Scholarly article on topic 'Amelioration of salmonellosis in pre-weaned dairy calves fed Saccharomyces cerevisiae fermentation products in feed and milk replacer'

Amelioration of salmonellosis in pre-weaned dairy calves fed Saccharomyces cerevisiae fermentation products in feed and milk replacer Academic research paper on "Veterinary science"

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Abstract of research paper on Veterinary science, author of scientific article — Matthew T. Brewer, Kristi L. Anderson, Ilkyu Yoon, Mark F. Scott, Steve A. Carlson

Abstract Salmonellosis is an insidious and potentially epidemic problem in pre-weaned dairy calves. Managing this disease, or any other diarrheal disease, is a financial burden to producers. Calf mortalities and medicinal treatments are overt costs of salmonellosis, while hidden costs include hampered weight gains and persistent intestinal colonization of the pathogen. In this study, we examined the anti-Salmonella effects of Saccharomyces cerevisiae fermentation products (SCFP) incorporated into both the milk replacer and the starter grain. In a blinded study, 2–8 day-old calves were fed SCFP (n =20 calves) or an SCFP-free Control (n =20 calves) for two weeks before and three weeks after experimental challenge with Salmonella enterica serotype Typhimurium. Following the challenge, calves were monitored for clinical signs and parameters associated with salmonellosis. Calves were then euthanized and examined for rumen development and intestinal Salmonella colonization. When compared to calves that received milk replacer and feed lacking SCFP, calves fed SCFP had fewer bouts of diarrhea and fever. Rumens from these calves were more developed, as measured by the length of papillae, which is consistent with the enhanced weight gain observed in this treatment group. Additionally, Salmonella intestinal colonization was reduced in SCFP-fed calves and Salmonella fecal shedding disappeared at an earlier stage in these calves. This study revealed that the combination of two proprietary S. cerevisiae fermentation products provide marked benefit for preventing the negative effects of salmonellosis in pre-weaned dairy calves, while also boosting productivity. The mechanism of action needs to be clarified, but it may be related to the observed decrease in colonization by the pathogen and increase in rumen development.

Academic research paper on topic "Amelioration of salmonellosis in pre-weaned dairy calves fed Saccharomyces cerevisiae fermentation products in feed and milk replacer"

Accepted Manuscript

Title: Amelioration of salmonellosis in pre-weaned dairy calves fed Saccharomyces cerevisiae fermentation products in feed and milk replacer

Author: Matthew T. Brewer Kristi L. Anderson Ilkyu Yoon Mark F. Scott Steve A. Carlson

veterinary microbiology

PII: DOI:

Reference:

S0378-1135(14)00272-7

http://dx.doi.org/doi:10.1016/j.vetmic.2014.05.026 VETMIC 6632

To appear in:

VETMIC

Received date: Revised date: Accepted date:

15-3-2014

16-5-2014 25-5-2014

Please cite this article as: Brewer, M.T., Anderson, K.L., Yoon, I., Scott, M.F., Carlson, S.A.,Amelioration of salmonellosis in pre-weaned dairy calves fed Saccharomyces cerevisiae fermentation products in feed and milk replacer, Veterinary Microbiology (2014), http://dx.doi.org/10.1016/j.vetmic.2014.05.026

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1 Highlights

2 (1) We examined the anti-Salmonella effects of Saccharomyces cerevisiae fermentation products

3 (SCFP) incorporated into both the milk replacer and the starter grain.

4 (2) Calves fed SCFP had fewer bouts of diarrhea and fever.

5 (3) Rumens from these calves were more developed

6 (4) Salmonella intestinal colonization was reduced in SCFP-fed calves

7 (5) The combination of two proprietary Saccharomyces cerevisiae fermentation products

8 provide marked benefit for preventing the negative effects of salmonellosis in pre-weaned dairy

9 calves, while also boosting productivity.

