EOI SUBMISSION

Expression of Interest (EOI)

Determination of the postbiotic effects of Saccharomyces cerevisiae fermentation product

(SCFP - Original XPC™) on the immune response post immunization of dairy cattle against

foot and mouth disease, anthrax, brucellosis and lumpy skin disease vaccines in dairy cows.

Submitted By:

Dr. Joseph Nginyi (PhD)

Veterinary Research Institute,
Kenya Agricultural and Livestock Research Organization,
P.O. Box 32-00902 Kikuyu, Kenya
Tel. +254-20-2524616/2519769
email: Joseph.Nginyi@kalro.org
Nairobi, Kenya.

Submitted to
Victor Nsereko
Research Manager
Cargill TRANSFORM
General objective
To determine the postbiotic effects of Saccharomyces cerevisiae fermentation product (SCFP -

Original XPC™) on the immune response post immunization of dairy cattle against foot and

mouth disease, anthrax, brucellosis and lumpy skin disease vaccines in dairy cows.

Specific objectives

i. To determine the modulative effect of feeding Saccharomyces cerevisiae

fermentation product (SCFP - Original XPC™) on stress and serum biochemical

profiles when fed to dairy cattle at various ages.

ii. To determine the modulative effect of feeding Saccharomyces cerevisiae

fermentation product (SCFP - Original XPC™) on the immunity of dairy cattle post

immunization with foot and mouth disease, anthrax, brucellosis and lumpy skin

disease vaccines.

iii. To determine the optimal levels of feeding Saccharomyces cerevisiae fermentation

product (SCFP - Original XPC™) that gives the best immune response outcome in

dairy cattle

Methodology

This study will be a completely randomized design (CRD) study it will involve various groups of

dairy cattle at different physiological groups involving weaners and heifers. The 64 animals will

be grouped into experimental and control groups of 4 animals each. All groups of animals will be

housed under the same conditions with the only varying component being the treatments.

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Common group treatments

The animals once moved to the experimental units and randomly assigned into the experimental

groups, they will be allowed to acclimatize to the new set up for 14 days with serum collection to

determine the baseline for the various variables - cortisol levels, FMD, LSD, brucella and

anthrax antibody titers as well as the various biochemical profiles. The animals will then be

dewormed and rested. After 14 days, the experiment will be initiated and the experimental

teams will be double blinded on the experiment – on the product under evaluation as well as the

experimental groups. 21 days into the start of the experiment, the animals will be immunized as

per the protocol shown below. All the various groups will then be samples every 14 days for the

various serological or biochemical tests for 36 weeks.

Vaccination of experimental groups

Paired sequential vaccination regimes will be implemented in the various experimental groups.

This means that the Foot and Mouth Disease vaccine will be paired with Brucellosis vaccine

while the Lumpy skin vaccine will be paired with the Anthrax vaccine. This will be done so to

combine a viral and bacterial vaccine to eliminate any chances of cross reactivity or interactions

as well as cut down on the numbers of animals to be used in the study. The vaccinations will be

carried out 12 weeks apart.

Animal groups and feeding of SCFP - Original XPC™.

The heifers will be grouped into; heifer group A (HA), heifer group B (HB), heifer group C (HC)

and heifer group D (HD) who will be fed on 14g/d throughout the experimental period (Zaworski

et al., 2014) . Heifer groups C and D will be the control groups for this cohort. This group will not

be fed on (SCFP - Original XPC™). Another group of Heifers will be heifer group 1 (H1), heifer

group 2 (H2), heifer group 3 (H3) and heifer group 4 (H4) who will be fed on 28g/d throughout
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the experimental period (Deters, Stokes, Genther-Schroeder, & Hansen, 2018; Zaworski et al.,

2014). Heifers group 3 and 4 will be the control groups for this cohort. Just like the other control

group, these groups will also not be fed on (SCFP - Original XPC™).

The weaners will be grouped into; weaner group A (WA), weaner group B (WB), weaner group

C (WC) and weaner group D (WD) who will be fed on 14g/d throughout the experimental period.

