prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/31/1/160S.pdf · To The Controller of Examinations, University of Agriculture, Faisalabad. We, the Supervisory Committee, certify

EVALUATION OF SOME NON-ANTIBIOTIC

ANTIBACTERIALS IN THE TREATMENT OF

BUBALINE MASTITIS

Muhammad Yousaf M.Sc. (Hons.) Vet. Med.

A thesis submitted in partial fulfilment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

IN

VETERINARY CLINICAL MEDICINE & SURGERY

Faculty of Veterinary Science,

University of Agriculture, Faisalabad-Pakistan.

2009

To The Controller of Examinations, University of Agriculture, Faisalabad.

We, the Supervisory Committee, certify that the contents and form of

thesis submitted by Mr. Muhammad Yousaf (Regd. No. 83-ag-998) have been

found satisfactory and recommend that it be processed for evaluation by the

External Examiner (s) for the award of the degree.

SUPERVISORY COMMITTEE:

Chairman: (Prof. Dr. Ghulam Muhammad)

Member: (Prof. Dr. Muhammad Zargham Khan)

Member:

(Dr. Sajjad-ur-Rahman)

Recommended and Forwarded:

Chairman, Department of Clinical Medicine & Surgery University of Agriculture, Faisalabad.

Dean, Faculty of Veterinary Science University of Agriculture, Faisalabad.

“THE FINAL OBJECTIVE OF THE VETERINARY MEDICINE DOES NOT LIE…. IN THE ANIMAL SPECIES THAT THE VETERINARIAN

COMMONLY TREATS. IT LIES VERY DEFINITELY IN MAN, AND ABOVE ALL IN HUMANITY”.

M. MARTINEZ BAEZ

“THERE IS NO FINER INVESTMENT FOR ANY COMMUNITY THAN PUTTING MILK INTO BABIES. HEALTHY CITIZENS ARE THE GREATEST

ASSET ANY COUNTRY CAN HAVE” SIR WINSTON CHURCHILL

“PUNJAB MIGHT HAVE TURNED SOCIALIST BUT FOR THE EXISTENCE OF BUFFALO WHICH PROVIDES SUSTENANCE TO MILLIONS OF LANDLESS

POORER SECTION OF OUR SOCIETY” KHAN AND REHMAN (1982)

DEDICATION

DEDICATED

To

The Memories of My Sweet & Beloved

Parents (Late)

My Loving & Caring

Wife,

&

Affectionate

Children

CONTENTS

Page #

• List of Abbreviations .............................................................. i

• Acknowledgements ............................................................... ii

• List of Tables .......................................................................... iv

• List of Figures .......................................................................... vi

• List of Appendices ................................................................. vii

Chapter 1 Introduction .............................................................. 1

Chapter 2 Review of Literature................................................ 7

Chapter 3 Materials and Methods ........................................... 75

Chapter 4 Results and Discussion ......................................... 95

Chapter 5 Summary .................................................................. 150

• Literature Cited .......................................................................... 156

• Appendices .................................................................................. 188

6

LIST OF ABBREVIATIONS

• ATCC = American Type Culture Collection

• C. pyogenes = Corynebacterium pyogenes

• C. bovis = Corynebacterium bovis

• C. ulcerans = Corynebacterium ulcerans

• CMT = California Mastitis Test

• CPZ = Chlorpromazine

• DMSO = Dimethylsulphoxide

• E. coli = Escherichia coli

• EUCAST = European Committee for Antimicrobial Susceptibility Testing

• G = Group

• HBSS = Hank’s Balanced Salt Solution

• Lid = Lidocaine

• MIC = Minimum Inhibitory Concentration

• NCCLS = National Committee for Clinical Laboratory Standards

• PE = Phagocytic efficiency

• PI = Povidone-iodine

• PMNLs = Polymorphonuclear leukocytes

• QSCC = Quarter Somatic Cell Count

• S. aureus = Staphylococcus aureus

• SCC = Somatic Cell Count

• SFMT = Surf Field Mastitis Test

• Str. agalactiae = Streptococcus agalactiae

7

ACKNOWLEDGEMENTS

First of all I am thankful to Allah who created every thing in this universe

for the benefit of mankind, and man has to find out its hidden secrets and facts in

the best interest of humanity.

There are many people who through their generosity, expertise and

knowledge have made important contributions to this dissertation work. It would

be virtually impossible to list everyone who contributed to resolve this research

enigma, or to appreciate adequately the extent of the contributions of those who are

mentioned.

First and foremost, I would like to express my heartiest gratitude and deep

sense of obligation to my worthy Supervisor and gracious mentor Dr. Ghulam

Muhammad, Professor Department of Veterinary Clinical Medicine and

Surgery, University of Agriculture, Faisalabad, for his sagacious guidance and

support that he provided all along the course of my Ph.D. degree program.

Without his insightful directions, many of the results presented in this

dissertation manuscript would not have been possible.

It gives me a momentous pleasure in transcribing my whole hearted thanks

to the members of my Supervisory Committee, Dr. Muhammad Zargham Khan,

Professor, Department of Veterinary Pathology, and Dr. Sajjad-ur-Rahman,

Associate Professor, Department of Microbiology, for their able guidance, keen

interest, unstinted help, constructive criticism and ever-encouraging attitude

throughout the course of research investigations and write-up of thesis

manuscript.

8

I extend my cordial thanks to Dr. Athar and Dr. M. Saqib for helping me

to conceive the idea of this research. They along with Dr. Abeera Naureen

provided the much needed assistance in the execution of research work, trouble

shooting and write up of the dissertation manuscript. Cooperation extended by

Dr. Tanveer Ahmad, Dr. Arfan Yousaf, Dr. M. Nadeem Asi, Dr. M.

Hammad Hussain, Dr. Abdul Hafeez, Dr. Farrah Deeba, Dr. Khurram

Ashfaq and Dr. Arif Zafar (lecturers) as well as support staff employees of the

Department of Clinical Medicine and Surgery is gratefully acknowledged. I would

remiss if I do not acknowledge the able assistance of Miss Sajida Perveen in

professional typing, incorporation of changes suggested by external examiners as

well as correcting the section on literature cited.

Last but not the leas, I have no words of thank for my family members

(Mrs. Najmun Nisa, Aaliya, Adnan, Usman, Bilal and Aaisha) and my

brother Dr. Aman Ullah (Medical Specialist) who wished to see me glittering

high on the sky of success during the entire period of my studies. Their day and

night prayers boosted me to fly high to accomplish my academic goals.

MUHAMMAD YOUSAF

9

LIST OF TABLES

Table No.

TITLE Page No.

1.1 Findings of some previous studies on prevalence of common mastitis pathogens in Pakistan (1966–2005) in buffaloes and cows

6

3.1 Stock solutions of chlorpromazine (CPZ), its concentration (μg/mL) and final concentration in petri dishes for determination of MIC

83

3.2 Stock solutions of lidocaine, its concentration (mg/mL) and final concentration in petri plates for the determination of MIC

84

3.3 Stock solutions of povidone-iodine, its concentration (mg/mL) and final concentration in petri plates for the determination of MIC

85

3.4 Evaluation of non-antibiotic antibacterials alone in the treatment of bubaline clinical mastitis

89

3.5 Evaluation of non-antibiotic antibacterials in combination with antibiotic (cephradine) in the treatment of bubaline clinical mastitis

92

4.1 Zones of inhibition (mm) of the non-antibiotics against field mastitis and control (American Type Culture Collection) isolates

103

4.2 Minimum inhibitory concentrations (MICs) of the non-antibiotics determined by agar dilution method

105

4.3.1 Analysis of variance table for viability (%) of polymorphonuclear leukocytes under the influence of different stock solutions of non-antibiotics and their ten fold dilutions

109

4.3.2 Comparison of means for viability (%) of polymorphonuclear leukocytes under the influence of different stock solutions of non-antibiotic antibacterials and their ten fold dilutions

109

4.4.1 Suppression of phagocytic efficiency of polymorphonuclear leukocytes by stock solutions of 4 non-antibiotic antibacterials and their ten fold dilutions

112

4.4.2

Analysis of variance table for influence of different stock solutions of non-antibiotic antibacterials and their ten fold dilutions on phagocytic efficiency of polymorphonuclear leukocytes

114

4.4.3 Comparison of means for influence of different stock solutions of non-antibiotic antibacterials and their ten fold dilutions on phagocytic efficiency of polymorphonuclear leukocytes

114

10

Table No.

TITLE Page No.

4.5.1 Surf Field Mastitis Test (SFMT) based percent quarters cure rates in various non-antibiotics antibacterials/antibiotic groups on day 14 post initiation of treatment

124

4.5.2 Surf Field Mastitis Test (SFMT) based percent quarter cure rates in various non-antibiotic antibacterials/antibiotic groups on day 28 post initiation of treatment

125

4.5.3 California Mastitis Test (CMT) based percent quarter cure rates in various non-antibiotic antibacterials/antibiotic groups on day 14 post initiation of treatment

126

4.5.4

California Mastitis Test (CMT) based percent quarter cure rates in various non-antibiotic antibacterials alone and in combination with cephradine on day 14 and day 28 post initiation of treatment

127

4.5.5

Surf Field Mastitis Test and California Mastitis Test based percent cure rates of non-antibiotic antibacterials alone and in combination with cephradine on day 14 and day 28 post initiation of treatment

128

4.6.1

Effect of intramammary infusions (n = 4) of non-antibiotic antibacterials alone and in combination with cephradine on quarter somatic cells count (QSCC; X 105/mL of milk) on day 14 post initiation of treatment

133

4.6.2

Effect of intramammary infusions (n = 4) of non-antibiotic anti-bacterials alone and in combination with cephradine on quarter somatic cells count (QSCC; X 105/mL of milk) on day 28 post initiation of treatment

134

4.6.3

Effect (% decrease or increase) of infusions (n = 4) of non-antibiotic antibacterials alone (groups G1 thru G4) and in combinations with cephradine (groups G6 thru G9) on quarter somatic cells count (QSCC) recorded on day 14 and day 28 post-initiation of treatment

135

4.6.4

Effect of infusions (n = 4) of non-antibiotic antibacterials alone (groups G1 thru G4) and in combination with cephradine (Groups 6 thru 9) on quarter somatic cells count (QSCC x 105) recorded on day 14 and day 28 post-initiation of treatment

136

4.7 Bacteriological cure rates of infusions (n = 4) of non-antibiotic antibacterials singly and in combinations with cephradine on day 14 and day 28 post-initiation of treatment

143

4.8.1

Effect of non-antibiotic antibacterials infusions (n = 4) alone and in combination with cephradine on milk yield (Loss of milk in liters/quarter/day) in buffaloes affected with clinical mastitis over a period of 4 weeks post initiation of treatment

147

4.8.2 Prioritization of different treatments on the basis of mean milk yield loss (mean ± SE) of buffalo mastitic quarters

149

11

LIST OF FIGURES

Figure No.

