Upaya pencegahan mastitis subklinik dengan menggunakan homeopatikum secara kombinasi dan pengaruhnya terhadap kualitas dan produksi susu

CONTROL OF SUBCLINICAL MASTITIS USING
CERTAIN HOMEOPATHIC COMBINATION
ITS INFLUENCEON MILK
DRUGS
QUALITY AND PRODUCTION

AGATHA WINNY SANJAYA

THE GRADUATE PRUGRAM
BOGOR AG;RliCULTW IJNWEMITY
BOGOR
2002

Just as my non-violence will never fail
homaeopathy never fails
But the followers of homeopathy may fail
owing to faulty application of the principles.

(Mahatma Gandhi, 1939)

ABSTRAK

AGATHA WINNY SANJAYA. Upaya Pencegahan Mastitis Subklinik dengan
Menggunakan Homeopatikum secara Kombinasi dan Pengaruhnya terhadap
Kualitas dan Produksi Susu: Dibimbing oleh 1 WAYAN TEGUH WIBAWAN,
MIRNAWATI SUDARWANTO, HEINRICH ENBERGS, SETYO WIDODO, MASDUKI
PARTADIREDJA (aim), HElNER SOMMER (alm)
Tigapuluhtiga ekor sapi perah penderita mastitis subklinis dikelompokkan
dalam grup A ( I 0 ekor), grup B (12 ekor) dan grup C (11 ekor). Sapi diobati dengan
homeopatika dalam bentuk kombinasi dan plasebo, diobati pada minggu ke-4 dan 3
sebelum partus (a-p.) dan setiap minggu selarna 4 kali berturut-turut setelah partus
(p.p.). Grup A diobati dengan Coenzyme camp@ (minggu ke-4 dan 3 a.p.), Lachesis
comp@dan ~raumeep(minggu ke-I dan 2 p.p.), Coenzyme camp@ dan Carduus
comp@(minggu ke-3 dan 4 p.p.). Grup B diobati dengan ~ r a u m e edan
l ~ Mucosa
comp@(minggu ke-4 dan 3 a.p.), Lachesis camp@ dan iraumeel@(mingguke-1 dan 2
p.p.), Coenzyme comp@dan Carduus comp@(minggu ke-3 dan 4 p.p.). Grup C
adalah plasebo.
Pengujian dengan reagens IPB-1 di Lapangan memperlihatkan bahwa insidensi
mastitis subklinis pada grup B adalah sebanyak 33,3% dan bertahan konstan
sampai akhir pengamatan, sedangkan grup A dan C kasus mastitis sangat
bervariasi. Kenaikan jumlah sel somatik susu dari grup A dan B satu minggu setelah

terapi terakhir selama 2 minggu (minggu ke 5 - 7 p.p.) menuryukkan adanya suatu
respon yang nyata terhadap terapi homeopathy kemudian diikuti penurunan jumlah
somatik sel mendekati nilai normal.
Respon setelah disuntik Coenzyme comp. (minggu ke-3 a$.) memperlihatkan
adanya peningkatan jumlah sel darah merah (RBC) untuk grup A (17,8%),
selanjutnya diikuti penurunan RBC sampai akhir pengamatan. Gambaran
haemoglobin dan pack cell volume tidak mengalami perubahan (konstan). Limfosit
grup A dan C meningkat menjelang proses kelahiran, ha1 sebaliknya terjadi bagi
grup B. Kadar haptoglobin (Hp) pada fase peripartal meningkat bagi grup B 0,86
mglml dan bagi grup A maupun plasebo 1,44 mglml serta 1,42 mglml. Pengamatan
5 minggu p.p. grup B mengalami penurunan jumlah Hp menjadi 0,09 mglml sampai
akhir pengamatan adalah 0,07 mglml. Kadar Hp darah grup A menurun secara
perlahan, kadar terendah dicapai pada minggu ke-5 p.p. yakni 0,18 mglml.
Pengamatan produksi susu selarna 5 bulan (bulan ke-3 sampai ke-7) dalam
masa laktasi normal menunjukkan adanya peningkatan 14,5% bagi grup B dan 5,0%
bagi grup A yang berbeda sangat nyata terhadap plasebo.

ABSTRACT
AGATHA WlNNY SANJAYA. Control of Subclinical Mastitis Using Certain
Homeopathic Combination Drugs and Its Influence on Milk Quality and

Production. Under the direction of I WAYAN TEGUH WIBAWAN, MlRNAWATl
SUDARWANTO, HEINRICH ENBERGS, SETYO WIDODO, MASDUKI
PARTADIREDJA (late), HEINER SOMMER (Late).
A total of 33 dairy lactating cows suffered from subclinical mastitis were
classified into group A (10 cows), group B (12 cows) and group C (11 cows)
which were treated with the combination of homeopathic drugs and placebo,
applied at the 4'h and 3* week antepartum (a.p.) and every week for four times
postpartum (p.p.). Group A received Coenzyme compQD
(given in the 4'h and 3'C'
week a.p.), Lachesis compQDwith ~raumeel' (I& and 2nd week p.p.) and
Coenzyme compQD
with Carduus compQD
(3rdand 4'h week p.p.). Group B received
(4" and 3rd week a. .), Lachesis comp@with
~ r a u m e ewith
l ~ Mucosa compQD
~raurneel~(1"
and 2ndweek p.p.) and Coenzyme comp with Carduus compQP
(3*
and 4'h week p.p.) and group C as a placebo.

Incidence of mastitis in group A and C appeared irregularly. In contrast,
group B showed a constant percentage (33.3%). Group A and B showed
significant response to the homeopathic drugs, expressed as an increasing of
week
the somatic cell count value after the last therapy for two weeks (5'h
p.p.) and at the last observation decreased nearly to the normal value.
At the puerperal phase, the red blood cell of group A increased (17.8%)
after the first therapy. Hemoglobine and pack cell volume showed no
comparable results. Lymphocyte of group A and placebo increased during
puerperal phase, contrary happened to group B. At peripartal phase haptoglobin
increased for group B 0.86 mglml and group A as well as placebo 1.44 mglml.
After birth, group B expressed a constant value (0.09 - 0.07 mg/ml), while group
A and placebo raised significantly.
The milk yield increased significantly for five month observation in normal
lactation period (the 3*-7'h month), with an increasing 14.5% and 5.0% for group
B and A respectively.

i?