Weaner groups C and D will be the control groups for this cohort. This group will not be fed on

(SCFP - Original XPC™). Another group of weaners will be weaners group 1 (W1), weaners

group 2 (W2) and weaners group 3 (W3) and weaner group 4 (W4) who will be fed on 28g/d

throughout the experimental period. Weaners group 3 and 4 will be the control groups for this

cohort. Just like the other control group, these groups will also not be fed on (SCFP - Original

XPC™). Animal groups will be on the same diets during the study.

Objective 1

Biochemical parameters of, aspartate amino transferase (AST) or serum glutamic oxaloacetic

transaminase (SGOT), alanine amino transferase (ALT) or serum glutamic-pyruvic

transaminase (SGPT), alkaline phosphatase (AP), total protein, albumin, direct bilirubin, total

bilirubin, creatine phosphokinase (CPK) and creatinine will be estimated in serum samples of

vaccinated animals using commercially available kits. Serum cortisol will also be assessed

every 14 days.

Objective 2

Foot and mouth disease (FMD): Vaccine serotypes A, O, SAT1 and SAT2. Will be assessed

in post-vaccination monitoring (PVM). Sera will be assessed for vaccine-induced antibodies as

outlined by using three commercial type ELISA. Paired t-test will be performed, and p < 0.05 will

be considered statistically significant.

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Anthrax: Both control and experimental animals will be immunized with Blanthrax® as per

manufacturer directions. Commercially available Indirect ELISA kits will be used to detect

protective antigen (PA) IgG antibody response post immunization. Slide agglutination test will be

carried out on day 0 and serially on day 30, 60 and 90 to determine serum antibody response.

Brucellosis: The experimental cattle will be vaccinated with either attenuated live vaccine

Brucella abortus S19 or RB51 as per manufacturers recommendations. Commercially available

indirect ELISA kits will be used to determine the amount of antibody specific to B. abortus

present in the sera of animals

Lumpy skin disease: Both control group and the experimental groups will be immunized using

quadrivalent vaccine LUMPIVAXTM as per manufacturer directions. Detection of LSD-specific

antibodies will be performed with the standard serological methods VNT and IFAT to determine

humoral immunity development.

Objective 3:

The outcomes of objective 1 and 2 will be compared across the weaner and heifer groups that

will be fed different quantities of SCFP - Original XPC™ to determine the best performing group

and what facors influence the performance through comparison of various parameters

Statistical analysis

Two-way analysis of variance (ANOVA) with Tukey’s multiple comparison posthoc test and

unpaired t-test will used to determine statistically significant differences within and across

groups.

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Annex 1: Vaccination groups

Group SCFP - Original First Second Number of

XPC™ g/d vaccine Vaccine animals

WA 14 FMDV Brucella Vaccine 4

WB 14 Anthrax LSDV 4

vaccine

WC 0 FMDV Brucella vaccine 4

WD 0 Anthrax LDDV 4

W1 28 LSD Anthrax vaccine 4

W2 28 Brucella FMDV 4

Vaccine

W3 0 FMDV Brucella vaccine 4

W4 0 Anthrax LDDV 4

HA 14 FMDV Brucella Vaccine 4

HB 14 Anthrax LSDV 4

vaccine

HC 0 FMDV Brucella vaccine 4

HD 0 Anthrax LDDV 4

H1 28 LSD Anthrax vaccine 4

H2 28 Brucella FMDV 4

Vaccine

H3 0 FMDV Brucella vaccine 4

H4 0 Anthrax LDDV 4

Total 64

*** red font = control groups

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Annex 2: Assessment of immunity to various vaccines

i. Foot and mouth disease (FMD)

Both control group and the experimental groups will be immunized using quadrivalent vaccine

FOTIVAX TM as per manufacturer directions. The vaccine is manufactured by the Kenya

Veterinary Vaccines institute. It has the serotypes A, O, SAT1 and SAT2. Post-vaccination

monitoring (PVM) to evaluate the performance of vaccination regimens and program is essential

for assessing vaccine efficacy and immunity development in vaccinates. For FMD vaccine

evaluation, Sera were assessed for vaccine-induced antibodies as outlined by WHO Manual 3