Title Page No.

4.1 Inhibitory zones produced by chlorpromazine at 25 and 50 μg per disc against S. aureus isolate

100

4.2 Inhibitory zones against S. aureus expanded as the disc potency of chlorpromazine was increased to 75 and 100 μg per disc

100

4.3 Inhibitory zones against S. aureus increased further as the disc potency of chlorpromazine was increased to 175 μg per disc

101

4.4 Inhibitory zones produced by Lidocaine impregnated discs at (10mg and 20mg per disc) against E. coli isolate

101

4.5 Increasing the disc potency of Lidocaine to 25mg per disc further axpanded the zones of inhibition of this non-antibiotic against E. coli isolate

102

4.3.1 Viability (%) of polymorphonuclear leukocytes under the influence of different stock solutions of non-antibiotic antibacterials and their ten fold dilutions

110

4.4.1 Percent inhibitory effect of different stock solutions of non antibiotic antibacterials and their ten fold dilutions on phagocytic efficiency of polymorphonuclear leukocytes

113

4.5.1 Effect of infusions (n = 4) non-antibiotic antibacterials alone and in combination with cephradine in reducing QSCC (means ± SE) after treatment

137

4.8

Mean effects of infusions (n = 4) of non-antibiotic antibacterials alone and in combination with cephradine on milk yield of the buffalo quarters affected with clinical mastitis over a period of 4 weeks post initiation of treatment

148

12

LIST OF APPENDICES

Appendix No.

TITLE Page No.

1

Guidelines for significance of colony numbers of specific microorganisms isolated in pure or with other colony types (based on 0.01ml quarter milk sample streaked on blood agar)

188

2 Viability (%) of polymorphonuclear leukocytes (PMNLs) under the influence of Chlorpromazine (CPZ) 2.5% and its ten fold dilutions

189

3 Viability (%) of polymorphonuclear leukocytes (PMNLs) under the influence of Lidocaine (Lid) 2% and its ten fold dilutions.

190

4 Viability (%) of polymorphonuclear leukocytes (PMNLs) under the influence of Povidone-iodine (PI) 10% and its ten fold dilutions.

191

5 Viability (%) of polymorphonuclear leukocytes (PMNLs) under the influence of dimethysulphoxide (DMSO) 30% and its ten fold dilutions.

192

6 Effect of Chlorpromazine (CPZ) 2.5% and its ten fold dilutions on phagocytic efficiency of polymorphonuclear leukocytes (PMNLs).

193

7 Effect of Lidocaine (Lid) 2% and its ten fold dilutions on phagocytic efficiency of polymorphonuclear leukocytes (PMNLs).

194

8 Effect of Povidone-iodine (PI) 10% and its ten fold dilutions on phagocytic efficiency of polymorphonuclear leukocytes (PMNLs).

195

9

Quarter SCC (X 105/ml of milk) of group G1 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of Chlorpromazine (CPZ) 50mg (2ml + 8ml normal saline) in dairy buffaloes

196

10

Quarter SCC (X 105/ml of milk) of group G2 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of Lidocaine (Lid) 4% (10ml + 30ml normal saline) in dairy buffaloes.

197

11

Quarter SCC (X 105/ml of milk) of group G3 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of Pivodine-iodine (PI) 10% (10ml + 30ml normal saline) in dairy buffaloes.

198

13

Appendix No.

TITLE Page No.

12

Quarter SCC (X 105/ml of milk) of group G4 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of dimethylsuphoxide (DMSO) (20ml + 20ml normal saline) in dairy buffaloes.

199

13

Quarter SCC (X 105/ml of milk) of group G5 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of Cephradine (Ceph) 500mg (in 40ml normal saline) in dairy buffaloes.

200

14

Quarter SCC (X 105/ml of milk) of group G6 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of Chlorpromazine (50mg = 2mL) + Cephradine (500mg) in 38ml normal saline in dairy buffaloes

201

15

Quarter SCC (X 105/ml of milk) of group G7 before treatment and at day 14 and day 28 post intiation of treatment with intramammary infusions (n = 5) of Lidocaine (4% = 10ml) + Cephradine (500mg) in 30ml normal saline in dairy buffaloes

202

16

Quarter SCC (X 105/ml of milk) of group G8 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of Povidone-iodine 10%(10ml) + Cephradine (500mg) in 30ml normal saline in dairy buffaloes.

203

17

Quarter SCC (X 105/ml of milk) of group G9 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of dimethylsulphoxide (20ml) + Cephradine (500mg) in 20ml normal saline in dairy buffaloes.

204

14

CHAPTER 1

INTRODUCTION

When antibiotic compounds were introduced into the therapeutic arsenal

of physicians and veterinarians, they naively and over optimistically believed to

have finally won the war against infectious bacterial diseases. It is now more

than 65 years since the first antibiotic was discovered but up until today only a

few infections have been overcome. A vast majority of bacterial infections are not

only still prevalent but some new infections have also emerged. One of the major

limiting factors in the control of infections is the development of resistance by

microorganisms against antibiotics. Needless to say that development of

resistance has outpaced the development of new antimicrobials and resistance is

now a buzzword with veterinary and medical communiteis. They are still

desperately trying various combinations of drugs to achieve success against even

simple infections. The use of antibiotics, actually, does not induce resistance but

rather eliminates the susceptible bacterial population in the host and spares the

resistant ones. The potential for genetic exchange between bacteria, combined

with their short generation times can rapidly produce resistant microbial

populations (Prescott and Baggot, 1993).

With a population of over 29 million heads of dairy buffalo (Bubalus

bubalis) and 31.8 million heads of cattle, Pakistan is one of the major dairying

CHAPTER 1 INTRODUCTION

15

countries in the World (Economic Survey of Pakistan, 2007-08). Despite this huge

number of cattle and buffalo, milk production is only 30 million tons per annum,

which translates into only 2-3 liters of milk/animal/day. This undesirably low

quantity of milk produced by a colossal dairy animal population can be ascribed

to poor genetic potential, poor nutrition and management and suboptimal health

of milk animals. The health of the milk-producing organ (udder) is of prime

importance especially for the production of wholesome milk.

Mastitis (inflammation of the milk producing organ of mammals) is the

most common and the most costly disease of the dairy industry all over the

World (DeGraves and Fetrow, 1993; Allert, 1995). At the very least, every fifth

dairy buffalo / cow is afflicted with this disease (Fazal-ur-Rehman, 1995). In

Pakistan, statistics of current losses due to this disease are not available although

it was estimated in 1978 that in Punjab Province alone, the total losses caused by

clinical mastitis amount to Rs.240 million per annum (Chaudhry and Khan,

1978). It is pertinent to mention that this survey did not take into account the

pecuniary losses associated with subclinical mastitis, the form of mastitis that is

15-40 times more prevalent than its clinical counterpart. These losses are mainly

contributed by Staphylococcus aureus, followed by Streptococcus agalactiae,

Corynebacterium pyogenes and Escherichia coli (Table 1.1). It is pertinent to mention

that these mastitogens can be controlled effectively by implementing the 5-point

mastitis control plan as advocated by National Mastitis Council, Inc., USA


16

(www.nmconlinbe.org). The components of this control program include (1) teat

dipping; (2) dry cow antibiotic therapy, (3) prompt treatment of clinical cases

with appropriate antibiotic(s), (4) proper milking hygiene/use of adequately

functioning milking equipment, and (5) culling chronically infected cows. Two of

these points involve the use of antibiotics therapy (Nickerson and Owens, 1990).

Cure rates in the treatment of clinical mastitis are appallingly poor (Sears

et al., 1990; Sol et al., 1994; Sol et al., 2000; Sears and McCarthy, 2003). The reasons

for failure of antibiotic therapy of mastitis have been reviewed by (Sandholm et

al., 1990; Sandholm et al., 1991). Failure of antibiotics to reach the site of infection

in adequate concentrations, development of resistance to antibiotics, bacterial

dormancy, L-form of bacteria (which are not sensitive to the β-lactam type of

antibiotics), detrimental nature of some antibiotics to phagocytosis,

incompatibility of antibacterials with milk, failure of the cooperative function of

endogenous and exogenous antibacterial factors, inadequate milking hygiene

and re-infections are some important reasons for suboptimal results obtained in

the therapy of mastitis with antibiotics.