-

Statement of originality

The studies described in this dissertation were performed in accordance with the
regulations for the degree of Doctor in the Bogor Agricultural University.
This dissertation consists entirely of original research which has not been
submitted in whole or in part for any other degree at this university or at any other
institution.

CONTROL OF SUBCLINICAL MASTITIS USING
CERTAtN HOMEOPATHIC COMBLNATION DRUGS AND
ITS INFLUENCE ON MILK QUALITY AND PRODUCTION

AGATHA WlNNY SANJAYA

Doctoral Dissertation
submitted in fulfilment of the requirements to achieve
a Doctor degree in Veterinary Sdence Study Program

THE GRADUATE PROGRAM

BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2002

CURRICULUM VITAE

Agatha W~nnySanjaya was born in MEDAN, June 7* 1946 as the fifth child from
the five children of lgnatius Dyan Kumiatan and Veronica Sukawati. The author
married with Dominicus Setyasusila Sanjaya and has two children Benedictus
Adhi and Martina Citra.
In 1964 entered the Faculty of Veterinary Medicine Bogor Agricultural
University and inaugurated as doctor of Veterinary Medicine in 1973. Master
degree was achieved in Veterinary Science in 1990 and in 1997 February,
entered Doctor Program at the Graduate Program in Bogor Agricultural University
and was funded by the Beasiswa Pendidikan Pascasarjana. A part of the
research was funded by the Project of University Research for Graduate
Education LOAN IBRD.

Title of Dissertation

Name of Student

: CONTROL OF SUBCLINICAL MASTITIS USING
CERTAIN HOMEOPATHIC COMBINATION DRUGS
AND ITS INFLUENCE ON MILK QUALITY AND
PRODUCTION
: Agatha Winny Sanjaya

Registration Number : 965104
Study Program

: Veterinary Science

Approved by:
1. Advisory Committee:

Dr.drh.1 Wavan Tenuh Wibawan M.S.
chairman

Prof.Dr.drh.Hi.Mimawati Sudawanto

member

Prof. Dr. Heinrich Enberns
member

2. Head of Study Program:

Date of examination : March 5'h2002

3. Director of the Graduate Program:

Acknowledgements
I would like to express my gratitude to my chairman advisor, Dr. drh. I

Wayan Teguh Wibawan, MS, Department of Animal Diseases and Veterinary
Public Health, member advisory, Prof. Dr. drh. Hj. Mimawati Sudawanto, Prof.
Dr. H. Enbergs, Dr. drh Setyo Widodo, for all his invaluable input, assistance and
support throughout the course of this work. Special my honour are due to the
late: Prof. Dr. Heiner Sommer and Prof. Dr. drh. H. Masduki Partadiredja for their
encouragement.

I am very grateful to the Beasiswa Pendidikan Pascasarjana, Project of

University Research for Graduate Education, for their sponsorship which enabled
me to study in Germany. My thanks also go to the Rector IPB, Director of PPSIPB, Dean of Veterinary Medicine Faculty, the head of Department of Animal
Diseases and Veterinary Public Health at IPB, Bogor, lnstitut fiir Anatomie,
Physiologie und Hygiene der Haustiere der Rheinischen-Friedrich-WilhelmsUniversiet at Bonn, Fa. HEEL Baden-Baden Germany and the chief of Animal
Quarantine Service, Jakarta International Airport, for their support.
I would also like thank to the director of TAURUS Dairy Farm, Ir. Nugroho

and his crew, all my co-workers, who were very helpful and sympathetic during
the completion of the work in the field and laboratory.
I am also grateful to the staff members of Veterinary Public Health

Laboratory, University of Bonn where I had done my research work and to all of
my colleagues who supported me in various ways: Dr D.W. Lukman; Drh R.K.
Achyadi MS; Ir E. Sudamika M.Si; Dr Fachrudin; Drh E.S. Pribadi, MS; Drh Lukas
Tonga, Ir Agustine Santosa MS, Dr Linscheid; Dr U. Miiller; Dr S. Knura and my
friend Fr. Rosemarie Schattevoy. Last but not least, I would like to thank my
families with their supporting and attention to my work.

LlST OF CONTENTS
LIST OF TABLES

vii

LIST OF FIGURES

vii

LlST OF APPENDICES

ix

LlST OF ABBREVIATION

X

1. INTRODUCTION
1.1 Background
1.2 General Objectives of this Study

1.3 Hypothesis

2. LITERATURE STUDY
2.1 Milk
2.2 Defence Mechanism of Mammary gland
2.3 lrnmunological Defence at Peripartal Phase
2.4 Subclinical Mastitis
2.4.1 Prevalence and Characteristic of Subclinical Mastitis
2.4.2 Diagnosis of Subclinical Mastitis
2.5 Homeopathy as an Alternative Therapy
2.6 Advantageous of using Combination Form of Homeopathic Drugs
2.7 Control of Subclinical Mastitis by Homeopathic Treatment
2.8 Homeopathica Substances
2.8.1 Coenzyme compositum
2.8.2 Traumeel
2.8.3 Mucosa compositum

3. MATERIAL AND METHODS
3.1 Materials
3.1.1 Milk and Blood Samples
3.1.2 Instruments
3.1.3 Chemicals.
3.2 Animal contingent
3.3 Methods

3.3.1 Determination of Health Status of Cows Concerning to
Subclinical Mastitis
3.3.2 Milk Quality Analysis
3.3.3 Blood Analysis
3.3.4 Clinical Chemistry
3.4 Data Collection
3.5 Research Design
3.6 Data Analysis