(2018) using three commercial type O SP antibody ELISAs: PrioCHECKTM FMDV Type O Ab

strip kit (Thermo Fisher Scientific) following manufacturer protocols

R software (www.r-project.org) and EXCEL will be used to compile the ELISA results. To

evaluate the effectiveness of the vaccine, and development of immunity. Sero-monitoring post-

vaccination will compared between controls and vaccinates. A comparison by age will be

analyzed to identify any differences in immunity development attributed to age. Paired t-test will

be performed, and p < 0.05 will be considered statistically significant. For the purpose of this

study, herd immunity was defined as seroprevalence with 95% confidence interval (CI)

estimated for FMDV serotype O.

ii. Anthrax

Both control and experimental animals will be immunized with Blanthrax® as per manufacturer

directions. The vaccine is manufactured by MSD Animal Health it is a combined vaccine for

black quarter and anthrax vaccine. Commercially available Indirect ELISA kits will be used to

detect protective antigen (PA) IgG antibody response post immunization. Further to this, slide

agglutination test will be carried out on day 0 and serially on day 30, 60 and 90 to determine

serum antibody response as described by Fatema, (2011).

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Results will be compared inter and intra groups. R software (www.r-project.org) and EXCEL will

be used to compile the ELISA and slide agglutination results. To evaluate the effectiveness of

the vaccine, and development of immunity. Sero-monitoring post-vaccination will compared

between controls and vaccinates. A comparison by age will be analyzed to identify any

differences in immunity development attributed to age. Paired t-test will be performed, and p <

0.05 will be considered statistically significant.

iii. Brucellosis

The experimental cattle will be vaccinated with either attenuated live vaccine Brucella

abortus S19 or RB51 as per manufacturers recommendations. Both vaccines are commonly

used in Kenya depending on their availability and farmer preference. Commercially available

indirect ELISA kits will be used to determine the degree of the color that develops (optical

density measured at 450 nm) is directly proportional to the amount of antibody specific to B.

abortus present in the sera of animals

Results will be compared inter and intra groups. R software (www.r-project.org) and EXCEL will

be used to compile the ELISA and slide agglutination results. To evaluate the effectiveness of

the vaccine, and development of immunity. Sero-monitoring post-vaccination will compared

between controls and vaccinates. A comparison by age will be analyzed to identify any

differences in immunity development attributed to age. Paired t-test will be performed, and p <

0.05 will be considered statistically significant.

iv. Lumpy skin disease

Both control group and the experimental groups will be immunized using quadrivalent vaccine

LUMPIVAXTM as per manufacturer directions. The vaccine is manufactured by the Kenya

Veterinary Vaccines institute. Detection of LSD-specific antibodies was performed with the

standard serological methods VNT and IFAT as well as a. Humoral immune response to
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vaccination will be investigated by using virus neutralization test (VNT), indirect fluorescent

antibody test (IFAT) and ELISA using commercially available Capripox double antigen multi-

species-ELISA. This is because VNT is the only serological test validated by the OIE (OIE,

2004) with a high specificity for detecting capripoxvirus-specific antibodies while the other tests

will be augmenting and validating the results as well as forming a basis of comparison across

groups.

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Annex 3: References
Deters, E. L., Stokes, R. S., Genther-Schroeder, O. N., & Hansen, S. L. (2018). Effects of a

Saccharomyces cerevisiae fermentation product in receiving diets of newly weaned beef

steers. I. Growth performance and antioxidant defense1. Journal of Animal Science, 96(9),

3897–3905. https://doi.org/10.1093/jas/sky246

Fatema, T. Z. (2011). Standerdization of an ELISA protocol for the detection of IgG antidody in

cattle against anthrax vaccine. BAU.

OIE. (2004). OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Mammals,

Birds and Bees) (5th ed.; M. S. J, Ed.). Cambridge: Cambridge.

WHO. (2018). Foot and Mouth Disease Vaccination and Post-Vaccination Monitoring.

https://doi.org/10.3389/fvets.2021.673820

Zaworski, E. M., Shriver-Munsch, C. M., Fadden, N. A., Sanchez, W. K., Yoon, I., & Bobe, G.

(2014). Effects of feeding various dosages of Saccharomyces cerevisiae fermentation

product in transition dairy cows. Journal of Dairy Science, 97(5), 3081–3098.

https://doi.org/10.3168/jds.2013-7692

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