The need for antibacterial substances other than antibiotics has since long

been felt. Recently, a variety of compounds commonly employed in the

treatment of pathological conditions of a non-infectious etiology have been

shown to modify cell permeability and to exhibit broad-spectrum antimicrobial

activity in vitro against bacteria and other micro-organisms (Kristiansen, 1990;


17

Amaral and Lorian, 1991; Cederiund and Mardh, 1993; Kramer et al., 1994). Such

compounds have been given the name 'non-antibiotics' (Kristiansen and Amaral,

1997). In addition, these compounds have been found to enhance the in vitro

activity of certain antibiotics against specific bacteria (Kristiansen, 1990,

Kristiansen, 1991; Amaral et al., 1992), to render in vitro antibiotic-resistant

bacteria susceptible to previously ineffective drugs (Amaral et al., 1992;

Kristiansen et al., 1993), and to exhibit strong in vitro antimycobacterial activity

against clinical strains resistant to one or more conventional antibiotics

(Kristiansen, 1990; Williams, 1995; Amaral et al., 1997). These compounds,

primarily phenothiazines, thioxanthenes and other agents with affinities for

cellular transport systems, are characterized by their effects on the plasma

membrane of eukaryotic cells (e.g., local anaesthetic activity), and have been

termed membrane stabilizers (Kristiansen, 1990). The proposed mechanism by

which they exert their in vitro antimicrobial activity is thought to be via their

effects on the inner membrane of bacteria (Molnar et al., 1992).

Several antibiotics are in use to treat and prevent intramammary

infections. Extra-label and unsupervised uses have led to the development of

resistance and in country like Pakistan, conventional antibiotics are prohibitively

expensive for the resource poor dairy farmers. The present study was designed

to ascertain whether chlorpromazine, lidocaine, DMSO and povidone-iodine


18

could be used to treat clinical form of bubaline (= buffalo) mastitis. The specific

objectives set forth were as follows:

1) To determine the in vitro antibacterial effect of non-antibiotic

antibacterials.

2) To evaluate the effect of non-antibiotic antibacterials on phagocytosis

by polymorphonuclear leukocytes under in vitro settings.

3) To evaluate the in vivo effect of non-antibiotic antibacterials in the

treatment of mastitis in buffaloes.

The overall objective was to determine whether the non-antibiotic

antibacterial could be used as alternative/adjunct to antibiotics in the treatment

of mastitis in dairy buffalo. Although, study zeroed in on mastitis pathogens, the

findings may potentially have some spin-off applications in the treatment of

other common animal and human bacterial infections.


19

Table 1.1. Findings of some previous studies on prevalence of common mastitis pathogens in Pakistan (1966–2005) in buffaloes and cows.

Workers Animals

Percent Prevalence of Common Mastitis Pathogens

S. aureus

Str. agalactiae

E. coli

Mixed1, Str. dysaga-lactiae2, Str. uberis3,Str. pyogenes4, Str. faecalis7, S. epidermidis5, CNS6

Others (C. pyogenes1, Pseud. aeruginosa2, Mycobac.3, Yeasts4, Klebsiella5, C. bovis6 P. vulgaris7, B. cereus8, A. aerogenes9 etc.)

Ahmad (1966) Buffaloes 44.00 – – – –

Ghumman (1967) Buffaloes 91.20 0.80 1.60 – 0.82

Ahmad (1968) Buffaloes 84.00 2.00 4.00 10.001 –

Hashmi (1978) Buffaloes and Cows

43.09 36.82 12.55 – 4.21, 2.92, 0.43

Hashmi & Munir (1981)

Buffaloes 34.55 34.44 20.00 – 2.02

Anwar & Chaudhari (1983)

Buffaloes 40.00 45.06 25.00 – 5.02

Sahi (1983) Buffaloes 45.45 33.57 12.59 – 4.21, 1.42, 0.74

Hussain et al. (1984)

Buffaloes 28.26 40.20 19.66 8.69 17.331, 0.262, 7.29

Cows 16.53 25.90 5.06 25.0 6.269

Shireen (1984) Buffaloes 54.54 10.60 12.12 12.122 3.01, 1.53

Cows 55.26 18.42 15.78 5.262

Iqbal (1991) Cows 32.09 3.01 16.08 7.03, 12.066 3.51, 7.52, 2.015,

3.018

Qamar (1992) Mixed 25.55 9.99 18.88 4.442, 2.24, 11.15 2.21, 4.46, 12.22,

3.37, 5.68

Fazal-ur-Rehman (1995)

Buffaloes 39.06 3.13 6.25 10.942 10.91, 3.14

Cows 47.87 1.06 1.06 7.452 3.21, 4.32

Razzaq (1998) Buffaloes 53.33 40.00 6.66 – –

Memon et al. (1999) Buffaloes 38.00 8.00 11.00 13.003 11.005

Ahmad (2001) Buffaloes 33.99 35.46 27.09 - 1.481, 1.972

Akram (2002) Buffaloes 40.32 14.52 17.74 4.842, 3.234, 4.845 –

Khan (2002) Buffaloes 45.00 23.00 18.00 – 14.008

Cows 49.00 30.00 13.00 – 8.008

Khan et al., (2004) Buffaloes 45.00 23.00 18.00 _ 148

Khan and Muhammad(2005)

Buffaloes 45 23 18 _ _

Cows 48 30 13 _ _

Mixed1 = Staphylococci + Streptococci Adapted and modified from Shakoor (2006)

20

CHAPTER 2

REVIEW OF LITERATURE

2.1 BUFFALO AS A DAIRY ANIMAL

The water buffalo (Bubalus bubalis) also called the ‘Black Gold’ of South

Asia, is the premier dairy animal of this region. Overall global population of

buffalo increased by 91% during the period from 1961 to 2001 while, it surged

upto 130 millions in 2005 (FAO, 2005). Water buffalo plays a vital role in Asian

and Far-Eastern countries where 96% of its total population in the world is

concentrated. At present, Pakistan has an estimated 28.4 million heads of

buffaloes and 25.5 million heads of cattle. In buffalo milk production, India

ranked first in the world, followed by Pakistan, China and Egypt, respectively

(FAO, 2006). Over 95% of world’s buffalo milk is produced in Asia (FAO, 2005).

Buffalo is recognized as the world’s second most important milk producing

species (McDowell et al., 1995).

Pakistan is the fifth largest milk producer in the world. According to

Economic Survey of Pakistan (2007-08), buffaloes and cattle respectively

contribute nearly 66% and 27% of the total milk produced in this country.

Pakistani buffaloes are among the best dairy buffaloes of the world based on the

fact that they constitute only 8.5% of the total world’s buffalo population but

produce 28% of the world’s total buffalo milk (FAO, 2006). For domestic buffalo

CHAPTER 2 REVIEW OF LITERATURE

21

major habitats are South and East Asian Pacific countries. In Europe, Italy is the

main producer of buffalo milk. Reasons for increasing interest (almost 2-fold) in

buffalo breeding during recent years owes to the increased demand of buffalo

milk with more butterfat, protein and lactose which is essential for buffalo

mozzarella cheese and makes it more valuable than bovine milk (Moroni et al.,

2006).

Buffalo milk has advantages in the preparation of processed products. The

ratio of butter (ghee) output is approximately three times greater than for cow

milk and the ratio of milk per kg of soft cheese is 5:1 vs. 8:1 from cow milk.

Buffalo milk is most desirable for soft cheeses, e.g., mozzarella or soft white

cheeses, but is less desirable for use in producing hard cheeses due to a slower

rate of acid production, lower retention of moisture in the curd and more loss of

fat in the whey (McDowell et al., 1995). Despite its usefulness, the dairy buffalo

remained neglected in the past. Numerous writers have reckoned buffalo as an

underutilized species with high potential for milk yield (Cockrill, 1994).

2.2 MASTITIS AND ITS IMPORTANT ETIOLOGIC AGENTS

Mastitis remains the most common and the most expensive disease of

dairy industry throughout the world (Kinabo and Assey, 1983; Lightner et al.,

1988; Kaneene and Hurd, 1990; Miller et al., 1993; Kossaibati et al., 1998). Among

major diseases of dairy animals, mastitis ranked first and is of major concern in

Pakistan (Akhtar, 1995). This disease not only affects the physical, chemical,

bacteriological, technological and organoleptic properties of milk but also affects


22

quantity of milk. It causes major economical losses in dairy animals and the

estimated economic loss to the US was found to be more than $ 2 billion per

annum (Jasper et al., 1982). The UK dairy industry faced a loss of £ 93 million per

year, while Australian dairy industry faced more than $ 100 million annually

(Hogeveen, 2005). In a recent study (Huijps et al., 2008), average economic losses

assessed by the Dutch dairy farmers were € 78/cow per year with a range of 17-

198 €/cow per year. According to Mayer (1990), the worldwide cost of this

disease was US $ 35 billions annually.

Buffaloes are susceptible to most of the pathogens that cause mastitis in

cattle (Adlakha and Sharma, 1992). However, as per Wanasinghe (1985),

buffaloes are more resistant to mastitis than cattle. Uppal et al. (1994) reported

that buffalo has a tighter teat opening than that of cow which may account for its

lower susceptibility to mastitis. Some researches have shown similar mastitis

frequencies for both the dairy species (Kalra and Dhanda, 1964; Badran, 1985;

Bansal et al. 1995). Similarly, Srinivasan and Singh (1988) proved that buffalo is as

susceptible to mastitis as cow. The finding of the Sri Lankan researchers (Silva

and Silva, 1994) are consistent to those of Wanasinghe (1985).These workers

concluded that the quality and/or quantity of somatic cells in response to

mammary infections in buffaloes may differ from that in cows. In India, the

incidence of clinical mastitis varied from 1 -10% (Kalra and Dhanda, 1964; Rao

and Ramaiah, 1982; Kumar, 1988), whereas that of subclinical mastitis varied

from 10–50% and 5-20% in cows and buffaloes, respectively (Singh, 1974;


23

Chander and Buxi, 1975; Sharma and Rai, 1977; Kumar, 1988). Thus from

literature cited it is clear that incidence of mastitis appears to be lesser in

buffaloes than in cows under similar managemental conditions. This might be

due to some genetical and anatomical factors providing higher resistance against

mastitis in buffaloes than in cows (Uppal et al., 1994). Moreover, Krishnaswamy

et al. (1965) studied histological difference in the teats of both species and

concluded that rich muscle fibers and vascular tissue in the buffalo teats may be

the factors responsible for lesser intramammary infections (IMI) in this dairy

animal.