10 Amelioration of salmonellosis in pre-weaned dairy calves fed Saccharomyces cerevisiae

11 fermentation products in feed and milk replacer

1 12 2 1*

13 Matthew T. Brewer , Kristi L. Anderson , Ilkyu Yoon , Mark F. Scott, and Steve A. Carlson

15 department of Biomedical Sciences, Iowa State University College of Veterinary Medicine,

16 Ames, IA 50011

17 2 Diamond V, 2525 60th Ave SW, Cedar Rapids, IA 52404

18 * Corresponding author: 2028 VetMed, Ames, IA 50011; 515-294-0912 (voice), 515-294-2315

19 (fax); stevec@iastate.edu

ABSTRACT: Salmonellosis is an insidious and potentially epidemic problem in pre-weaned dairy calves. Managing this disease, or any other diarrheal disease, is a financial burden to producers. Calf mortalities and medicinal treatments are overt costs of salmonellosis, while hidden costs include hampered weight gains and persistent intestinal colonization of the pathogen. In this study, we examined the anti-Salmonella effects of Saccharomyces cerevisiae fermentation products (SCFP) incorporated into both the milk replacer and the starter grain. In a blinded study, 2 to 8 day-old calves were fed SCFP (n = 20 calves) or an SCFP-free Control (n = 20 calves) for two weeks before and three weeks after experimental challenge with Salmonella enterica serotype Typhimurium. Following the challenge, calves were monitored for clinical signs and parameters associated with salmonellosis. Calves were then euthanized and examined for rumen development and intestinal Salmonella colonization. When compared to calves that received milk replacer and feed lacking SCFP, calves fed SCFP had fewer bouts of diarrhea and fever. Rumens from these calves were more developed, as measured by the length of papillae, which is consistent with the 'enhanced weight gain observed in this treatment group. Additionally, Salmonella intestinal colonization was reduced in SCFP-fed calves and Salmonella fecal shedding disappeared at an earlier stage in these calves. This study revealed that the combination of two proprietary Saccharomyces cerevisiae fermentation products provide marked benefit for preventing the negative effects of salmonellosis in pre-weaned dairy calves, while also boosting productivity. The mechanism of action needs to be clarified, but it may be related to the observed decrease in colonization by the pathogen and increase in rumen development. Key words: Salmonella infection, dairy calf, yeast culture

43 1. Introduction

45 Salmonellosis is one of the many diarrheal diseases affecting pre-weaned dairy calves.

46 Salmonella organisms are commonly isolated from dairy farms (Fossler et al., 2004) and the

47 fecal-oral transmission route can occur from dam to offspring. Calves can also acquire the

48 organism from fecal-contaminated fomites or the environment. Calves manifest the disease as

49 diarrhea, fever, anorexia, and dehydration all of which significantly compromise the

50 development and maturation of the animal. Further costs include treatment with electrolytes or

51 antibiotics or both, and some calves still perish because of the increasing prevalence of antibiotic

52 resistance in Salmonella (Cummings et al., 2013) and hypervirulence associated with multi-

53 resistant strains (Rasmussen et al., 2004). Furthermore, calves that survive salmonellosis can be

54 long-term carriers of the pathogen (Nielsen et al., 2012) and these adult animals can serve as a

55 persistent source for new infections in the herd (Cobbold et al., 2006). Environmental

56 persistence also contributes to this problem (Cobbold et al., 2006).

57 Preventing Salmonella infections currently focuses on a vaccine technology (Hermesch et

58 al., 2008). Unfortunately, this vaccine is only for cattle that are six months or older thus pre-

59 weaned calves are dependent upon colostral passive immunization from the vaccinated dam.

60 Anti-Salmonella bacterins have been tried but are frequently unsuccessful because of the

61 immunodominance of the Salmonella O-antigen (Barat et al., 2012) and serovar specificity

62 (House et al., 2001). Anti-lipopolysaccharide antiserum and lipopolysaccharide toxoids are

63 available but the anti-Salmonella benefits have not been clearly established. Thus Salmonella

64 prophylaxis is not optimal at this time in the pre-weaned calf (Lanzas et al., 2008), although

65 vaccinating the dam will reduce the environmental exposure of the calf.

