Proposal Research Internship and Minor, Master Farm Animals
and Veterinary Human Health
Personalia
Student-number:
Name:
Curriculum:
Phonenumber:
E-mailadress:
3383415
T.M. Vingerhoets, BSc
Master
06-18083419
T.M.Vingerhoets@uu.nl
Subject and place of performance
Title of research:
Allicin as a treatment for subclinical
mastitis in dairy cows.
Field-research of functionality of a
short-term bolus (STB) based on allicin
26 August 2013
Dept. Gezondheidszorg
Landbouwhuisdieren, afdeling Voeding
Dr J.T. Schonewille
Dr A. van Vuuren, Wageningen UR
Livestock
J.T.schonewille@uu.nl
ad.vanvuuren@wur.nl
Description
Planned start date
Departement:
Supervisor at faculty
External supervisor
E-mailadresses supervisors
Introduction
Bovine subclinical mastitis is an inflammation of the udder with which cows do not show any
clinical symptoms, but a decreased milk yield and a decrease in quality of the milk
composition. (L. Forsback, 2009) Farmers should be aware that subclinical mastitis spreads
easily over the herd and can evolve into a clinical mastitis with clinical symptoms. Especially
because of the decreased milk yield subclinical mastitis is an important problem for the
economics and the health of cows at dairy farms. Subclinical mastitis can be caused by a
large diverse group of bacteria. These can be contagious as well as environmental. The most
important pathogen concerning subclinical mastitis is Staphylococcus aureus, however also
Streptococci spp., Escherichia coli and Klebsiella spp. play their parts. (Radostitis, 2007) In
the last decennia different types of antibiotics have been developed to treat subclinical
mastitis and to avoid an increase in the prevalence of clinical mastitis. During-lactation
antibiotic therapies, however, are not always successful. There are many different factors and
causes affecting these therapies. One of them is the development of resistance against
antibiotics by pathogenic bacteria. (M. Sandholm, 1990), (Ayma El Behiry, 2012) Therefore
there is a strong need for a new kind of treatment.
The “producer” wants to provide an answer to this need by the production of two types of
boluses based on allicin. Allicin is the active substance in garlic, which amongst others is
responsible for its characteristic odour. Research showed us that allicin has antibacterial,
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antiviral, antifungal and anti-parasitic properties. It is stored, in a bulb of garlic, in particles
as the precursor protein alliin. In other particles of the bulb the enzyme alliinase or alliinlyase is present. When the alliin and alliinase come together, a volatile reaction takes place
and allicin is produced. (Serge Ankri, 1999) In vitro research has proven that allicin has
antibacterial properties against mastitis-causing bacteria. (M. Safithri, December 2011). Also
reports have been published stating the success of a liquid form allicin.
The “producer” plans to develop two kinds of boluses, of which the first will be a short-term
bolus (STB), that will administer the cow with allicin for 7 days, where the second, a longterm bolus (LTB), will administer the cow with allicin for 21 days. Both boluses will be
administered orally, after which they will reach the rumen. It is assumed that they will remain
in the rumen for the whole period. Furthermore it is assumed that in the rumen the active
allicin will come free from the bolus and that the allicin will reach the udder via the
circulatory system. In the udder it will kill the pathogens responsible for the subclinical
mastitis. This way, the allicin boluses should reduce the prevalence of subclinical mastitis in
dairy cows.
It should be questioned however, whether the allicin would not also attack the ruminal flora.
The effect of allicin on the composition of the different volatile fatty acids produced by the
ruminal flora was studied. It was shown that allicin has no significant influence on the
composition of the volatile fatty acids, but other components of garlic extract do. (M.
Busquet, December 2005) Therefore the allicin boluses should contain allicin only, or at least
as few of the other components as possible. If the boluses consist of merely allicin, they will
be safe for use in the bovine rumen.
In this research proposal a description will be given for a field-research concerning the
functionality of the STB in subclinical mastitis dairy cows.
Research goal
In this field-research the functionality of the STB on subclinical mastitis dairy cows will be
studied. After oral administration of subclinical mastitis cows with a STB the cowSCC will
be screened at every milking by an Automatic Milking System (AMS) from Lely Industries
N.V. and be expressed as semi-quantitative values. Also, milk samples will be taken from the
infected quarters of the udder at t=0, t=7 and t=28. Herein t is time in days. The presence of
pathogenic bacteria will be analyzed by PCR, performed at the GD Deventer. The species of
the present bacteria will be determined and the number of present bacteria will be expressed
as a semi-quantitative value. As a golden standard, the milk samples from four randomly
selected cows will be analyzed at VMDC of the Utrecht University as well. From all milk
samples, also the cowSCCs will be measured and expressed as a quantitative value. When the
allicin boluses are functional, the SCC and the presence of pathogenic bacteria will be
reduced.
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Materials
Dairy farms will be contacted until five farms are found willing to participate in the study.
The dairy farms will be selected for having access to an AMS from Lely Industries N.V.