Mastitis exists wherever there are cows and buffaloes. It is frequently said

that every cow/buffalo develops mastitis before she dies. Indeed, it is doubtful

that there is a single herd of dairy cows [and buffaloes as well] anywhere,

regardless of size, that is truly free of the disease. It has been estimated that one

third of all dairy cows are infected with some form of mastitis in one or more

quarters (Philpot and Nickerson, 1991). Staphylococcus aureus is often considered

to be the most common contagious mastitis pathogen in US dairy herds (Fox et

al., 2000).

A score of studies have been carried out in the South Asian countries on

mastitis in water buffaloes and its etiological agents. In one such study in India

(Prabhakar et al.1995), of 421 mastitis cases in buffaloes herds, Staphylococcus spp.

(47.37%) were the major causative organisms (S. aureus 34.21% and coagulase

negative 13.16%) followed by Streptococcus agalactiae, (14.47%), E. coli (10.53%)


24

Pseudomonas spp. (7.89%), S. pyogenes (3.95), Str. dysgalactiae (2.63%), and mixed

infections (1.31%).

In a previous study on the etiological organisms of buffalo mastitis

conducted in Lahore (Pakistan), of the total of 740 mastitic milk samples, 44%

yielded S. aureus, 30% Str. agalactiae, 8.7% Str. dysgalactiae, 12.4% coliforms, 5.1%

Corynebacterium pyogenes, 3.2% Pseudomonas aeruginosa, and 0.4% yeast cells

(Jaffery and Rizvi, 1975).

Similarly, findings of some previous studies conducted on prevalence of

common mastitis pathogens in buffaloes and cows in Pakistan during 1966-2004

(Table 1.1) indicate that S. aureus and Str. agalactiae are the most frequently

isolated organisms from clinical and subclinical mastitis.

Chander and Baxi (1975) studied 304 quarter milk samples of 78

apparently healthy cows. The principal causative organisms included

staphylococci (68.8%) and streptococci (16.2%). Leukocyte count showed the

highest percent agreement 82.8% with bacteriological examination. Nag (1975)

conducted a study on the incidence of mastitis in buffaloes, cows and goats. The

microorganisms isolated from mastitic milk were S. aureus 35% in buffaloes milk,

20% in cow milk and 11.1% in goat milk. Streptococci were 15% each in buffaloes

and cows milk and 22% in goats milk, while E. coli was 15% in buffaloes milk and

10.5% in cow milk.

Gonzalez et al. (1989) isolated microorganisms from subclinical cases of

bovine mastitis. The percentage of isolates was S. aureus (43%), S. epidermidis


25

(21%), Str. uberis (19%), Str. agalactiae (13%), Str. dysgalactiae (9%), Corynebacterium

bovis (7%), Corynebacterium pyogenes (13%), and coliforms (1.7%). Mixed infections

of Staphylococcus and Streptococci were also noticed.

Hodges et al. (1984) conducted a taxonomic study on 900 isolates of

Staphylococci from bovine milk sample. Of these, 831 were coagulase positive

staphylococci (810 S. aureus and 21 S. intermedius). Of 65 coagulase negative

staphylococci isolates, 19 could not be identified by identification system used.

Langoni and coworkers (1984) investigated the etiology of bovine mastitis

in Brazil and confirmed that 88% of cases of mastitis were caused by

Staphylococci, Streptococci and E. coli. Staphylococcus alone accounted for 56% of

cases. The pyogenic microorganisms were isolated by Slee and McOrist (1985)

from cows affected with summer mastitis. Of the 31 isolates, Actinomyces pyogenes

was present in 25, Fusobacterium necrophorum in 23, Microaerophilic coccus in 16,

Peptostreptococcus indolicus in 15 and Bacteroides spp. in 7. The remainders were

identified as S. hyicus (29), S. haemolyticus (17), S. hominis (3), S. epidermidis (4), S.

capitis (1), S. warneri (1). Four other isolates could not be assigned to the

Staphylococci or Micrococci and were designated as irregular strains. A study

conducted on bovine acute mastitis by Pyörälä and Syväjärvi (1987) found that

26.7% of quarters were infected by S. aureus, 19.4% by Streptococci, 17% by

coliforms and 3.8% by Actinomyces pyogenes.

El-Bayomi and Mahmood (1987) isolated Str. agalactiae (55.26%), S. aureus

(28.95%). Str. dysgalactiae (10.35%) and E. coli (5.26%) from cases of bovine


26

mastitis. Similarly, Dutch workers Schukken et al. (1989) isolated E. coli (13%), S.

aureus (62.8%), and Str. uberis (8%) from clinical mastitis cases in cows. While

Chanda et al. (1989) isolated 57.72 percent Staphylococci, 35.40 percent Streptococci,

5.3 percent Corynebacterium and 1.7 percent E. coli from cases positive for

subclinical mastitis. Trinidad et al. (1990) isolated S. aureus (31%) from the teat

canal of heifers and 12.3% of quarters, while from mammary secretions of 57.1%

of heifers and 14.5% of quarters with clinical symptoms.

Ahmad et al. (1991) investigated the prevalence of subclinical mastitis in

4620 lactating animals comprising of 2216 Sahiwal cows, 917 crossbred cows and

1483 buffaloes. Staphylococci were isolated from 72.32% of the affected udder of 3

types of dairy animals, followed by streptococci (20.96%) and other

microorganisms (6.75%). Iqbal (1991) compared Whiteside test and pH indicator

paper technique on 3980 quarter milk samples. Microorganisms were isolated

from 190 samples. Different pathogenic bacteria isolated were S. aureus (32.60%),

Str. agalactiae (16.58%), E. coli (16.08%) coagulase negative staphylococci (12.06%).

Str. uberis (3.01%), Str. dysgalactiae (3.01%), and Bacillus spp. (3.01%). Pseudomonas

aeruginosa (7.50%), Klebsiella (2.01%), and Corynebacterium pyogenes (3.51%).

Kapur et al. (1992) determined the bacteriology of clincial mastitis in buffaloes.

Out of 868 clinical mastitic quarters samples from 597 buffaloes, 642 (73.96%) were

culturally positive. Mixed infection was present in 96 (11.06%). The micro-organisms

and their prevalence were as follows: Staphylococcus aureus (26.30%), S. epidermidis

(19.10%), Micrococci (1.89%), Str. agalactiae (8.13%), Str.dysgalactiae (17.00%),


27

Str.uberis (0.54%), Str. bovis (1.62%), Streptococcus spp.(2.84%), Enterococci (0.81%),

Corynebacterium pyogenes (5.96%), Corynebacterium spp. (12.87%), Escherichia coli

(6.50%), Klebsiella spp. (2.16%), Pseudomonas aeruginosa (0.67%), Proteus spp.

(0.27%), Bacillus spp. (1.21%), Diptococcus pneumoniae (0.54%), Candida spp. (1.35%)

and Aspergillus spp. (0.13%). Str. agalactiae was of serotypes II and II/R, and included

some non-typable strains. This is probably the first report on serotype distribution of Str.

agalactiae in buffaloes. E. coli O. serotypes were 017, 020, 028, 029, 030, 036, 039, 061,

068, 073, 074, 075, and 0116. Rough and untypable strains were also encountered.

Lafi et al. (1994) reported the findings of a National Cross Sectional study

of mastitis in Jordon. Between July 1991 and August 1992, 63 Jordanian dairy

farms were visited to identify the major causes and prevalence of intramammary

infection in dairy cows. Of 773 cows, the most common isolate from clinical cases

was S. aureus (37.5%). Mitra et al. (1995) studied the prevalence of subclinical

mastitis in an-organized buffalo farm. Of 132 lactating buffaloes, 528 milk

samples were screened and 116 were found positive for subclinical mastitis. The

percent occurrence of the microorganisms was Staphylococci (41.73%), Streptococci

(27.58%), E. coli (24.13%), and Corynebacterium (6.89%). Radostits et al.(2007)

described four Mycobacterium species causing bovine mastitis namely

Mycobacterium lacticola, Mycobacterium fortuitum, Mycobacterium smegmatis and

Mycobacterium chelonei. Infections of these occured after the intramammary

infusions of therapeutic agents in oil. Clinically there was tremeudons

hypertrophy of the quarter with appearance of clots in discoulored milk but


28

there was no systemic reaction. Affected animals did not show sensitivity to

avian or mammalian tuberculin. These cases were refractory to different

treatments.

2.3 CURRENT STATUS OF ANTIBIOTICS EFFICACIES IN THE TREATMENT OF MASTITIS

Bovine mastitis is the single most common reason for antibacterials use in

lactating dairy animals (Erskine et al., 2004). Therapy is a major method for

reducing infection levels. The basic therapeutic principles for lactating and dry

cow treatment should actually be equivalent, but different spontaneous cure and

new infection rates would apparently influence the outcome. The cure rate in

mastitis is often <50% (Deluyker et al., 2005) Penicillin is the drug of choice in the

therapy of mastitis caused by Streptococcus agalactiae or Str. dysgalactiae,

bacteriological cure rate being high i.e. about 50-90% (Sandholm et al.1990).

Staphylococcus aureus remains the most difficult of the major mastitis

pathogens to treat. It is an intracellular contagious pathogen that responds

poorly to conventional antibiotic therapy. Because of the nature of S. aureus

intramammary infection (IMI), traditional antibiotic therapy during lactation is

only marginally effective with cure rates ranging from 15-30% for chronic cases.