It has been shown that soluble components present in Saccharomyces cerevisiae fermentation products (SCFP) enhance gut health (Jensen et al., 2008a) and promote immune function (Jensen et al. 2007). When supplemented to the starter grain, SCFP improved rumen development, starter grain intake, and BW gain of non-challenged calves (Lesmeister et al., 2004). Additionally, SCFP was shown to improve the gastrointestinal health of calves in a Salmonella endemic herd (Magalhaes et al., 2008). Because of these benefits associated with SCFP, we examined its anti-Salmonella effects when fed to pre-weaned dairy calves experimentally infected with Salmonella. The specific aims of this study were to determine the effects of the combination of two proprietary SCFP (Diamond V SmartCare™ and Diamond V Original XPC™) on the growth and rumen development, clinical signs of salmonellosis, Salmonella shedding, and intestinal colonization of Salmonella in pre-weaned dairy calves experimentally infected with Salmonella.

2. Materials and methods

2.1. Calves andPre-infection Treatments

Animal experiments were approved by the Animal Care and Use Committee at Iowa State University. Forty Holstein or Holstein-cross calves (32 females and 8 males) were purchased from a local supplier in northwest Iowa. Calves were fed colostrum for the first two days after birth and then fed a standard milk replacer until shipment to Iowa State University at 2 to 8 days of age. Upon arrival at an animal biosafety level-2 building at Iowa State University, calves were weighed (28 to 47 kg, with Holstein-Jersey crosses representing the lower weights)

89 and randomly assigned (without redistribution) to one of two separate but adjacent rooms. Each

90 room had constant ambient temperatures (about 22oC) and humidity (about 40%) and was

91 ventilated by negative pressure through HEPA filters. Calves were housed in individual 18 m2

92 pens on Tenderfoot-type flooring without bedding.

93 Two separate experiments were performed each using 20 calves (10 per group) of similar

94 ages (2-4 days in one experiment and 6-8 days in the other experiment), and treatment groups

95 were alternated in the two different rooms in each experiment in order to avoid a "room effect".

96 Each room was fed either SCFP or the Control to avoid the potential for inappropriate

97 administration of a treatment within a room. Rooms were alternated between the two

98 experiments, i.e., in the first experiment calves in "Room A" received SCFP while calves in this

99 same room received the Control in the second experiment.

100 Calves were randomly assigned to one of two treatments: Control (no additive in milk

101 replacer or starter grain) or diet that contained two proprietary Saccharomyces cerevisiae

102 fermentation products delivered separately (SCFP; 1 g/head/d SmartCare™ [0.15% inclusion

103 rate in conventional milk replacer] and 3.5 g/head/d Original XPC™ administered orally via

104 gelatin capsule; Diamond V, Cedar Rapids, Iowa). SmartCare is a water dispersible product that

105 can be added directly to milk or milk replacer as a supplement for pre-weaning liquid calf diets

106 (starting at d 1). Original XPC is dry feed product commonly used in pre-weaning calf starter

107 diets. The combination of these products is the basis for Diamond V's dairy calf program during

108 the pre-weaning phase. A gelatin capsule containing 3.5 g/head/d grain matrix used to produce

109 XPC was given to Control calves to equalize the nutrients, although minimal, contributed by

110 XPC.

All calves were fed a non-medicated milk replacer (20% all-milk protein, 20% fat; Land O'Lakes Animal Milk Products, Shoreview, MN) at a volume equivalent rate of approximately 10% of arrival BW twice per day (i.e., 5% of BW each feeding) for the duration of the trial. Milk replacer was mixed in single batches using warm water and a cordless drill-driven stirrer. Specifically, each calf received 6 oz of milk replacer in 1 qt water bid, in which the milk replacer was 18.8% w/v of the solution.

Calves were fed calf starter (Calf Startena™, 18% crude protein, 0.005% decoquinate, Purina Mills, LLC, St. Louis, MO) and water ad libitum, although it was not feasible to measure intake of either because of spillage and other uncontrollable factors. The Iowa State University investigators (M.T.B., K.L.A., and S.A.C.) were blinded as to which calves received the Control or SCFP treatments. Specifically, the Diamond V investigators (I.Y. and M.F.S.) notified a third party (scientists at the Office of Intellectual Property at Iowa State University) as to the identity of the treatment groups prior to the onset of the studies. Once the studies were completed, the Iowa State University investigators revealed the data to the Office of Intellectual Property who then revealed the identity of the treatment groups.