4. RESULTS AND DISCUSSION
4.1 Milk
4.1.1 Incidence of Subclinical Mastitis
4.1.2 Somatic Cell Count
4.1.3 pH of Milk
4.1.4 Milk Electrical Conductivity
4.1.5 Fat Content
4.1.6 Protein Content
4.1.7 Lactate dehydrogenase Enzyme in the Milk
4.2 Blood Profile
4.2.1 Red Blood Cells and Pack Cell Volume
4.2.2 Hemoglobin
4.2.3 Leukocyte Profiles.
4.2.4 Lactate dehydrogenase and Aspartate Aminotransferase
Enzymes
4.2.5 Cholesterol
4.2.6 Urea in Blood
4.3 Haptoglobin

4.4 Milk Yield
5. CONCLUSIONS AND RECOMMENDATIONS

A. Conclusions
B. Recommendations

6. REFERENCES

LlST OF TABLE
1. Relationship between somatic cell count-score, somatic cell count
and daily production loss of dairy cows

2. Estimated infection prevalence and looses in milk production
Associated with elevated bulk tank somatic cell counts

3. Determination of CMT-score associated with an average somatic
cell count

4. A comparison of methods used in subclinical mastitis test and their
correlation with somatic cell counts
5. An overview of the use homeopathic drugs in mastitis control

6. Combination of homeopathic drugs used in the treatments
7. Application schedule of respective homeopathic combination
(A, B and C) and time of milk and blood sampling

LlST OF FIGURE
I.The incidence of subclinical mastitis by cows treated with a
respective homeopathic drug combinations determined with
IPB-I test
2. The effect of homeopathic treatments to the profile of somatic cell
counts with a respective homeopathic drug combinations
3. The pH value of milk from subclinical mastitis treated with a
respective homeopathic drug combinaiions

4. Milk electrical conductivity of subclinical matitis cows treated with
a respective homeopathic drug combinations

5. Fat contents of milk from subclinical mastitis cows which pretreated
with difference homeopathic combinations
6. Protein content of milk from subclinical mastitis cows treated with a
respective homeopathic drug combination

7. Lactate dehydrogenase in milk of subclinical mastitis cows before
and after treatments
8. Red blood cell profile of milk from subclinical mastitis cows treated
with a respective combination of homeopathic drugs
9. Hemoglobin profile of subclinical mastitis cows treated with respective
combination of homeopathic drugs

10. The leukocyte profile of subclinical mastitis cows treated with respective
combinations of homeopathic drugs
48

II.The neutrophil profile of subclinical mastitis cows treated with
respective combination of homeopathic drugs

49

12. The lymphocyte profile of subclinical mastitis cows treated with
respective combination of homeopathic drugs

49

13. The eosinophil profile of subclinical mastitis cows treated with
respective combination of homeopathic drugs

50

14. The LDH profiles of blood from subclinical mastitis cows treated with
respective combination of homeopathic drugs

52

15. The ASAT profiles of blood from subclinical mastitis cows treated with
respective combination of homeopathic drugs

52-

16. The cholesterol profile of blood from subclinical mastitis cows treated
with respective combination of homeopathic drugs

53

17. The urea profile of blood from subclinical mastitis cows treated with
respective combination of homeopathic drugs

54

18. Haptoglobin profile of subclinical mastitis cows treated with respective
combination of homeopathic drugs

56

19. Milk yield observed in normal lactation at the 3'Cl-pmonth before
and after treatment

57

,LIST OF APPENDICES
1. Mean values of somatic cell count in milk of subclinical mastitis
cows treated with respective combination of homeopathic drugs
2. Mean values of pH milk of subclinical mastitis cows treated with
respective combination of homeopathic
3. Mean values of electrical conductivity of subclinical mastitis cows
treated with respective combination of homeopathic drugs

4. Mean values of milk fat from subclinical mastitis cows treated with

respective combination of homeopathic drugs
5. Mean values of milk protein from subclinical mastitis cows treated
with respective combination of homeopathic drugs

68

6. Mean values observations of red blood cell, pack cell volume and
hemoglobin from subclinical mastitis cows

69

7. Mean values observation of leukocyte and its differentiation
picture from subclinical mastitis cows
8. Mean values observation of blood enzyme and metabolite from
subclinical mastitis cows

71

9. Mean values of haptoglobin concentration in blood plasma from
subclinical mastitis cows

72

10. Total 5 months production from cows before and after treatment
with respective combination of homeopathic drugs

72

11. Statistical Analysis of Varians of 5 months milk production in normal
lactation before and after treatment

73

LIST OF ABBREVIATION

= degree Celsius
= registered
= weighheight
= unitllitre
= ante partum
= post partum
= Aspartate Aminotransferase enzyme
APRP

= Acute Phase-Reactant Protein

C

DHlA

= centesimal scale
= California Mastitis Test
= compositum
= C-Reactive Protein
= remedy made from the products of a disease
= decimal scale
= Dairy Herd ImprovementAssociation

Dl

= decilitre

DNA
et a!.

= Desoxyribonucleicacid
= et alia

Fig.

= Figure

Hb

= hemoglobin

HP
IU

= haptoglobin

CMT
comp.
CRP
de n o w
D

mg
ml

= international unit
= Lactate dehydrogenase
= minute
= milligram
= millilitre

mS

= millimetre Siemens

NAD

= Nicotine Adenine Denucleotide
= Pack Cell Volume
= pro analyse
= hydrogen ion concentration
= Polymorphonuclear Neutrophil
= rotation pro minute
= red blood cell

LDH
min.

PCV
p.a.
pH
PMN
rpm
RBC

sec
SCC

= second
= Somatic cell counts contains leukocyte cells and mammary
epithel cell debris

VLDL

= very low density lipoprotein

WMT

= Wisconsin Mastitis Test

WST

= Whiteside Test

IJL

= microlitre

CONTROL OF SUBCLINICAL MASTITIS USING
CERTAIN HOMEOPATHIC COMBINATION DRUGS AND
ITS INFLUENCE ON MILK QUALITY AND PRODUCTION

1. INTRODUCTION

Subclinical mastitis is one of the most important diseases in dairy cattle in
lndonesia with the prevalence of 85-90% (Sudarwanto, 1995). It shows no
significant changes of udder and in milk appearance but causes a significant
economic loss (10-18%) mainly because of losses of milk production (15-40%)
per day, poor quality of milk, milk discard, drug costs and veterinary fee (Kirk et
a/., 1994; Lee, 1996; Sudarwanto, 1999). Similar results had also shown by Hirst

et a/. (1984)

.