Novel therapy regimens using multiple doses of antibiotics and combination of

intramammary and parenteral administration have been shown to increase cure

rates (Owens et al., 1988; Nickerson and Owens, 1989). However, these

therapeutic procedures constitute extralable use of antibiotics and require close


29

veterinary supervision and careful monitoring of milk for antibiotic residues. In

countries where antibiotics have been widely used, Str. agalactiae has been

eradicated from most herds (Sandholm et al., 1990) and S. aureus is gradually

replacing Str. agalactiae as the major dominant isolate, while Gram negative

bacteria, Str. uberis and coagulase-negative staphylococci represent an increasing

percentage of infection in herds where lactation therapy, dry cow therapy, and

teat dipping are being practiced.

Pirilimycin hydrochloride is a newly developed lincosamide antibiotic

labeled for use against staphylococcal and streptococcal organisms causing

mastitis. In induced S. aureus mastitis, the cure rate for pirlimycin (50 mg dose)

was 63.6% of quarters in cows, when an extended duration of treatment was

adopted (Owens et al., 1994).

In a large British study, intramammary cloxacillin produced a

bacteriological cure in 61% of treated quarters (Griffin et al., 1982). In infection

caused by certain pathogens, bacteriological self-cure following clinical episode

may contribute substantially to the apparent efficacy of the treatment given. The

high cure rate of about 90% of coliforms infections in quarters treated with

cloxacillin is significant and reflects the high spontaneous recovery rate which

occurs in coliform mastitis, once the udder defense mechanisms are stimulated

(Craven , 1987)

The combination of intramammary infusion along with intramuscular

injection of antibiotics resulted in a higher bacteriological cures than did


30

intramammary infusion alone. About 48% of cows and 51% of quarters treated

by infusion plus injection were bacteriologically negative 21 days post-treatment

compared with 30% of cows and 25% of quarters treated by intramammary

infusion only (Owens et al., 1988). The cure rate after parenteral norfloxacillin

nicotinate therapy is significantly better (Soback, 1988). Pharmacokinetically,

norfloxacin and oxytetracycline are well distributed into the body fluids and

should be able to reach the site of infection but only norfloxacillin can kill

phagocytosed intracellular staphylococci. The third generation fluoroquinolones

are excreted well into the milk, which may indicate high mammary tissue

concentrations (Soback, 1988) Antibiotics currently widely used include

penicillin, amoxicillin, ampicillin, cephapirin, tetracycline, novobiocin,

erythromycin, fluoroquinolones and streptomycin in combination with

penicillin.

Bacteriological cure rates of S. aureus IMI during lactation are influenced

by several important factors. Cows with lower parity have a better likelihood of

cure compared with cows with higher parity (Sol et al., 1997). Bacteriologic cure

rates are also lower in cows with multiple infected quarters and in those with

higher somatic cell counts. Another factor that affects cure rate is the

susceptibility of the isolate to antibiotic. In a Scandanavian study, bacteriological

cure rate for S. aureus IMIs was 76% after a combination of parenteral and

intramammary therapy if the bacterial isolates were susceptible to penicillin but


31

only 29% for quarters infected with penicillin-resistant bacteria (Taponen et al.,

2003).

2.4 THERAPEUTIC FAILURE IN THE TREATMENT OF MASTITIS

Treatment of mastitis has historically been limited to the use of broad-

range antibiotics such s tetracycline, pencilline, and pirlimycin, which are often

<50% successful (Deluyker et al., 2005) Cure rate in S. aureus mastitis is generally

15-30%.

The poor response of S. aureus mastitis to antibiotic therapy is a major area

of concern for veterinarians, dairy farmers, and mastitis researchers. Therapy

success ranges from 15 to 70%, with less than 50% efficacy in general (Owens,

1988). Reasons for this low success rate include poor penetration of antibiotics

into areas of scarring and inflammation, inactivation of antibiotics by milk and

serum components, intracellular or metabolically inactive organisms, bacterial L-

forms, resistance to antibiotics and improper treatment procedures. Some of the

major causes of mastitis therapy failure are discussed as under:

2.4.1. Tissue invading nature of pathogens

Tissue invaders such as staphylococci become walled-off in the udder

parenchyma by thick, fibrous scar tissue. Thus, antibiotics cannot reach the

pathogen. Staphylococcus aureus udder infection promotes development of

localized scar tissue which does not have blood vessels, so that intramuscular

and intravenous injections probably do not provide the desirable benefit (du

Preez, 1988). Therapy may kill the bacteria that are not walled off, but at a later


32

date, the bacteria within the scar tissue can break out, multiply, cause additional

damage to the udder secretory tissue and promote further formation of scar

tissue.

The interaction of bacteria with milk leukocytes, particularly neutrophilic

leukocytes greatly influences the establishment of intramammay infections. The

phagocytosis is insufficient to cure infections in the udder due to lack of energy

source, low opsonic activity, and interference caused by casein and butter fat

(Nickerson and Owens, 1994). In addition, some staphylococci survive inside

neutrophils after they have been phagocytosed, and most antibiotics can not

penetrate the neutrophil cell membrane to contact intracellular bacteria. Bacteria

later escape from the cell upon lysis, and multiply within the gland as the

concentration of antibiotic decreases.

2.4.2. L-form of bacteria

Cell wall defects in S. aureus and other Gram-positive organisms have

been reported in bovine mastitis and other infections of unstable L-form,

protoplast phase, spheroplast phase, transitional phase or stable L-forms (Sear et

al., 1987). These may be associated with the use of such antibiotics as penicillins

and cephalosporins which kill bacteria by preventing synthesis of new cell walls

as new bacterial cells are being formed. The induction of an L-form phase or

spheroplast of bacteria might follow routine treatment. These forms might

survive and re-establish an infection after reduction in the concentration of

antibiotics in the gland (Sears et al., 1987).


33

2.4.3. Development of resistance to antimicrobials

Bovine mastitis is the major cause of illegal antibacterial residues in

marketed milk (Erskine, 1996). Antibacterial therapy of mastitis has been

incriminated as a catalyst for developing resistance in pathogenic bacteria both in

treated and healthy individuals within a herd (Berghash et al., 1983; Griggs et al.,

1994; Livermore, 2000; Teuber, 2001).

Staphylococcus aureus is the most important mastitis pathogen against

which antimicrobial resistance has been studied (Jones and Heath, 1985; Watts

and Salmon, 1997) i.e., resistance against penicillin, ampicillin, methicillin and

vancomycin (Livermore, 2000; Hiramatsu et al., 2001). An increased resistance

from 38% to 80% was observed in S. aureus mastitis isolates by Devriese et al.

(1997).

McDonald et al. (1976) found no resistance against the strains of Str.

agalactiae. However, Berghash et al. (1983) reiterated that some strains of Str.

agalactiae showed resistance to β-lactam antibiotics. Both selective pressure due

to antimicrobial use and resistance genes in bacteria like S. aureus and E. coli

strains mediate the development of antimicrobial resistance.

In subsistence economies such as developing countries, resistance

frequently occurs in microorganisms having the ability to cause disease in

healthy individuals. As a result of antimicrobial resistance, patients infected with

resistant organisms do not improve in response to conventional chemotherapy


34

unless alternative treatment options are executed. This problem of antibiotic

resistance has also been noticed in many developed countries (EARSS, 2003).

2.4.4. Drug factors

The degree of transfer of drugs from blood into milk is directly

proportional to the concentration gradient across the membrane and inversely

proportional to the extent to which the drug in ionized, since ionized molecules

pass through biological membranes poorly. Effective passage of drugs from

blood to the udder is best achieved with the macrolide and lincosamides

antibiotics (erythromycin, lincomycin, spiromycin, and tylosin) due to their high

lipid solubility. These drugs become trapped in the milk phase due to ionization

but their antibacterial spectra are limited to Gram-positive pathogens.

Selection of an effective drug and maintaining adequate antibiotic

concentration of that drug at the site of infection is considered to be one of the

prerequisites for effective antimicrobial therapy. Aminoglycosides, such as,

streptomycin, neomycin, kenamycin, and gentamicin have extremely poor

distribution due to low lipid solubility. Antibiotic concentration, as measured in

the milk phase may not be relevant for S. aureus which is an invasive organism

and may be present intracellularly as well as extracellularly (Sandholm et al.,

1990). Mastitis organisms like S. aureus, which cause udder tissue necrosis

leading to a poor blood supply of the affected areas and consequently a

decreased redox potential favour anaerobic mastitogenic bacteria (du Preez,

1988). There is no effective passage of drugs into necrotic areas. In all cases of


35

mastitis, oedema and inflammatory products to a certain extent, obstruct the

diffusion of antibiotics by compression or blockage of the milk duct system. The

diffusion of antibiotic solutions throughout the gland is impaired and for this

reasons, it is often very difficult to bring antibiotics into contact with mastitis

causing bacteria, especially with intramammary therapy alone.

2.4.5. Treatment factors

In staphylococcal mastitis and in some streptococcal mastitis, therapy

usually results in a very low bacteriologic cure rate (du Preez, 1988). One of the

main problems in the selection of antibiotic for mastitis therapy is that in vitro

susceptibility tests carried out either by minimum inhibitory concentration (MIC)

determination or by Kirby-Bauer radial agar diffusion method (Sandholm et al.,

1990), show extremely poor correlation with the outcome of the therapy and so

without knowledge of pharmacokinetic behavior of the drugs, the concentration

at the site of infection remains unknown.

Tissue lining the teat duct is very delicate and any unnatural

manipulation of this structure, such as cannula insertion or intamammary

infusion, may jeopardize its antibacterial functions. Although, a mastitic quarter

is treated with antibiotics, trauma predisposes the quarters to infection or re-

infection. Secondary pathogens (e.g., fungi, yeasts), may be introduced into the

udder when, for example, a contaminated cannula is inserted into the teat with

intramammary drug infusion where teats are not thoroughly cleaned and

cannula sterilized before insertion. No or minimal non-antimicrobial supportive


36

treatment when warranted (e.g., in shock) in peracute coliform mastitis, is one of

the causes of failures. Other treatment factors may involve delayed treatment,

inappropriate selection of antibacterial drugs and stopping treatment too soon.