In the two-week pre-infection phase, six to seven calves from each group were orally treated with one dose of sulfamethazine (356 mg/kg; Sustain™, Bimeda, Oakbrook Terrace, IL) for veterinarian diagnosed coccidial infections manifested by blood in the feces. This treatment alleviated the bloody feces within 3 d of treatment. Treatment with sulfamethazine was deemed to not have a negative effect on the outcome of the trial since the Salmonella strain used in this study is resistant to sulfonamides and equivalent numbers of calves from each experimental group were subjected to sulfamethazine treatment.

134 2.2. Salmonella Infection of Calves

136 Calves were confirmed to be Salmonella-free by fecal culture on arrival and on d 7 and

137 12 post-arrival. Specifically, 1 g of freshly voided feces was diluted in 20 mL of Lennox L broth

138 (Invitrogen, Carlsbad, CA). After settling, an aliquot (100 ^L) of this mixture was streaked onto

139 and then incubated overnight at 37oC on XLD agar (Fisher Scientific, Pittsburgh, PA) selective

140 for Salmonella that appear as red colonies with black centers. All pre-infection fecal samples

141 were free of Salmonella. Sulfamethazine was not used in the XLD agar for pre-infection

142 assessment. SL1344 was plated on XLD as a positive control during the experiments.

143 At d 14 post-arrival (d 0 post-infection), calves were orally inoculated with Salmonella

144 enterica serotype Typhimurium strain SL1344 (Wray and Sojka, 1978; personal collection of

145 S.A.C.) at the dose of 2 x 106 CFU/kg BW (Xiong et al., 2013). The Salmonella inoculum was

146 prepared and dosed as described previously by the Carlson laboratory (Xiong et al., 2013). The

147 inoculum was placed in the gelatin capsule containing the Control or SCFP, which was

148 administered using a small balling gun. Our empirical studies revealed that the strain SL1344

149 was viable after incubation with either the Original XPCTM or the Control treatment. That is,

150 pre-inoculated experiments revealed that SL1344 was 100% viable and recoverable after

151 remaining in the Control- or SCFP-containing gelatin capsule for 24 hrs. Additionally, the

152 virulence of SL1344 was retained (as determined by a tissue culture invasion assay) following

153 recovery from the Control- or SCFP-containing capsule (data not shown).

2.3. Assessment of Clinical Parameters in Calves

Pyrexia and diarrhea are frequently observed components of salmonellosis in calves (Smith et al., 1979). On d 0, 1, 2, 3, 4, 5, 6, 7, 10, and 21 post-infection, rectal temperatures were measured and diarrhea was assessed on an ordinal scale. Diarrhea scoring was as follows: 0, no diarrhea; 1, mild diarrhea; 2, profuse diarrhea; or 3, profuse diarrhea with blood. This determination was performed by one investigator (S.A.C.), who was blinded to the treatment groups, with vast experience with experimental salmonellosis.

2.4. Assessment of Salmonella Shedding in Calves

Fecal shedding of Salmonella is a highly variable and sporadic occurrence in calves infected with Salmonella (Kirchner et al., 2012). Nonetheless, this event is of importance for the spread of this pathogen (Lanzas et al., 2010). On d 0, 1, 2, 3, 4, 5, 6, 7, 10, and 21 post-infection, 1 g of freshly voided feces was briefly vortexed in 20 mL of Lennox L broth (Invitrogen, Carlsbad, CA). An aliquot of this mixture (100 ^L) was incubated overnight at 37oC on XLD agar containing the Committee on Laboratory Standards Institute-derived (CLSI, 2008) breakpoint concentration (512 ^.g/mL) of sulfamethazine (Sigma Aldrich, St. Louis, MO), i.e., a concentration that will enable the growth of SL1344 but will inhibit the growth of many other enteric bacteria. The following day red colonies with black centers were enumerated and CFU/g of feces was calculated based on a dilution factor equal to 200.