The decrease of milk production is felt to be more important than that
caused by clinical mastitis. The recent data show that the amount of dairy cattle
in Indonesia is 343,000 heads from which 260,000 are in lactation. They
produced 405,000 tons of milk/ lactation period. By assumption that 60% of the
lactated group suffer from subclinical mastitis, the economic loss due to the
decrease of milk production is approximately 108 billion rupiahs per year
(Sudarwanto, 1999).

1.I Background
Antibiotics have been used in controlling mastitis. The use of inappropriate
antibiotics cause bacterial resistance, residue problems, allergy, metabolic
disorders, toxicity and influenced in milk processing (Kiehvein, 1976). Previous
studies indicated that 32.52% of pasteurised milk and 31.10% of fresh milk in

Jakarta, Bogor and Bandung contained significantly high antibiotic residues
(Sudarwanto et a/. 1992).
Nowadays, greater emphasis has been placed on antibiotic residue
avoidance to ensure a residue-free milk for the consuming public. The major
change in this direction is the reduction in the use of antibiotics in the treatment
of mastitis.
As an alternative, homeopathic drugs are to be taken into consideration.
Their advantages are that they cause no toxic side effects and no residues in
animal's products. Missing corresponding placebo-controlled studies it was the
aim of the following studies to test the effect of certain homeopathic drugs in
prevention of subclinical mastitis in dairy cows.

1.2 General Objectives of this Study

1). To study the most effective combination of the homeopathic drugs in
prevention and therapy of subclinical mastitis.
2). To study the effect of the homeopathic drugs in enhancing milk production
and quality.
3). To study the influence of homeopathic drugs to the blood profile of subclinical
mastitis-treated cows.

1.3 Hypothesis
1). Homeopathic drugs depress the incidence of subclinical mastitis.
2). Homeopathic drugs enhance the milk quality and milk yield.
3). The effective combination of homeopathic drugs for subclinical mastitis
prevention and therapy could be determined.

2. LITERATURE STUDY

2.1 Milk
Milk is composed of water, carbohydrate (lactose), fat, protein, minerals
and vitamins and is secreted as a complex mixture of these components. The
properties and importance of milk are greater and more complex than the sum of
its individual component parts.
Water content of milk is about 87% and dependent upon the synthesis of
lactose. If water is added to the milk, the additional water is easily detectable by
several methods. These methods are based upon changes in freezing point of
the milk (cryoscopic method) or on changes in refraction light of the whey
component of milk after precipitation and removal of the casein and fat
components. These methods and other standard methods of milk testing are
described in the Association of Official Agricultural Chemists (AOAC), Official
Laboratory Analytical Procedures (National Academy of Science).
Carbohydrate substance of milk is mostly lactose. Lactose is a disaccharide composed of the monosaccharides D-glucose and D-galactose, joined in a
13-1,4-glycosidic linkage. The chemical name of lactose is 4-0-13-D-galacto-

pyranosyl-D-glucopyranose and is essentially unique to milk. Lactose plays an
important role in milk synthesis. Lactose is responsible for maintaining the
osmolarity of milk and for drawing water in milk synthesis. Lactose is not as
sweet as other disaccharides such as sucrose or monosaccharides fructose or
glucose. The lactose cleaved to glucose and galactose in the neonate intestine
is done by an enzyme activity called lactase or 13-galactosidase. Other carbohydrates are found in milk at low concentrations, such as free glucose (0.1 mM)
and free galactose (0.2 mM).

The fat component of milk is composed of a complex mixture of lipids.
Triglycerides or triacylglycerides are the major type of lipid in milk fat, 98% of the
total milk fat (wtw). Triglycerides are composed of 3 fatty acids covalently bound
to a glycerol molecule by ester bonds. Milk fat is secreted from mammary
epithelial cells as fat globules which are primarily composed of a globule of
triglyceride surrounded by a lipid bilayer membrane similar to the apical
membrane of the epithelial cells. This fat globule membranes helps to stabilize
the fat globules in an emulsion within the aqueous environment of milk. Other
milk lipids include diacylglycerides (0.25-0.48%), monoacylglycerides (0.020.04%), phospholipids (0.6-1.0%), cholesterol (0.2-0.4%), glycolipids (0.006%)
and free fatty acids (0.1-0.4%).
The fatty acids used for the synthesis of triglycerides arise from the break
down of blood lipids and from de novo synthesis within the mammary epithelial
cells. About 40-60% of the fatty acids come from the blood. These are primarily
derived from very low density lipoproteins (VLDL), which are synthesized in the
intestine or liver. VLDL are composed of 90-95% on the inner core and 5-10%
protein at the outer surface. Chylomicrons, containing ingested fatty acids from
the intestine, also can act as a source of blood-derived fatty acids for the
mammary gland. Synthesis of short or medium chain of fatty acids occurs by de
novo synthesis in the cytoplasm of the mammary epithelial cells. The sources of
carbon the acetate and 13-hydroxybutyrate are mainly used.
The total protein component of milk is composed of numerous specific
proteins. The primary group of milk proteins are the caseins. Caseins have an
appropriate amino acid composition which is important for growth and development of the nursing young. This high quality protein in cow milk is one of the key
reasons why milk is such an important human food. Casein is one of the most
abundant organic components of milk, in addition to the lactose and milk fat. An

other protein component of milk is whey or plasma phase of milk. The major
whey protein in cow milk are a-lactalbumin and 13-lactoglobulin. Alpha lactalbumin is an important protein in the synthesis of lactose and its presence is
central to the process of milk synthesis. Other whey proteins are immunoglobulins (especially high in colostrum), serum albumin, enzymes, hormones,
growth factors, nutrien transporters, and disease resistance factors.
The major minerals found in milk are calcium and phosphorus. These
minerals are required in large quantities by the rapidly growing neonate for bone
growth and development of soft tissues. They are both mostly associated with
the casein micelle structure. Milk also contains most other minerals found in the
body.
Milk contain all the major vitamins. The fat soluble vitamins A, D, E, and K
are found primarily in the milk fat, and has limited amounts of vitamin K. The B
vitamins are found in the aqueous phase of milk. Milk always contains leukocyte
cells and mammary epithelial cell debris, also known as somatic cells in cow
milk. The concentration of leukocytes in milk varies with the species, infection
status of the gland and the stage of lactation.