2.5. POSSIBLE SOLUTIONS TO THERAPEUTIC FAILURE IN MASTITIS TREATMENT

2.5.1. Selection of appropriate antimicrobials

The 5- point plan for mastitis control include (i) teat dipping, (ii) dry cow

therapy, (iii) prompt treatment of clinical cases (iv) proper milking hygiene/use

of adequately functioning milking machine; and (v) culling chronically infected

animals (Nickerson and Owens, 1990). Two of these points involve the use of

antimicrobial therapy to eliminate and/or to prevent udder infections. Thus

antimicrobials play a major role in attempts to control the incidence of

intramammary infections. The goal of mastitis therapy is to maintain

concentrations of antimicrobials at or above the MIC for the offending pathogen

at the site of infections for sufficient time periods to eliminate the pathogens. The

ultimate purpose is to allow rapid and complete return of the gland to normal

function and milk production.

The extent of penetration of antimicrobial into milk from the systemic

circulation is largely dependent upon the properties of the drug molecules i.e.

lipid solubility, degree of ionization and extent of protein binding in the serum

and udder. Most drugs are weak organic acid and bases with dissociation

constant (pKa value) 3-10 and exist in various proportions of ionized and


37

unionized molecules. The antibiotics, which are weak organic acid, tend to

accumulate to limited extent in normal milk. However, in mastitis, with its

increase in milk pH, concentration in milk may approach those in plasma

(Anderson, 1988). The additional factors like the ability of divalent cations i.e.,

calcium to bind and inactivate the drugs such as tetracyclines also affects the

potency of the drugs.

Anderson (1988) described the principles of clinical mastitis therapy as

follows: (a) Early detection/treatment of clinical cases, (b) frequent milking out

to remove bacteria and toxins, (c) appropriate use of antibiotics susceptibility

data, (d) for intramammary therapy preference to drugs which are absorbed and

well distributed to the mammary tissue and should be used at least 2-4 times at

12-24 hours intervals, (e) for systemic therapy, preference should be given to

those drugs which are distributed to the mammary glands to a sufficient degree,

(f) adequate dose and duration of treatment (g) one should consider the

interaction with intramammary infused drugs, and (h) effect on mammary

phagocytes.

2.5.2. Combination Therapy

Until recently, the recommendation has been to treat mastitis without

systemic reaction by intramammary infusions only. Parenteral treatment was

recommended in cases showing systemic reactions. Combination of antibiotics

(especially triples) have been found effective against strains of S. aureus, which

were highly resistant to the individual antibiotic (Rodriguez et al., 1990). The


38

combination therapy i.e., systemic injection coupled with intramammary

infusion over several days may achieve greater drug concentrations for sufficient

period of time and be more effective than conventional intramammary therapy

or systemic therapy alone (Soback et al., 1989). The combination of

intramammary (62.5 mg amoxycillin for 6 consecutive milkings) and

intramuscular (9 x 106 units procaine penicillin G for 3 days) cured 51% of

chronic S. aureus quarters compared with 25% of quarters treated with

intramammary infusion alone (Owens et al., 1988).

It is believed that infused antibiotics could contact bacteria growing in the

cisternal areas and large ducts, and systemic antibiotic could function

synergistically with infused drugs by combating bacteria residing deeper in

gland. A combination therapy by ceftiofur (a third generation cephalosporin) 100

mg infused and 500 mg injected markedly reduced S. aureus numbers in milk and

tissues. In addition, the use of combination therapy resulted in higher percentage

of alveolar luminal areas and lower percentage of inter-alveolar stromal area

compared with treatment by infusion or injection alone, suggesting that quarters

exposed to higher concentrations of antibiotics (combined therapy) regained

secretory activity at a greater rate than those treated by injection or infusion

alone (Nickerson and Owens, 1994).

2.5.3. Drugs against β-Lactamases

There are many different β-lactamases, some mainly directed against

penicillins and some against cephalosporins. Clavulanic acid (a component


39

produced by Streptomyces clavuligerus) is predominantly active against

penicillinases and these are predominant type of β-lactamases produced by

amoxycillin resistant staphylococci, Escherichia coli, and Salmonella.

AugmentinnTM (Beecham, England) is the formulation of amoxycillin plus

potassium clavulanate (in a 4:1 ratio). Amoxycillin is selected to accompany

clavulanic acid because of its inherent broad spectrum of activity, its good

absorption and its rapid bacterial action. The two compounds have a similar half

life in bovine (amoxycillin 1.5 hour; clavulanic acid 1.2 hour after intravenous

injection) and are protein bound to a similar extent 14 and 22%, respectively

(Marshal et al., 1982).

2.5.4. Use of Liposomal Drugs

Liposomal drugs are useful in the treatment of infectious diseases caused

by bacteria like S. aureus, Mycobacterium and Brucellae which are not only able to

resist killing by phagocytic cells but are also protected from drugs by their

intracellular location. Liposomes which are readily phagocytosed by

macrophages and neutrophils, are the potential drug carriers and administration

of liposomally entrapped drugs provides a means of delivering to the site of

inbtection those antimicrobial drug that penetrate cells poorly directly to the site

of infection. Liposomal entrapment may be useful in the treatment of bovine

mastitis with those drugs which penetrate phagocytic cells poorly but which

have high intraphagocytic antimicrobial activity (Yancey et al. 1991). A liposomal


40

suspension of gentamicin is available for veterinary use in Pakistan under name

of PlasmacolinTM (Cenavisa Lab., France).

2.5.5. Use of immunoactivators

Levamisole is an anthelmintic drug, which also possesses

immunomodulatory properties. In the light of present understanding of the

pathogenesis of mastitis, it is thought that the prospect of using levamisole

beneficially for mastitis treatment is limited. However, drying off and parturition

are two critical times at which levamisole may have rational application in

mastitis prevention. The use of levamisole per os reduced somatic cell count and

number of intramammary pathogens and was also found to restore the depleted

B-lymphocytes in mastitis milk (Nickerson and Owens, 1994). An

immunostimulant containing Propionibacterium acnes was found to decrease the

number of colony forming units shed from S. aureus infected quarters but had no

effect on somatic cell counts or cure rate (Nickerson et al., 1994).

2.5.6. Use of vitamin E and Selenium

The nutrition programme of a dairy herd is believed to exert a major

influence on cow’s productivity and health. Serum selenium (Se) concentration in

healthy cows falls in the range of from 0.05 to 0.40 mg of Se per liter of serum

(Gyang et al., 1984). The Se-vitamin E injection improves the ability of

polymorphonuclear leukocytes (PMNLs) to phagocytose the bacteria. The

function of Se lies in its involvement with glutathione peroxidase (GSH-PX). As

part of this enzyme, Se is required to remove hydrogen peroxide (H2O2) by


41

oxidation of glutathione. The inability of PMNLs from selenium deficient

animals to kill phagocytized microorganisms may be a result of damage to

neutrophil lipids and proteins by toxic peroxide normally destroyed by a

mechanism associated with GSH-PX activity. Selenium supplementation plays a

role to increase the amounts of immunoglobulins. Killing of engulfed bacteria by

PMNLs involves a multiplicity of mechanisms that set into motion the

deregulation process and initiation of respiratory burst, which results in the

production of anti bacterial agents like H2O2 and superoxides. Production of

these compounds is dependent upon the availability of dihydronicotinamide

adenine dinucleotide phosphate (NADP+), which is supplied by oxygen

formation reaction and GSH-PX reductase system (Gyang et al., 1984). As per

Nickerson (1990), researchers at the Ohio State University (USA) experimented

by supplementation the diets of heifers with vitamin E (50-100 ppm) and

selenium (Vetamix®) (0.30 ppm) sixty days pre partum and throughout lactation

and achieved reductions of staphylococcal and coliform infection at calving by

42%.

2.5.7. Use of enzymes to promote diffusion of antibacterials

The use of enzymes like hyaluronidase, tryspin, chemotrypsin,

streptokinase, or streptodernase (Varidose®, Lederle) to digest pus, is very

valuable adjunct to treatment in certain cases to promote the diffusion of

antibiotics. Administration of antibiotics in combination with enzymes has been

shown to give higher bacteriologic cure rates than those obtained with the use of


42

antibiotics alone. Muhammad and coworkers (2000) at University of Agriculture

Faisalabad (Pakistan) instilled TenazymeTM (Veyx Pharma, D-34639

Schwarzenborn, Germany; Each intramammary tube of 10ml contains 30mg

tetracycline hydrochloride, 22.5mg neomycin sulphate, 2mg prednisolone 21-

acetate, 0.4mg trypsin, 0.4mg chymotrypsin, 1mg papain per ml) by

intramammary route to two Nili-Rawi buffaloes and one indigenous cow

affected with mastitis with fibrosis in the teats. The treatment was repeated daily

for six consecutive days. No mastitis causing organism was present in milk

samples taken on day 14 and 21 post-treatment. A noticeable diminution of

fibrosis and an increase in quarters yield was recorded.

2.5.8. Other ancilliary agents

The use of pitocin (oxytocin) to produce a complete let down of milk, and

assisting to flush out inflammatory debris, is an assistance to precede stripping

and then intramammary infusion. Arazyme is an exocellular metalloprotease

isolated from Serratia proteamaculans culture medium. The preliminary work of

Bersanetti and coworkers (2005) indicated a favourable effect of this bacterial

protease in bovine mastits. Power-cellR is a proprietary probiotic preparation

(Insect Biotech Co. Ltd. Daejon, Korea) recently introduced into the Pakistani

market for the prevention of mastitis and reduction of somatic cell counts.