180 2.5. Assessment of Intestinal Colonization by Salmonella

182 Intestinal colonization by Salmonella contributes to the persistence of the pathogen

183 (Cobbold et al., 2006) and the fecal shedding that transmits the microbe to other cattle. On d 21

184 post-infection, all calves were euthanized using xylazine (0.5 mg/kg, intramuscular, Lloyd

185 Laboratories, Walnut, CA) and pentobarbital (100 mg/kg, intravenous, Fort Dodge Laboratories,

186 Fort Dodge, IA). A 2 cm section (approximately 1 g) of distal ileum was aseptically removed

187 from each calf and cut longitudinally. Each section was placed in 20 mL Lennox L broth

188 (Invitrogen, Carlsbad, CA) and briefly vortexed to dislodge the Salmonella. An aliquot (100 ^L)

189 of this mixture was then dispersed onto XLD agar containing sulfamethazine (to prevent the

190 growth of other bacteria) that were incubated overnight at 37oC. The following day red colonies

191 with black centers were enumerated and CFU/g of ileum was calculated based on a dilution

192 factor equal to 200.

194 2.6. Assessment of Rumen Development in Calves

196 Salmonella, and any other enteric pathogen, can have a negative impact upon

197 performance in the pre-weaned calf. It was hypothesized that SCFP may abrogate salmonellosis

198 by promoting overall gastrointestinal health and improving rumen development in pre-weaned

199 calves. Therefore, both weight gain and the size of rumen papillae were assessed in Salmonella-

200 infected calves fed SCFP. Following euthanasia, a 4 cm2 section of a ventrolateral portion of the

201 rumen was removed and placed in 10% buffered-neutral formalin. Rumen tissues were

202 submitted to the Histopathology Laboratory at the College of Veterinary Medicine at Iowa State

University. Tissues were prepared using standard hematoxylin and eosin staining. Length and width of rumen papillae were measured using an intra-ocular ruler. Measurements were collected from 10 randomly selected papillae present on two different sections (i.e., five papillae from each section). Widths were measured at mid-shaft.

2.7. Statistical Analyses

For data in which assessments were performed on multiple days (rectal temperatures, diarrhea scores, and fecal shedding), statistical comparisons were made using a repeated measures analysis of variance with Tukey's ad hoc test for multiple comparisons (GraphPad Prism, Version 6, La Jolla, CA). For data involving single measurements from each calf (intestinal colonization and rumen papillae length), statistical comparisons were performed using a student's t-test (GraphPad). Significant differences were defined at P < 0.05. Statistical trends were consistent when the two sets of experiments were examined independently (data not shown).

3. Results

3.1. Assessment of Clinical Parameters in Calves

Rectal temperatures and diarrhea we monitored in the calves on d 0, 1 through 7, 10, and 21 post-infection. As shown in Figures 1 and 2, pyrexia (rectal temperature > 39.2oC) and diarrhea were observed less frequently (P < 0.05) in calves fed SCFP. The largest differences

between calves supplemented with SCFP and Control calves were observed on d 3 to 5 postinfection for rectal temperatures (38.8 to 39.0oC versus 39.5 to 39.7oC, respectively), and d 3 through 7 post-infection for diarrhea scores (0.10 to 0.25 versus 0.61 to 0.86 arbitrary units, respectively). None of the SCFP-fed calves exhibited pyrexia at anytime throughout the study, while nearly all Control calves exhibited pyrexia on d 2 through 6 post-infection. The relative incidence of diarrhea was less in SCFP-fed calves on all days except day 0 (Fig. 2b).

3.2. Assessment of Salmonella Shedding in the Calves

Salmonella fecal shedding was monitored on d 0, 1 through 7, 10, and 21 post-infection. As shown in Figure 3 a, there was a quantitative difference in fecal shedding between the two groups of calves on d 6 post-infection in which shedding was less (P < 0.05) in calves fed SCFP (undetectable versus 955 CFU/g of feces). There also was a qualitative difference in fecal shedding of Salmonella on d 3 through 7 post-infection (Fig. 3b).