2.2

Defence Mechanism of Mammary gland
Physical barriers of mammary glands play an important role in the defence

mechanisms of the mammary glands. This included the teat shape, teat
sphincter, streak diameter, streak canal, the length and the diameter of streak
canal and the keratin lining in streak canal, which contains factors that seem to
be bacteriostatic. Keratin is a meshlike substance, formed from descquamated
epithelial cells, fatty acids and cationic proteins and play as a physical
obstruction to bacteria and by the adsorption of bacteria. The keratin lining is

desquamated during milking which removes bacteria in the streak canal. The
keratin's fatty acids are bactericidal and bacteriostatic and it has proteins which
bind to and cause lysis of Gram positive bacteria. Furstenberg's rosette is
situated at the internal end of the streak canal, contain a protective leukocytes
population, which are thought to leave teat wall and enter the cistern via
Furstenberg's rosette. This rosette contains also ubiquitin a cationic protein
which has bactericidal activities.
lmrnunological defence of mammary glands includes all physiological
mechanisms allowing the body to identify and neutralise foreign bodies and is
comprised of: (1) cellular Immunity and (2) humoral Immunity. The immune cells
include the role of leukocytes such as granulocytes (neutrophils, basophils and
eosinophils) and lymphocytes, especially T-killer cells, and monocytes or macrophages. These cells are mainly responsible for the phagocytosis process in milk
and are considered as a second line of defence in the mammary gland. Humoral
immunity includes the role of antibody as opsonin, neutralisation of antigens or
activation of the complement system.

2.3

lmrnunological Defence at Peripartal Phase

lmmunological defence must be understood as an integrated system of
non-specific and specific immune responses, which has an important function to
maintain the physiological homeostasis. Peripartal period (about 4 weeks before
and after birth) is considered as a critical point and unstable physiological
condition of the dairy cows due to the sudden changes or switch over of many
processes including hormonal, metabolic, stress and cytologic performance.
Especially during the early lactation stage problems may arise to maintain the
cow in a physiological balance. This might influence the whole system of
defence mechanism. A negative balance of energy with the following reactions

(1) exhaustion of reserves of carbohydrates, (2) conversion of the body's protein
into sugars, (3) mobilisation of lipid depots and decomposition of the lipids to
produce energy with a risk of a fatty fiver and fmlly (4) increase of so called
aceton-bodies. Interference between aceton bodies and leukocyte function
decreased the interferon (IFN-Y) production. Interferon involved in the regulation
of T cells, 6 cells, macrophages and granulocytes, so that the disturbance of IFN
production

caused the reduction of phagocytic activity of the -phages

as

well as of the granulocytes which are considered as the first line of defense of
the bovine mammary gland against infections.

This unstable physiological

condition might lead to the incidence of mastitis facilitated by postpartal
immunosupression (Kandefer-Szerszen et a/., 1992; Klucinski et al., 1988). The
stress-situation and damages of tissues connected with the birth process,
especially accrued in the soft tissue of the birth ways.

2.4 Subclinical Mastitis

2.4.1 Prevslence and Charadetfsticsof Subclinical MasWs

Mastitis from the Greek mastos-breast and itis is a general term used to
refer to any inflammation of the mammary gland The previous studies indicated
that subclinical mastitis in dairy cattle is thought to be a multicomplex disease.
Subclinical mastitis could appear as an infectious disease. The non infectious
mastitis might occur due to the inadequate of feeding, milking-management and
handling (Sphor and Schulze-Wartenhorst, 2991). Deficiencies of the following
vitamins, such as vitamin A, 6-carotene, minerals i.e. Se, Zn, Cu, Co and some
others have been shown to be related to increased incidence of subclinical
mastitis, increased severity of infection and elevated somatic cell counts.
Selenium protects mammary tissue from oxidative damage and augments
phagocytic function of macmphages as well as of microphages. For the

infectious subclinical mastitis the Streptococcus agalaciiae, Staphyilocioccus
aueus and Escherichia mli are well known as the main causal agents (Wibawan
et al., 1999). Bacteria could enter the mammary glands and multiply sufficiently
to trigger the inflammatory response. Vasodilatation occurred and results in
increased blood flow to the mammary glands. Inflammatory products such as
prostaglandins, leucotrienes, pmteases, and oxygen radicals increased the
vascular permeability. Leukocytes, initially polymorphonuclear neutrophils (PMN)
leaved the blood vessels and diapedicaly enter the surrounding tissue, after
which macrophages predominate.
Subclinical mastitis has remained the most economically important
problem in dairy cattle in Indonesia with high prevalence ranging fmm 85 to 90%
(Sudarwanto, 1995; Wibawan et a1.,1999). The information about the pathogenesis of subclinical mastitis is very limited. In subclinical mastitis, the milk appears
to be normal. Bacteria usually, but not always, can be isolated in milk. Milk yield
is depressed, and composition may be altered. No pathological changes of
mammary gland could be observed but the histopathological examination
showed that the inflammation of the mammary gland had occurred. Inflammation
of mammary gland caused by colonisation as well as by invading pathogens is
common among lactating dairy cows and is a major cause of economic losses.
The negative influence of subclinical mastitis on milk production is
substantial. Estimates put the cost of one case of subclinical mastitis per cow in
the range of $ 200 per year in Virginia, America. Generally producers put an
emphasis on clinical mastitis cases and underestimate the significance of
subclinical mastitis not relizing that for every one clinical case in the herd there
are 40 subclinical cases contributing to an elevated somatic cell count.
Subclinical mastitis may contribute 70% of the total milk loss. A mastitis control
program should begin with determining the level of subclinical mastitis. Mastitis