Participating farms must have proper record-keeping, have proper cow identification and
have to be officially free of notifiable diseases. From these five farms a total of sixty
subclinical mastitic dairy cows (based on a power-analysis for the paired t-test as performed
in figure 1) will be selected. The selection will be based on the two last MPR cowSCC
scores, as well as on the cowSCC screens performed by the AMS of Lely Industries N.V. in
time within these two MPR scores. A cow will be selected if the last two MPR cowSCC
scores are at least >250,000 cells/mL milk and the mean cowSCC in the time within the two
MPR scores is at least category 2 (200,000 cells/mL milk – 400,000 cells/mL milk). A cow
will however be excluded from the research if it has teat lesions, is systematically ill, has
clinical mastitis, has been treated with antibacterial / anti-inflammatory drugs in the last 30
days, has reached a low milk yield, when it has an alarming MPR feeding score or when it is
to be dried off in the next 30 days. Duo’s will be made from semi-identical selected cows,
based on their cowSCC score in the second MPR.
Methods
The cows will be divided over two research groups both containing 30 cows. The first
research group, the control group, contains subclinical mastitis cows, that will not be treated.
The second group will be treated with the STB. The STB will be orally administered, From
this moment (t=0) the cowSCC will be screened at a semi-quantitative scale at every milking
by the AMS of Lely Industries N.V. At t=0, t=7 and t=28 milk samples will manually be
taken from the infected quarter. Which quarters are infected will be analyzed by electric
conductivity measurements, also performed by the AMS from Lely Industries N.V.. The milk
samples, taken from the infected quarters, will be analyzed for the presence of pathogenic
bacteria. The species will be determined and the number of present bacteria will be expressed
as a semi-quantitative value. From the milk samples, also a cowSCC will be measured and
expressed as a quantitative value. The presence of pathogenic bacteria will be analyzed by
PCR performed at the GD Deventer. Twelve milk samples from four randomly selected cows
(two pairs) will be analyzed at the VMDC from the Utrcht University as well as at the GD
Deventer. The PCR, performed at the VMDC from the Utrecht University will be seen as a
golden standard.
n≥8(σLnSCC/D)2
σLnSCC=0,92
D=Ln(250**)-Ln(150**)=0,51
n≥8(0,92/0,51)2
n≥26
** x1,000 cells/mL milk
Figure 1. The power-analysis for a paired t-test. (Schluter, 2009)*
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Hypotheses:
H0: The STB is not an effective treatment against subclinical mastitis in dairy cows. The
cowSCC and bacterial activity have not significantly been decreased within 28 days.
H1: The STB is an effective treatment against subclinical mastitis in dairy cows. The
cowSCC and bacterial activity have significantly been decreased within 28 days.
Time schedule
What
Orientation subject
Research proposal for “producer”
Writing literature study
Preparations Field-Research
Field Research farm visits
Start
Until
26 August
16 September
16 September 21 October
21 October
31 March
4 November
6 January
20 January
31 March
5 May
26 May
3 March
31 March
5 free weeks will be planned
for completing this research
in May and / or June 2014.
11 November 31 January
Writing rapport and article for publication
Parttime “chosen classes”
References
Ayma El Behiry, G. S. (2012). In vitro susceptibility of Staphylococcus aureus strains isolated from
cows with subclinical mastitis to different antimicrobial agents. J. Vet. Sci. 13(2), 153-161.
Craven, N. (1987). Efficacy and Financial Value of Antibiotic Treatment of Bovine Clinical Mastitis
During Lactation - a Review. Br. vet. J., 143, 410-422.
Dr. Tom Vanholder, D. M. (2012, maart). Mastitis bij melkkoeien. Diergeneeskundig Memorandium,
pp. 15-19.
H. A. Deluyker, S. N. (2005). Factors Affecting Cure and Somatic Cell Count After Pirlimycin
Treatment Of Subclinical Mastitis in Lactating Cows. J. Dairy Sci. 88, 604-614.
L. Forsback, H. L.-M.-S. (2009). Udder quarter milk composition at different levels of somatic cell
count in cow composite milk. Animal, 710-717.
M. Busquet, S. C. (December 2005). Effect of Garlic Oil and Four of its Compounds on Rumen
Microbial Fermentation. Journal of Dairy Science, vol. 88, iss. 12 , 4393-4404.
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M. Safithri, M. B. (December 2011). Antibacterial Activity of Garlic Extract Against some Pathogenic
Animal Bacteria. Media Peternakan, 155-158.
M. Sandholm, L. K. (1990). Bovine mastitis - why does antibiotic therapy not always work? An
overview. J. Vet. Pharmacol. Ther. 13, 248-260.
N. Sharma, N. S. (2011). Relationship of Somatic Cell Count and Mastitis: An overview. Asian-Aust. J.
Anim. Sci. Vol. 24, No. 3, 429-438.
Radostitis, C. (2007). Diseases of the mammary gland, bovine mastitis. In C. Radostits, Veterinary
Medicine, A textbook of diseases of cattle, horses, sheep, pigs and goats (pp. 673-696).
Edinburgh, London, New York, Oxford, Philadelphia, St Louis, Sydney, Toronto : Elsevier
Limited.
Schluter, M. C. (2009). Designing experiments. In M. C. Schluter, The Analysis of Biological Data (p.
383). Greenwood Village, Colorado: Roberts and Company Publishers.
Serge Ankri, D. M. (1999). Antimicrobial properties of allicin from garlic. Microbes and infection, 2.
Elsevier Paris, 125-129.
Sol, J. O. (1997). Factors associated with bacteriological cure during lactation after therapy for
subclinical mastitis caused by Staphylococcus aureus. J. Dairy Sci. 80, 2803-2808.
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