3.3. Assessment of Adherent Salmonella in the Intestines of the Calves

Upon euthanasia, ileal sections were excised and subjected to Salmonella culture and enumeration that quantifies intestinal colonization of Salmonella. As shown in Figure 4, fewer (P < 0.05) adherent Salmonella were present in the ilea of calves fed SCFP (1,620 versus 9,289 CFU/g of ileum). Gross examinations of the ilea revealed no apparent differences between the two groups.

249 3.4. Assessment of Rumen Development and Weight Gain in the Calves

251 As shown in Figures 5 and 6, rumen papillae length was greater (P < 0.05) in calves fed

252 SCFP (236 versus 203 ^.m) while papillae width was indistinguishable (50 versus 40 |j,m). Table

253 1 shows the body weight at different stages of experiment and Figure 7 reveals the superior (P <

254 0.05) weight gain in Salmonella-infected calves fed SCFP (23.8 versus 17.2%).

256 4. Discussion

258 Salmonella is an insidious problem for the dairy industry. This problem represents a

259 critical animal health issue since cattle of all ages are affected by the pathogen. Adult cattle will

260 exhibit diarrhea and anorexia, both of which compromise the performance of the animal. In the

261 pre-weaned calf, Salmonella can cause diarrhea and malaise that will also hamper performance

262 and expose caretakers to the pathogen. Furthermore, salmonellosis in the pre-weaned calf can

263 lead to persistent infection and the carrier state. These animals become an asymptomatic source

264 of Salmonella for the herd, while some animals may perish because of the infection.

265 Identifying Salmonella mitigation strategies is of critical significance yet the progress is

266 very slow. In this study, the anti-Salmonella effects of SCFP were examined and three critical

267 indicators of salmonellosis (pyrexia, diarrhea, and intestinal colonization) were significantly

268 reduced by SCFP. The absence of pyrexia and the diminished diarrhea in the calves fed SCFP

269 are consistent with the reduced intestinal colonization by Salmonella. Both pyrexia and diarrhea

270 are dependent upon pathogen burden and it appears that Salmonella may be less efficient at

271 attaching to the intestinal tract in the presence of SCFP. This diminished intestinal colonization

was manifested by a cessation of Salmonella shedding in calves fed SCFP at 4 d prior to the last day of fecal shedding in calves fed the Control. In Control-fed calves, a higher number of adherent Salmonella were present in the intestinal tract thus extending the overall shedding period, which ultimately increased the risk of disease transmission. While we did not measure intracellular bacteria in the gut epithelium, we do not envision that differences in intracellular bacteria would account for a five-fold difference in the recovery of the Salmonella in the two groups.

Our results are consistent with a previous study (Magalhaes et al., 2008) in which feeding SCFP led to an improvement of gastrointestinal health in pre-weaned dairy calves naturally exposed to Salmonella. Although the mechanism of action is yet to be clarified, the effect of SCFP on intestinal colonization (Ibukic et al., 2012) and growth (Broomhead, et al., 2012; Nsereko et al., 2013) of Salmonella has also been reported in poultry.

Other significant findings in this study are the improved weight gain and rumen papillae maturation in calves fed SCFP, which is consistent with a non-infectious study whereby supplemental SCFP improved pre-weaning calf growth, feed intake, and corresponding rumen development parameters (Lesmeister et al., 2004). Although it is unclear how these parameters were improved by feeding the Saccharomyces cerevisiae fermentation products, these findings suggest an economic benefit for inclusion in milk replacer and starter grain as demonstrated previously (Magalhaes et al., 2008). It is likely that these benefits extend to calves in herds even in which Salmonella is not endemic. Furthermore, it is possible that these benefits extend to protection from related enteric pathogens such as E. coli since previously reported studies suggested SCFP could inhibit the growth of E. coli (Jenson et al., 2008b). Future studies will assess this possibility.

