Bowersock TL, Martin S. Vaccine delivery to animals. Advanced Drug Delivery Reviews 1999;38(July (2)):167–94. [2] Savini G, MacLaclalan NJ, Sanchez-Vinaino JM, Zientara S. Vaccines against bluetongue in Europe. Comparative Immunology Microbiology and Infectious Diseases 2008;31(March (2–3)):101–20. [3] Mamak N, Aytekin I. Fatal adverse reactions of enterotoxaemia vaccine in a sheep flock. Journal of Animal and Veterinary Advances 2009;8(November (11)):2388–91. [4] Murphy D, Corner LAL, Gormley E. Adverse reactions to Mycobacterium bovis bacille Calmette-Guerin (BCG) vaccination against tuberculosis in humans, veterinary animals and wildlife species. Tuberculosis 2008;88(July (4)):344–57. [5] Giudice EL, Campbell JD. Needle-free vaccine delivery. Advanced Drug Delivery Reviews 2006;58(April (1)):68–89. [6] Vermeulen B, De Backer P, Remon JP. Drug administration to poultry. Advanced Drug Delivery Reviews 2002;54(October (6)):795–803. [7] Walter D, Knittel J, Schwartz K, Kroll J, Roof M. Treatment and control of porcine proliferative enteropathy using different tiamulin delivery methods. Journal of Swine Health and Production 2001;9(May–June (3)):109–15. [8] Kolb JR. Swine production management – vaccination and medication via drinking water. Compendium on Continuing Education for the Practicing Veterinarian 1996;18(February (2)):S75–9. [9] Agerso H, Friis C, Haugegaard J. Water medication of a swine herd with amoxycillin. Journal of Veterinary Pharmacology and Therapeutics 1998;21(June (3)):199–202.

V.L. Mohler et al. / Vaccine 30 (2012) 1481–1491 [10] Bergman D, Bender S, Wenning K, Slate D, Rupprecht C, Heuser C, et al. Bait acceptability for delivery of oral rabies vaccine to free-ranging dogs on the Navajo and Hopi Nations. In: Dodet B, Fooks AR, Miller T, Tordo N, editors. Towards the elimination of rabies in Eurasia. 2008. p. 145–50. [11] Cliquet F, Barrat J, Guiot AL, Cael N, Boutrand S, Maki J, et al. Efficacy and bait acceptance of vaccinia vectored rabies glycoprotein vaccine in captive foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and dogs (Canis familiaris). Vaccine 2008;26(August (36)):4627–38. [12] Niin E, Laine M, Guiot AL, Demerson JM, Cliquet F. Rabies in Estonia: situation before and after the first campaigns of oral vaccination of wildlife with SAG2 vaccine bait. Vaccine 2008;26(July (29–30)):3556–65. [13] Nol P, Palmer MV, Waters WR, Aldwell FE, Buddle BM, Triantis JM, et al. Efficacy of oral and parenteral routes of Mycobacterium bovis bacille CalmetteGuerin vaccination against experimental bovine tuberculosis in white-tailed deer (Odocoileus virginianus): a feasibility study. Journal of Wildlife Diseases 2008;44(April (2)):247–59. [14] Begg AP, Walker KH, Love DN, Mukkur TKS. Evaluation of protection against experimental salmonellosis in sheep immunized with 1 or 2 doses of live aromatic-dependent Salmonella-Typhimurium. Australian Veterinary Journal 1990;67(August (8)):294–8. [15] Hassan JO, Curtiss 3rd R. Development and evaluation of an experimental vaccination program using a live avirulent Salmonella typhimurium strain to protect immunized chickens against challenge with homologous and heterologous Salmonella serotypes. Infection and Immunity 1994;62(December (12)):5519–27. [16] Heithoff DM, Enioutina EY, Daynes RA, Sinsheimer RL, Low DA, Mahan MJ. Salmonella DNA adenine methylase mutants confer cross-protective immunity. Infection and Immunity 2001;69:6725–30. [17] Heithoff DM, Enioutina EY, Bareyan D, Daynes RA, Mahan MJ. Conditions that diminish myeloid-derived suppressor cell activities stimulate cross-protective immunity. Infection and Immunity 2008;76(November (11)):5191–9. [18] Jelinek PD, Robertson GM, Millar C. Vaccination of sheep with a live Gal-E-Mutant of Salmonella-Typhimurium. Australian Veterinary Journal 1982;59(1):31–2. [19] Nagy G, Dobrindt U, Hacker J, Emody L. Oral immunization with an RfaH mutant elicits protection against salmonellosis in mice. Infection and Immunity 2004;72(July (7)):4297–301. [20] Smith BP, Reina-Guerra M, Hoiseth SK, Stocker BA, Habasha F, Johnson E, et al. Aromatic-dependent Salmonella typhimurium as modified live vaccines for calves. American Journal of Veterinary Research 1984;45(January (1)): 59–66. [21] Garcia-Del Portillo F, Pucciarelli MG, Casadesus J. DNA adenine methylase mutants of Salmonella typhimurium show defects in protein secretion, cell invasion, and M cell cytotoxicity. Proceedings of the National Academy of Sciences of the United States of America 1999;96(20):11578–83. [22] Heithoff DM, Sinsheimer RL, Low DA, Mahan MJ. An essential role for DNA adenine methylation in bacterial virulence [see comments]. Science 1999;284(5416):967–70. [23] Dueger EL, House JK, Heithoff DM, Mahan MJ. Salmonella DNA adenine methylase mutants elicit protective immune responses to homologous and heterologous serovars in chickens. Infection and Immunity 2001;69(December (12)):7950–4. [24] Dueger EL, House JK, Heithoff DM, Mahan MJ. Salmonella DNA adenine methylase mutants prevent colonization of newly hatched chickens by