contributes to the loss of income in the following ways. 70% from lost of milk
production, 14% from death and premature culling, 8% miUc discarded at
treatment and 8% for drugs and veterinary (Bailey, 1996).
S. aureus and S. agalactiae as the main causal agents of infectious

subclinical mastitis often start with an acute phase and generally become chronic
and subclinical. In contrast, E. coli caused an extensive damage of mammary
gland tissue. This opposite characteristics are likely to results from the difference
of pathogenic mechanisms used by those bacteria. By S. agaladiae and S,
aureus the adhesion is thought to be the most important and critical step of

infection and followed by the colonisation of bacteria on the surface of the
mammary epithelial cells wbawan

et al., 1999). The differences in the

pathogenesis and clinical reaction of causal agents offer the opportunity to
contrast the inflammatory responses and to study the early host responses to the
respective causal agents by measuring the inflammatory markers such as
haptoglobin, C-reactive protein (CRP) or other inflammatory mediators which
might be used for the early screening test of subclinical mastitis in dairy cattle
(Sandholm et a/., 1995). In case of acute mastitis, the clinical symptomes are
obvious and laboratory tests are not required to detect inflammation. Detection of
subclinical mastitis is a challenging problem. Subclinical mastitis reduces milk
production, milk composition and quality of milk and also maintains infection foci
in the herd (reservoir). The extent to which various compositional changes occur
depends on the inflammatory response. This depends on the virulence factor of
the pathogen and the amount of affected tissue within the mammary gland.
Inflammation should be analysed by monitoring various inflammatory mediators.
According to Kielwein (1976), disturbances of milk synthesis could
influence the milk secretion and milk composition, especially increasing the
concentration of Na, CI,immunoglobulin, serum albumin and decreasing of K, P,

Ca, Mg concentration. Disturbance of the milk synthesis caused the decreasing
of casein-, p ladoglobulin-, a lactalbumine-, lactose concentration and fat in milk.

The high concentration of Na and CI in the milk caused a salty taste and alkalic
property of milk. The milk becomes heat unstable and was not completely
coagulated by rennin and caused failure in cheese ripening. According to Munm

in Hamann and Rund (1994) mastitis effect on milk and milk product are : (1)
fresh milk changes very easy in a rancid taste and the protein are heat unstable,

(2) pasteurised milk has an unfavourable flavour and with an unstable quality
and (3) effect on making cheese are: a reduction in starter activation and
delayed coagulation time, breaking firmness decrease, lost of fat and casein in
milk at last there is a low profit and (4) problem in processing butter would
influenced the taste with an oxidation taste, present a weak aroma, prolonged
rippening butter time and inhibition of diacetyl product.

2.4.2 Diagnosis of Subclinical Mastitis
2.4.2.1 Somatic Cell Count (SCC)
Somatic cell counts (SCC) from a day's milk is the best indicator of the
extent to which the gland is involved in fighting a mastitis infection. Milk contains
cells which derive from the cow, these are somatic cells. The number of somatic
cells is immediately high after calving, but it drops rapidly during the first week of
lactation and increases again towards end-lactation. Therefore, the number of
cells depends on two factors: 1). a real increase in their quantity as a
consequence of udder initation and attraction to the inflammatory site or 2). The
dilution factor, i.e. the daily milk yield shows an inverse relation with the somatic
cell count Somatic cells are present in the milk in high number only when and
where they needed, therefore high SCC might indicate mammary infection

(Sandholm et a/., 1995). The quality of raw milk produced in Germany is
guaranteed by a milk quality control scheme conducted in accordance with
European Committee standards. In addition, because of its effect on the price
paid for milk, the somatic cell count in bulk milk is an indicator of udder health.
Udder health can only be maintained by strict adherence to accepted control
measures during milking, hygiene and prophylactic treatment (Fehlings and
Deneke, 2000). The cells found in milk consist of about 75% leukocytes and
about 25% epithelial cells. Normally SCC are 60-70% epithelial cell, 10%
lymphocyte and 2030% leukocytes (Sinell and Neuschulz, 1965). Leukocyte
numbers increase in response to bacterial infection, tissue injury and stress.
Increasing somatic cell numbers is a result of an increase in the number of
leukocytes.
The somatic cell count of milk serves as an indirect method to measure the
level of irritation in the mammary gland. There are 3 methods of evaluating SCC:
(1) Bulk Tank SCC (BTSCC), (2) Weighted SCC (VVTSCC) and Somatic Cell
Count Score (SCCS). The BTSCC is usually based on single sample of milk
from the bulk tank. The WTSCC is calculated on samples from individual cows
and is weighted or adjusted based on each cow's level of milk production. The
WTSCC for all milking cows in a herd should approximate the BTSCC. The
SCCS is based on the logarithmic conversion of the actual SCC to a linear score
as shown in Table 1.
The loss in milk production between scores 2 and 3 is estimated at 1.5
Ibs/cow/day based on an actual SCC difference of about 50,000 cells. Cows with
a SCCS of 0-3 a= generally considered uninfected while cows with SCCS of 7 9 are considered infected.

Table I . Relationship between somatic cell count-score, somatic ceH count range
and daily production loss of dsmy cows (Shook and Saeman in Smith et

a/. 2000)

The value of SCCS is associated with the ladation period, milk yield, the
dens@ of cows in a herd and the season. The first lactation have a Iower SCCS
with a h i r percentage in the 0-3 category compared to older cows. This is not
directty associated with the age but mused by increased rate of udder infections.
Unmfected cows,regardless of age generally have low SCC (Ebehart et a/.,
1979). High producing cows as well as high density herds trends to have a

higher SCC compared to low producing cows or lower dens@ herds. A high
SCCS are generally observed during the summer time, in the months of July,
August and September and the towest during the winter and spring months.