295 5. Conclusions

297 In summary, Salmonella-infected dairy calves were significantly less likely to exhibit

298 clinical signs associated with salmonellosis when fed SCFP. Specifically, these calves were less

299 likely to exhibit pyrexia and diarrhea, possibly as a direct result of diminished intestinal

300 colonization by Salmonella. Ultimately, these protective effects augmented growth and

301 improved rumen development in the calves infected with a serious enteric pathogen.

303 Conflict of interest

305 This work was funded by Diamond V, along with partial matching funds from the Institute of

306 Physical Research and Technology at Iowa State University. Salary support was provided to the

307 corresponding author in exchange for his time devoted to the project, but no other financial

308 compensation was or will be awarded. Thus the authors declare no conflict of interest.

310 Acknowledgments

312 The authors thank Dale Hinderaker and Diane McDonald for animal husbandry. We also

313 would like to thank Dr. Michael Yaeger (Diplomate ACVP) for the rumen papillae studies.

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Table 1. Effect of Saccharomyces cerevisiae fermentation products (SCFP) on body weight of calves before and after Salmonella challenge1.

Body Weights (mean + SEM) (kg) % Growth (mean + SEM) d 0 (start) d 14 (at d 35 % growth % growth % growth Treatment2 challenge) (end) from d 0 from d 14 from d 0 to _to d 14 to d 35_d 35

Control 42.8 + 0.7 45.1 + 0.7 50 + 0.8 5.3 + 0.2 10.9 + 0.2 16.7 + 0.5 SCFP 40.3 + 1 42.5 + 1.1 50 + 1.4 5.5 + 0.5 17.4 + 0.9* 23.8 + 0.8*

Salmonella enterica serotype Typhimurium strain SL1344 at the dose of 2 x 106 CFU/kg BW.

Control - no additive in milk replacer or starter grain; SCFP - diet that contained two proprietary

SCFP products (1 g/head/d SmartCare™ [0.15% inclusion rate in conventional milk replacer] and 3.5

g/head/d Original XPC™ administered orally via gelatin capsule; Diamond V, Cedar Rapids, Iowa)

*P <0.05 versus Control when comparing growth rates.

39.9-1

o> 39.7 о

ф 39.5 ■o

§_39.1

138.9 (6

0 1 2 3 4 5 6 7 10 21

Days Post-Infection

Figure 1. Time course of rectal temperatures in Salmonella-infected calves fed SCFP or the Control. Treatments include Control (no additive in milk replacer or starter grain) or SCFP (diet that contained two proprietary Saccharomyces cerevisiae fermentation products (1 g/hd/d SmartCare™ [0.15% inclusion rate in conventional milk replacer] and 3.5 g/hd/d Original XPC™ administered orally via gelatin capsule; Diamond V, Cedar Rapids, Iowa). Data represent the mean + SEM for single daily measurements on 20 calves in each group. The dashed line represents the upper limit of the normal rectal temperature for a calf. *P <0.05 versus Control.

m- scfp

Control

Days Post-Infection

Figure 2a. Time course of diarrhea scores in Salmonella-infected calves fed SCFP or the Control. Treatments include Control (no additive in milk replacer or starter grain) or SCFP (diet that contained two proprietary Saccharomyces cerevisiae fermentation products (1 g/hd/d SmartCare™ [0.15% inclusion rate in conventional milk replacer] and 3.5 g/hd/d Original XPC™ administered orally via gelatin capsule; Diamond V, Cedar Rapids, Iowa). Diarrhea scoring was as follows: 0, no diarrhea; 1, mild to moderate diarrhea; 2, profuse diarrhea; or 3, profuse diarrhea with blood. Data represent the mean + SEM for single daily measurements on 20 calves in each group. *P <0.05 versus the Control.

7 10 21

Days Post-Infection

Figure 2b. Time course of diarrhea incidences in Salmonella-infected calves fed SCFP or the Control. Treatments include Control (no additive in milk replacer or starter grain) or SCFP (diet that contained two proprietary Saccharomyces cerevisiae fermentation products (1 g/hd/d SmartCare™ [0.15% inclusion rate in conventional milk replacer] and 3.5 g/hd/d Original XPC™ administered orally via gelatin capsule; Diamond V, Cedar Rapids, Iowa). Percentages are based on the number of calves exhibiting any type of diarrhea on a given day, with n=20 per group on all days.