43

2.5.9. Role of biotechnology in improving the cure rates in mastitis therapy

2.5.9.1. Endolysins as a novel class of antibacterial agents

According to Borysowski et al. (2006), endolysins (also termed lysins) are

double strandard DNA bacteriophage-encoded enzymes produced during the

late phase of gene expression in the lytic cycle to degrade peptidoglycan, (the

main constituent of the bacterial cell wall) thereby enabling progeny virions to be

liberated. The capability of lysins to digest the cell wall (especially in Gram-

positive bacteria) when applied exogenously (as recombinant proteins) to

bacterial cells has enabled their use as alternative antibacterials. Because of their

unique ability to cleave peptidoglycan in a nearly species-specific manner,

endolysins represent a novel class of antibacterial agents and provide a means of

selective and rapid killing of pathogenic bacteria with no effect on the normal

microflora.

The feasibility of using endolysins as antibacterial agents arises from the

fact that they display muralytic activity (especially to Gram-positive bacteria).As

per Borysowski et al. (2006), capability of a partially purified lysin to kill bacteria

was first reported back in 1959. However, it was not until 2001 that it was

demonstrated that purified recombinant endolysins may constitute highly

effective topical antibacterial agents. The most likely reason for this delay was

the fact that earlier antibiotic resistance was not a problem serious enough to

compel the development of alternative antibacterial agents. Several basic


44

applications have been reported for endolysins, including: (i) the elimination of

bacterial colonization of mucous membranes, (ii) the treatment of bacterial

infections, (iii) the biocontrol of bacteria in food and feed, and (iv) the protection

of plants against phytopathogenic bacteria.

As is evident from narration in previous section of this chapter, treatment

of mastitis has historically been limited to the use of broad-range antibiotics such

as tetracycline, penicillin, and pirlimycin, which are often < 50% successful

(Deluyker et al., 2005). In the quest for more specific agents, alternative

antimicrobials such as bacteriophages and phage endolysins are receiving more

interest. Donovan et al. (2006) produced two novel antimicrobial from

bacteriophage. The (i) full-length and (ii) 182-aminoacid, C-terminally truncated

Str. agalactiae bacteriophage B30 endolysins were fused to the mature lysostaphin

protein of Staphylococcus simulans. Both fusions display lytic specificity for

streptococcal pathogens and S. aureus. The full lytic ability of the truncated B30

protein also suggests that the SH3b domain at the C terminus is dispensable. The

fusions are active in a milk-like environment. They are also active against some

lactic acid bacteria used to make cheese and yogurt, but their lytic activity is

destroyed by pasteurization (63˚C for 30 min). Imunohistochemical studies

indicated that the fusion proteins can be expressed in cultured mammalian cells

with no obvious deleterious effects on the cells, making it a strong candidate for

use in future transgenic mice and cattle.


45

2.5.9.2. Lysostaphin

Bramely and Dodd (1984) at the Institute of Animal Health in Compton,

England attempted to link a cloned lysostaphin gene to beta lactoglobulin, a

protein normally found in milk. Lysostaphin is a cell wall degrading enzyme

secreted by certain Staphylococcus species (S. simulins and S. staphylolyticus). It

cleaves the peptidoglycan of the staphylococcal cell wall at pentaglycine cross

links; death results from rapture of the bacterial spheroplast (Blackburn, 1991).

The lysostaphin gene has been successfully cloned in Bacillus subtilis and

Escherichia coli resulting in the production of recombinant lysostaphin (Ambicin-

LR) which has been shown to be effective in vivo in mouse and guinea pig against

experimental S. aureus mastitis indicating both prophylactic and therapeutic

potential (Nickerson and Owens, 1994). Lysostaphin is synergistic with nisin,

another bacteriocin (Oldham and Daley, 1991).

2.5.9.3. Interleukin-1

Interleukin-1 (IL-1) is a term defining a group of low molecular weight

(~17 kd) glycoprotein cytokines produced during inflammatory responses by

many types of cells. IL-1 is chemotatic for monocytes and lymphocytes, promotes

adherence of neutrophils, monocytes and lymphocytes to endothelial cells, and

activates T- and B-lymphocytes.

The numerous biological activities of IL-1 make it a logical candidate for

studies on bovine mastitis. A vaccine using capsular antigen of S. aureus and a

vaccine developed from a mutant strain of E. coli in conjunction with


46

recombinant bovine IL-1 as an adjunct, may prove more effective in controlling

mastitis.

2.5.9.4. Interleukin-2

Interleukin-2 (IL-2) often described as T-cell growth factor, is a

lymphokine which plays a crucial role in regulation of T-and B-cells response to

antigens. It is produced by a subset of T-helper cells in response to mutagenic or

antigenic stimulation. Bovine IL-2 has been recombinantly derived from yeast

and E. coli (Oliver et al., 1990). Nickerson et al. (1994) demonstrated that

recombinant bovine IL-2 administered intramammarily via slow release

miniosmotic pumps resulted in an enhanced cellular response and found

increased percentages of macrophages and lymphocytes in treated quarters,

while neutrophil percentages decreased. There is increase of IgG1 and IgG2. The

data indicated that intramammary treatment with bovine IL-2 stimulates a

cellular response and antibodies synthesis, and so has both therapeutic and

prophylactic action against bovine mastitis. The use of rbIL-2 as an adjunct to

antibiotics to treat subclinical mastitis during lactation improved the

bacteriological cure rates of experimentally induced S. aureus IMI by 20 to 30%

over use of antibiotic therapy alone (Daley et al., 1991). Another study (Daley et

al., 1993) reported that mammary glands infused with rbIL-2 were two to five

fold more resistant to intramammary challenge from S. aureus than were

untreated controls. More recently, Ohio State University (USA) Workers (Hogan


47

et al., 1995) showed that intramammary infusion of rbIL-2 was not effective as an

adjunct to dry period antibiotic therapy in three commercial dairy herds.

2.5.9.5. Interferons

Interferon α is produced by almost all cells in response to stimulation by

virus, bacteria or bacterial product, and foreign nucleic acid while interferon β is

a T-lymphocyte product which can be stimulated by mitogen, antigen or IL-2

(Oliver et al., 1990). Biological activities of interferon include suppression of cell

proliferation, regulation of antibody production, and regulation of macrophage

differentiation and phagocytic capabilities. Mammary gland neutrophils are

considered to be a major line of defense once bacteria breach the teat canal

(Paape et al, 1981). However, it is well documented that mammary gland

neutrophils have lower phagocytic and bacterial activities than peripheral blood

leukocytes (Weber et al., 1983). The diminished antibacterial capacity of

mammary gland neutriphils is attributable to several factors. Indiscriminate

ingestion of milk constituents, degranulation, and lower glucose metabolism has

been correlated with compromised neutrophil activity (Paape et al., 1981; Weber

et al., 1983). Elevated levels of glucocorticoids associated with parturition and the

onset of copious milk production can contribute to lower chemotactic responses,

inhibit phagocytosis, and intraleukocytic killing of phagocytosed bacteria by

neutrophils during the immediate post partum period (Nagahata et al., 1988).

Recombinant bovine interferon gamma can partially overcome inhibitory effects

of mammary secretion on bovine mammary neutrophil phagocytic and


48

bactericidal capabilities (Sardillo et al, 1991). This cytokine has been found to

have a beneficial effect in the treatment of E. coli mastitis and this effect may be

mediated through reduced local and systemic concentration of tissue necrosis

factor (Paape et al., 1985).

2.5.9.6. Bovine Somatotropin

Bovine somatotropin (BST) possesses immunomodulatory properties.

Studies have shown that somatotropin reversed thymic aging and augmented IL-

2 synthesis in old rats, enhanced immune cell function of hypophysectomized

rats, and enhanced production of super oxide anion from porcine blood

mononuclear cells in vitro (Edward, 1988). Short term (10 days) treatments with

BST in combination with an antibiotic therapy has been shown to have a

beneficial effect upon the recovery of yield and composition of milk in non-

infected mammary gland during experimentally induced E. coli mastitis and in

infected glands (Messom et al., 1988). Burvenich et al. (1989) concluded that

elevated blood levels of BST might favour proliferation and generation of

neutrophil population that is capable of enhancing the oxidative response.

2.5.9.7. Colony Stimulating Factors

Colony stimulating factors (CSF) comprise a family of proteins which

stimulate differentiation of immature haemopoietic cells and stimulate functions

of mature cells. The use of granulocyte-CSF (G-CSF) and granulocyte

macrophage-CSF (GM-CSF) increases the numbers of neutrophils and

macrophages in controlling mastitis (Oliver et al., 1990).


49

2.5.10. The role of Mucolytic/Secretolytic Agents

Mucolytic/secretolytic agents such as bromhexin (Bisolvon®;

Mucosolvon® @ 0.6-1.2 mg kg) when co-administered intramuscularly with

antibiotics to lactating ruminants have been shown to result in the significant

elevation of serum and milk antibiotic levels (Ziv, 1988). The use of these agents

in mastitis treatment could be expected to improve the cure rate by dissolving

the fibrin threads present in the mastitis secretion and thus facilitate the diffusion

of antibiotics into the inflamed areas.

2.5.11. Change of pH

Infection of mammary glands tends to raise the pH of milk towards

alkaline side (Radostits et al., 2007) and there are many antibiotics, which cannot

work properly in such conditions. If the pH is lowered by using different

acidifying compounds like ammonium chloride, orally or sodium acid phosphate

IV, the diffusion of drugs into udder may be facilitated which can improve

microbiological as well as clinical cure rate. Although, antibiotics have played a

major role in dry cow therapy and clinical cure during lactation, there is a need

to improve their effectiveness. Future treatment regimens should be geared

towards maintaining higher drug levels in milk and mammary tissues for a

sufficient period of time to successfully eliminate infection and reduce somatic

cell count.