Herds with butk tank SCC above 200,000wilt have varying degrees of subclinical
mastitis present.
In the Table 2, data from the National Mastitis Council (1987) show that 6%
of the quarters in a herd could be expected to be infected in a herd with a bulk
tank SCC of 200,000. At 500,000 SCC, 16% of tho quarters may be infected
with a 6% reduction in milk produdion. Ttds information lead to the conclusion
that the monitoring of udder heal# is very important in order to control and
reduce the level of mastitis in a herd. Somatic d l count measurement provide
the opportunity to monitor and evaluate udder health status.
Tabk 2.Estimated infedion prevalence and t m s in milk produdion associated

with elevated bulk tank somatic ceH counts

* Pmduction loss cabMed as a percent d production expected at 200,000 cell1 ml.
National M a s t i Council (1981).

Somatic celt count under 400,000 dldmt are typical of herds that have

good management practices, but no partiwlar emphasis on mastitis control. In
contrast, somatic cell caunts greater than 500,000 cellslml indicates that one
third of the mammary gtands are infected and the lost of milk due to subclinical
mastitis is at bast 10 % (Keown, 1987)

2.4.22 California Mastitis Test ( C m and Mliscmin Mast&% Test (WIUT)

The CMT test is a simple methodto estimate the DNA content of milk. This
test bases on an anionic detergent, Na-iauryt sulphate (Dodecyl Sulfate Sodium),

which dissolves eel membranes and nuclei. Consequently DNA is released and

it forms a transient gel with the detergent The more DNA in the milk sample,the
higher the viscosity of the get (Sandholm et aL,1995). The v a w ~ o n
of scoring of
CMT, depend upon the skill of the person reading the result and the method
used to conduct the test. Uniformity in techniques are necessary if results are to
be comparable. The use of the CMT on the entire herd at monthly intervals can

be extremely useful as an aid in detecting herd mastitis problems. individual and
total quarter infedions can be determined and, with proper records, the tevel of
herd mastitis can be monitored. The relation between CW-Score and the

average somatic cell count is presented in Table 3
Table 3. Determination of CMT-Score associated with an average somatic cell
count.
CMT-Scored

Average somatic cell count (cells/ml)

Negative

300,OOO

Trace

m,m
~,~

2

2,700,000

3

8,100,000

fairty
) accurate in measuring somatic
The advantages of the CMT are: (I

ceH concentratin in mitk, meiating well with ather test, (2) it is sensitive and
can be used for sampling quafters, bucket and buk tank milk samples, (3)
foreign material does not interfere with the test, (4) simple and a little equipment

is needed, (5) environmentat temperature changes have only a lime effed on the
CMT as Long as the milk has been refrigemfed and is not over two days OMand
(5) herd mastitis levels can be estimated from bulk €ank A CMT d 2 or 3 in tank
milk indicates a probable percent of infected cows.
The Wisconsin Mastitis Test (WMT) is an other method, which frequently is
used as a screening test for subclinical mastitis. The principle af the WAM is the
same as that of the CMT except that the amount of gel formed is measured in a
tube calibrated in miltimeters (mm) instead of being visually scmed. The
advantageous of WMT is conducted under precise procedures and standard
temperatwe conditions. A more accurate and precise method of measuring

somatic cetls in milk is the ekctronically m - d

somatic cell counts used by

Dairy Herd ImprovementAssociation (DHIA).
Somatic cell counts is expressed in thousands of cells per milliliter,
sometimes referred to as raw score, and converted to a Enear score ranging
from 0 until 9. Linear scores have been shown to be highly correlated with milk
losses resulting from mastitis. A comparison of the CMT, W T and SCC in cells
per milliliter is shown in Table 4.

TaMe 4. A comparison of methods used in subdinical mastitis test and their
correlation with somatic cell counts (Eastridge and HoMet, 1992).

2.4.23 IPE1 Pest

The determinatk#1of mastitis irt the btd can be done by using diagnostical
reagent. Recently, fieid subdical mastitis tests which are available such as
CMT are rarely done due to the

price and the avaitabifi of the reagents.

Another reagent called IPB-1 reagent, is devetoped for a field test that is
relatively easy to use, inexpensive, and consttucted of ingredients readily are
available in domestic market. The reliability value of IP8-I is better than CMT or
WST, the sensitivrty of IPB-I is 0.99and CMT 0.92,the spectficity 0.92with 0.36
and the prect'~divevalue 0.95 with 0.97 respectively. The prdictive value still
needs improvement (Sudarwanto, 1998).

2.4.2.4 Lac-

dehydrogenase

Lactate dehydrogenase (LDH) is an enzyme widely distributed among the
various species of the animal kingdom and ubiquitous in the tissue of man and
other vertebrates. This enzyme catalyses the oxidation of lactate to pyruvate and
the reverse reaction, the reduction of pyruvate to lactate. LDH plays an important
role in the intermediary metabolism of various tissues. For the catalytic activities
of LDH the presence of a coenzyme nicotine amide adenine denucleotide (NAD)
is essential.
The LDH activity in milk might be used as an indicator to determine the
early stage of inflammation in the mammary glands and is recommended for the
screening technique (Hambitzer

and Sommer, 1987). The value of this enzyme

in the milk is not more than 85 UA and when this value exceeded, mastitis is
present (Andersson, 1991). Accoding to Sommer et a/. (1986), LDH activity in
milk is higher in samples containing pathogenic bacteria (S. aureus) compared to
the non infectious mastitis milk. A high positive correlation was found between
cell count and LDH activity in milk containing pathogenic bacteria. The LDH
activlty increased from about 130 to 260 Un as cell count increased from
250,000 to 1.5 millionlml. No comparable results were shown by the samples
containing non-pathogenic bacteria, where the activity of LDH remained fairly
constant about 100 UII as cell count increased from about 250,000 to 1.25
millionlml.

2.4.2.5 Acute P h s e Response and Subclinical Mastitis
The maintenance of a physiologic homeostasis during ups and downs of
daily living is assured by a number of physiological mechanism. Tissue injury
and infection which represent a threat to the integrity of the organism, and
appear to require readjustments in the usual metabolic and physiologic

eqicilibrium. The local response of tissue to such injury or infection is acute
inflammation. It's major clinical maniksWons resuit from changes in vascular
caliber and flow, increased vascular permeability and attraction of leukocytes.
Durtng the first few days €&tawing insult, a vast number of systemic and
metabolic changes occur, that is the acute response. The acute response should
be understood as a value in helping to pemtit survival during the period
immediately foflowing iniuty and in achieving the same goals as We local
inflammatory response does, containment or destruction of infedious agents,
removal of damaged tissue and repair of the affected organ.