Days Post-Infection

Figure 3a. Time course of Salmonella fecal shedding in Salmonella-infected calves fed SCFP or the Control. Treatments include Control (no additive in milk replacer or starter grain) or SCFP (diet that contained two proprietary Saccharomyces cerevisiae fermentation products (1 g/hd/d SmartCare™ [0.15% inclusion rate in conventional milk replacer] and 3.5 g/hd/d Original XPC™ administered orally via gelatin capsule; Diamond V, Cedar Rapids, Iowa). Data represent the mean + SEM for single daily measurements on 20 calves in each group. *P < 0.05 versus SCFP.

Days Post-Infection

Figure 3b. Time course of fecal shedding incidences in Salmonella-infected calves fed SCFP or the Control. Treatments include Control (no additive in milk replacer or starter grain) or SCFP (diet that contained two proprietary Saccharomyces cerevisiae fermentation products (1 g/hd/d SmartCare™ [0.15% inclusion rate in conventional milk replacer] and 3.5 g/hd/d Original XPC™ administered orally via gelatin capsule; Diamond V, Cedar Rapids, Iowa). Percentages are based on the number of calves shedding any detectable amount of Salmonella on a given day, with n=20 per group on all days.

? 11000! 10000

O 8000

| 7000 | 6000^

| 5000-

5? 4000-

1 3000-m 200010000-

Control

Figure 4. Salmonella ileal colonization in Salmonella-infected calves fed SCFP (filled column) or the Control (open column). Treatments include Control (no additive in milk replacer or starter grain) or SCFP (diet that contained two proprietary Saccharomyces cerevisiae fermentation products (1 g/hd/d SmartCare™ [0.15% inclusion rate in conventional milk replacer] and 3.5 g/hd/d Original XPC™ administered orally via gelatin capsule; Diamond V, Cedar Rapids, Iowa). Data represent the mean + SEM for single measurements on 20 calves in each group. *P <0.05 versus the Control.

c o to c CD

Length

Figure 5. Rumen papillae dimensions in Salmonella-infected calves fed SCFP or the Control. Treatments include Control (no additive in milk replacer or starter grain) or SCFP (diet that contained two proprietary Saccharomyces cerevisiae fermentation products (1 g/hd/d SmartCare™ [0.15% inclusion rate in conventional milk replacer] and 3.5 g/hd/d Original XPC™ administered orally via gelatin capsule; Diamond V, Cedar Rapids, Iowa). Data represent the mean + SEM for measurements on 20 calves in each group. Rumen papillae were measured ten times for each animal. Widths were measured at the mid-shaft of the papillae. *P <0.05 versus the Control.

499 Figure 6. Representative rumen papillae photomicrographs (40X) of hematoxylin-eosin stained sections

500 from Salmonella-infected calves SCFP (top) or the Control (bottom). Treatments include Control (no

501 additive in milk replacer or starter grain) or SCFP (diet that contained two proprietary Saccharomyces

502 cerevisiae fermentation products (1 g/hd/d SmartCare™ [0.15% inclusion rate in conventional milk

503 replacer] and 3.5 g/hd/d Original XPC™ administered orally via gelatin capsule; Diamond V, Cedar

504 Rapids, Iowa). Papillae are longer in calves fed SCFP, whereas papillae widths are indistinct. A 100^,m

505 scale bar is present on the bottom right of each photo.

Q d 15 through 35

Control

Figure 7. Percent body weight gains in Salmonella-infected calves fed SCFP or the Control. Treatments include Control (no additive in milk replacer or starter grain) or SCFP (diet that contained two proprietary Saccharomyces cerevisiae fermentation products (1 g/hd/d SmartCare™ [0.15% inclusion rate in conventional milk replacer] and 3.5 g/hd/d Original XPC™ administered orally via gelatin capsule; Diamond V, Cedar Rapids, Iowa). Data represent the mean + SEM for measurements on 20 calves in each group. Body weights were measured on d 0, 14, and 35. *P <0.05 versus the Control.