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32] [33]

[34]

[35]

[36]

[37]

[38] [39] [40]

1491

homologous and heterologous serovars. International Journal of Food Microbiology 2003;80(January (2)):153–9. Mohler VL, Heithoff DM, Mahan MJ, Walker KH, Hornitzky MA, Gabor L, et al. Protective immunity conferred by a DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine when delivered in-water to sheep challenged with Salmonella enterica serovar Typhimurium. Vaccine 2011;29(April (19)):3571–82. Dueger EL, House JK, Heithoff DM, Mahan MJ. Salmonella DNA adenine methylase mutants elicit early and late onset protective immune responses in calves. Vaccine 2003;21(July (23)):3249–58. Mohler VL, Heithoff DM, Mahan MJ, Walker KH, Hornitzky MA, McConnell CS, et al. Cross-protective immunity in calves conferred by a DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine. Vaccine 2006;24(9):1339. Mohler VL, Heithoff DM, Mahan MJ, Walker KH, Hornitzky MA, Shum LWC, et al. Cross-protective immunity conferred by a DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine in calves challenged with Salmonella serovar Newport. Vaccine 2008;26:1751–8. Conner CP, Heithoff DM, Julio SM, Sinsheiner RL, Mahan MJ. Differential patterns of acquired virulence genes distinguish Salmonella strains. Proceedings of the National Academy of Sciences of the United States of America 1998;95:4641–5. Radostits OM, Gay CG, Hinchcliff KW, Constable PD. Veterinary medicine: a textbook of the diseases of cattle, sheep, pigs, and goats. 10th ed. Saunders Elsevier; 2007. Terra RL. History, physical examination, and medical records: ruminant history, physical examination, and records. In: Smith BP, editor. Large animal internal medicine. 4th ed. St. Louis, Missouri: Mosby, Inc.; 2009. p. 3–14. Buchan IE. The development of a statistical software resource for medical research. Liverpool, England: University of Liverpool; 2000. Savage DB, Nolan JV, Godwin IR, Mayer DG, Aoetpah A, Nguyen T, et al. Water and feed intake responses of sheep to drinking water temperature in hot conditions. Australian Journal of Experimental Agriculture 2008;48(6–7):1044–7. Singh NP, More T, Sahni KL. Effect of water deprivation on feed-intake, nutrient digestibility and nitrogen retention in sheep. Journal of Agricultural Science 1976;86(APR):431–3. Holley RA, Arrus KM, Ominski KH, Tenuta M, Blank G. Salmonella survival in manure-treated soils during simulated seasonal temperature exposure. Journal of Environmental Quality 2006;35(July–August (4)):1170–80. Arrus KM, Holley RA, Ominski KH, Tenuta M, Blank G. Influence of temperature on Salmonella survival in hog manure slurry and seasonal temperature profiles in farm manure storage reservoirs. Livestock Science 2006;102(July (3)):226–36. Leigh SA, Evans JD, Branton SL, Collier SD. The effects of increasing sodium chloride concentration on Mycoplasma gallisepticum vaccine survival in solution. Avian Diseases 2008;52(March (1)):136–8. Chambers PG, Lysons RJ. The inhibitory effect of bovine rumen fluid on Salmonella typhimurium. Research Veterinary Science 1979;26(3):273–6. Mattila T, Frost AJ, O’Boyle D. The growth of salmonella in rumen fluid from cattle at slaughter. Epidemiology and Infection 1988;101(2):337–45. Branton SL, Leigh SA, Roush WB, Purswell JL, Olanrewaju HA, Collier SD. Effect of selected water temperatures used in Mycoplasma gallisepticum vaccine reconstitution on titer at selected time intervals. Avian Diseases 2008;52(June (2)):291–6.