In mammals, tissue damage or inflammation at a localized site lead to
systemic changes known as the acute phase response. The acute phase
reaction is an earty body defense mechanism tfiggered in response to cellular
injury manifested through a variety of adverse conditions, such as infection,
inflammation and advanced matignancies. This reaction provides substantial
immediate protection before humoraCmedited immunity becomes effective.
Among the varied physiological atteratian, which together produce this response,
is a change in the circulating levels of a number of liver derived proteins,
collectively known as acute phase reactant p m f e h (APRP) (Koj, $974; Saini et

a/.,1998). Acute phae-pmtetns are serum proteins, whose concentrations
increase during the acute phase response to inflammation or infedion. The
respanse occurs in alt animals, but it is s ~ i m t t different
y
in various species
(Eckersall et a/., 1988). APRP in man and animals indude C-reactive protein
(CRP), serum amyloid A (SAA), haptogtobi (Hp), cenrioplasmin, orosomucoid,
al-antitrypsin, a2-moglobuIirtl comptement and coagulation proteins (Peppys
et a/., 1989). In cattle the Hp and SAA are We major APRP. Bovine Haptoglobin
is a Glycoprotein from a-2 Globulin fraction of serum, with a molecular weight

100 kDa find in blood and fomred in the Cver of mammals (Knura, 2000). The
assay of serum Hp in bovine serum or plasma is now a routine biochemical test
for veterinary diagnostic in Eurppe and USA. This is due to the ability of Hp to
bind hemoglobin. It binds free hemoglobin, thus p r e s e ~ n gthe body, which is
liberated from damaged erythmcytes. SAA and Hp concentration were
associated only with acute and sub awte lesions but not for animals with chronic

infhmmation (EckersaH, 1995). The possibility of using Hp for the detection of
mastitis has been reported by Comer et a!. (I-),

which showed that the

change in haptoglobin level from zero to 70 mg HpflOO ml by cows with clinical
mastitis. Until now, there is no information available about the using of H p
biological activity for early detection of subclinical mastitis.

2.4.2.6 Ekctrical Conductivity in Milk
A precise diagnose of udder health status is the base for effedive mastitis
control. Cell count and electricai conductivity are used to indicate inflammatory
changes in the bovine mammary gland. The basis of mastitis diagnosis consists

of the combination of the detection of the inflammatory changes, but for
economic reasons very often the measurement of the number of somatic cells
and the ekctrkai conductwit)c in milk performed withwt parallel bacteriological
examination (Hamann and Gyod, 1999). Accocdfng to Linzetl and Peaker (1975),
canductivlty is expressed as the molarity of a sodium chloride solution of the
same electrical conductivity as the milk sample. Physiological studies have

shown that in minfected glands the major ions responsible for milk conductivity
pass into milk onky through h secretory cells and that the relatively high
potassium and low sodium concentrations are mainted by the integrity and active
metabolism of the cells. The larger ducts where much of the milk is stored
between miking are normally impermeabie to these ions and lactose. Thus

factors which interfere with cell metabolism will tend to alter the ionic
composition and more severe damage to the ducts or the secretory epithelium

. will have a more drastic effect because the milk will be allowed to partially
equilibrate with extracellular fluid which is rich in sodium and chloride but low in
potassium and these ions will rapidly move across the damaged epithelium. Two
relevant facts are well established. Firstly, milk fat concentration rises steadily
during milking and secondly conductivity decreases as the fat concentration is
increased.

2.5

Homeopathy as an Alternative Therapy

The main aim of homeopathic treatment is to maintain the physiological
homeostasis, which is based on natural healing. The use of homeopathic drugs
in "organic livestock fanning'' has been recommended by Europe Union.
Homeopathy is an alternative treatment, which is effective against bacterial as
well as viral infections. Contrary, antibiotic treatment had no effect on viral
infections, only avoiding the bacterial secondary infections and caused residue
problems (Wheeler, 1978). Their effect follows a physiological principle, have no
negative side effect and are used mostly in small dose at LOIN concentrations

without side effects. They give no additional toxic burden for the liver and
kidneys and cause no residue, which is very important for food producing
animals. They cause no additional burden to the environment and are also
ecologically friendly.
Homeopathy can be defined as a therapy, which activates the natural cure
and defense potential of the organism (Gebhardt, 1977). Homeopathy is a
system of medicat therapeutics for treating people and animals on the basis of
the principle "Similia similibus curentur" which means 'Yreats likes with likes". The
theoretical basis states, that any substance which can cause, at toxic dose, a

range of clinical symptoms in a healthy individual, can be used, prepared as
homeopathic remedy, to cure individuals showing similar symptoms. The precise
mechanism of action of a homeopathic on the molecular level is not yet fully
understood.
The homeopathic drugs are prepared from natural substances derived
from plants, animals, mineral, inorganic salts and organic substances.
Preparations we made on either a centisimal (1C) or decimal scale (ID).
Repeated dilution and succussion result in higher potencies, releasing more
energy in the process. Therefore, homeopathy is a system of medicine, which
concern itself with energy and not with material doses of a drug (Hamann, 1992).
Based on the potencies grade homeopathic drugs could be divided into 3
criteria: Lower potencies (D1-D6), the drugs should be used to treat chronic
cases with or without pathological changes being present, middle potencies (D7D30), and higher potencies (>D30) which are more energized should be
employed in acute infections (McLeod, 1991). Prepamtion of minerals and
metals are made with the 'Yrituration" method. More refined the fragment size of
the drugs caused a better "dynamic curative power"1efficacy (K6hler' 1999).
Pathological tissues are more sensitive to the drugs. Using small amount
of drugs enhances the viability of cells, but in high dose of drugs it caused cell

necrosis (Wheeler, 1978).

2.6

Advantageous of Using Combination Form of Homeopathic Drugs
Manifestation of a disease is not