Dhillon et al. (1989) successfully treated 10 Zebu crossbred cows and 10

buffaloes by administering trisodium citrate (12gm in 250 ml water, PO) to each


50

animal once daily till recovery. In another experiment, Dhillon et al. (1995)

administered tri-sodium citrate (12 gm in 250 mL water/ animal, PO) to 6

buffaloes. This brought about cure in these cases. There was decrease in bacterial

count, milk pH dropped to normal (6.5) and citric acid was replenished. A

similar study was conducted by Singh et al. (1997) on 10 crossbred cows suffering

from mastitis. Mastitic milk consistency was abnormal with high pH and low

percentage of citrate, total proteins, lactose and fat compared to normal milk.

Following treatment with trisodium citrate, milk consistency, pH and percentage

of above constituents was restored to nearly normal.

2.5.12. Therapeutic cessation of lactation

Middleton and Fox (2001) compared the efficacies of povidone-iodine

(5%) and chlorhexadine (71%) in therapeutic cessation of lactation in 14 Holstein-

Friesian cows in the Washington State University dairy herd. Following

experimentally induced S. aureus mastitis in two groups of cows after complete

milk out 24 hours apart, one group was treated with povidone-iodine (5% in

120mL), while other group was treated with chlorhexadine (71%) through

intramammary route. Povidone-iodine caused permanent cessation of lactation,

while in chlorhexadine treated group, cows returned to function in the

subsequent lactation. Hence, if the primary objective is to eliminate mammary

quarters from lactation and thereby presumably lower the risk of herdmates

acquiring new S. aureus IMI then povidone-iodine appears to be the better of two

these methods.


51

2.5.13. Udder lavage as a mastitis treatment

Udder lavage refers to infusing a large volume of a weak antiseptic

solution into the quarter and then withdrawing it. For this purpose, acriflavine

solution (1:10000 in boiled water) is generally employed. The treatment may be

applied to lactating or dry animals. In lactating animals, the infected quarters are

completely milked out, the solution infused until the quarter is fairly tense (a

pint i.e. approximately 480 mL or so of fluid being required) and after 5 minutes

the chemical is then withdrawn. The treatment should be repeated two weeks

later. In animals to be dried, the fluid may be allowed to remain in the infused

quarters for 24 hours. By this method, permanent cures have been reported in 60

or 70 % of suitable cases. Advanced cases with much udder induration should

not be treated, and in any case some quarters resist sterilization even on repeated

infusions (Shirlaw, 1949). The affected quarter can also be lavaged with edentate

trisodium (EDTA-tris) and then infused with a suitable antibacterial (Wooley,

1983).

2.5.14. Use of vaccines in the treatment of mastitis

Despite the implementation of standard mastitis control programs,

mastitis is still economically the most important disease of dairy animals even in

technologically advanced countries. In a bid to improve the level of mastitis

control, several attempts have been made to develop and evaluate mono and

polyvalent vaccines against the most prevalent mastitis pathogens like S. aureus,

Str. agalactiae, E. coli and Str. uberis. In general, commercially available S. aureus


52

vaccines have a limited to moderate ability to prevent new infections. On the

other hand, it is fairly well established that these vaccines enhance the

spontaneous cure rates and are effective in reduction of chronic infections

(Widel, 1994).

Israeli workers (Leitner et al., 2004) reported that 6 (30%) of the 20 S. aureus

infected cows when vaccinated with a commercial S. aureus vaccine (Mastivac-1R,

Vireo Biological Lab, Israel) remained infection free till the end of 348-day trial

compared with 1 (6.25%) out of 14 cows non-vaccinated. These findings suggest a

curative role of this vaccine. Mastitis vaccines intended for treatment should be

able to raise antibody titers in the shortest possible period and this lag period is

often a function of the adjuvant. Sear (2002) cited a US study that compared an

oil-based adjuvant with aluminium hydroxide Al(OH)3 adjuvant in a mastitis

vaccination-treatment protocol. Cure rates for the oil-based vaccine was far lower

(25%) as compared to that noted with Al(OH)3 vaccine (62%). Although, Al(OH)3

adjuvanted vaccine does not produce a prolonged antibody response or protect

as well against new intramammary infections, it produces a more rapid antibody

response that makes it a better choice for the treatment. A recent study (Athar,

2007) conducted at Department of Clinical Medicine & Surgery, University of

Agriculture, Faisalabad (Pakistan) demonstrated that locally prepared polyvalent

bacterin-toxoid mastitis vaccines (containing killed S. aureus, Str. agalactiae, and

E. coli with various adjuvants) not only prevented new infections but also cured

existing infections of these organisms in dairy buffaloes. Similar observations


53

were made with locally prepared S. aureus vaccines (live attenuated vaccine,

plain bacterin, dextran sulphate adjuvanted bacterin and oil-adjuvanted bacterin)

(Muhammad, 2004).

Therapeutic efficacy of autogenous S. aureus vaccines has been reported in

some studies. Korean workers (Hwang et al., 2000) investigated therapeutic

efficacy of an autogenous S. aureus toxoid-bacterin in lactating cows suffering

from subclinical S. aureus mastitis. Twenty two cows which had at least one S.

aureus infected quarter were selected. Eleven cows were injected with their own

autogenous toxoid-bacterin and the other 11 were maintained as non-injected

control. In the toxoid-bacterin group, 27% of infected quarters were cured during

the 12 weeks long trial compared to 5% in the control. New intramammary

infections with S. aureus were only detected in 3 quarters of the control group.

Analysis of the data showed that autogenous toxoid-bacterin treatment against S.

aureus subclinical mastitis in lactating cows may increase the cure rate of existing

infections, reduce their severity and also prevent occurrence of the new

infections.

In order to improve the cure rate of antibiotic therapy, protocols based on

the use of antibiotic + vaccine have also been evaluated in the treatment of S.

aureus mastitis. In 4 herds, of 61 cows infected in 131 quarters, 42 cows received

the trivalent vaccine and 37 cows were vaccinated with autogenous + Smith

strain. Each cow was vaccinated twice before the initial treatment and again 7

days after second vaccination. Each infected quarter was treated with 6


54

intramammary infusions of pirlimycin at 24 hours intervals. There was no

significant difference between the two vaccines with 76% quarters cured for the

trivalent vaccine and 67% cured for the autogenous + Smith strain. In the cows

treated with antibiotic only, S. aureus was eliminated from 22% of the quarters. In

18 cows that were vaccinated but were not treated, all of the quarters remained

infected (Sears, 2002).

To make an effective use of a mastitis vaccine in treatment of a mastitic

animal, it is important to determine the nature of mastitis pathogen. If this is not

known and some organism other than the one contained in the vaccine is the

cause of mastitis, vaccine obviously would not have any curative value.

Nonetheless, it may work as a prophylactic agent. It is interesting to note that

most S. aureus vaccines developed for the prevention of infection act as better

curative than preventative agents.

2.6 USE OF NON-ANTIBIOTIC ANTIBACTERIALS IN THE TREATMENT OF HUMAN AND ANIMAL BACTERIAL DISEASES A review of literature, coupled with a more recent investigations, suggests

that some of non–antibiotics and other membrane active compounds enhance the

activity of conventional antibiotics, and eliminate natural resistance to specific

antibiotics (Kristiansen and Amaral, 1997).

There is evidence that certain non-antibiotics compounds alone or in

combination with conventional antibiotics, may play a useful role in the

management of specific bacterial infections associated with high risk of


55

resistance to conventional antibiotics (Kristiansen, 1990; Kristiansen, 1993;

Schmidt and Rosenkranz, 1970).

2.6.1 Synergy between antibiotics and non-antibiotic antibacterials

Synergy between conventional antibiotics and non-antibiotics has been

suggested and supported by many investigators, (Kristiansen, 1990; Amaral et al.

1992; Molnar et al. 1990; Manion et al. 1969). The compounds that have produced

such synergy against a wide range of bacterial species are phenothiazines

(Chlorpromazine, methidilazine etc) and tricyclic anti depressant, (Kristiansen,

1990; Amaral et al. 1992; Manion et al. 1969; Molnar et al. 1990; Chattopadhyay et

al., 1988; Heller and Sevag, 1966) and also membrane stabilizing compounds

employed in the management of psychosis, pain etc. (Kristiansen, 1991;

Domenico et al., 1989; Parry and Neu, 1977; Ehrlich, 1913; Roberts and Cole,

1981).

The synergy produced by chlorpromazine in combination with

aminoglycosides, β-lactams, quinolones etc. has been generally limited to

bacteria that are sensitive either to antibiotics or to chlorpromazine (Amaral et al.,

1992). Kolaczkowski et al. (2003) reported that phenothiozine were synergistic

with the antifungal ketoconazole at micromolar concentrations.

As stated in the foregoing, drugs not designed as antibiotics, and whose

primary mode of action is modulation of active and passive ionic transport

mechanisms in the eukaryotic cell, also act on prokaryotic cell-walls, and the

action is antimicrobial. The drugs may be classified as non antibiotics (Bender


56

and Kristiansen, 1999). Anesthetic gases are bactericidal in the fluid state, and in

the vaporous state at high concentrations. Local anesthetics of the ester type have

stronger antimicrobial actions than the amide type, and synergy is found

between local anesthetics and antibiotics. Barbiturates show antimicrobial action

at high concentrations, and there is a possible synergy with antibiotics. Synthetic

analogs of morphine have stronger antimicrobial action than the natural

derivatives. Aspirin (Acetyl salicylic acid) inhibits the growth of Kelebsiella

pneumoniae at concentrations within the range of that in plasma in normal clinical

usage; but induces non genetical resistance to antibiotics (Bender and

Kristiansen, 1999).

The emergence of multiple antibiotic resistant organisms in the general

community is a potentially serious threat to public health. The emergence of

antibiotic resistance has not yet prompted a radical revision of antibiotic

utilization. Instead, it has prompted the development of additional antibiotics.

Unfortunately, this does not relieve the underlying selection pressure that drives

the development of resistance. A paradigm shift in the treatment of infectious

disease is necessary to prevent antibiotics becoming obsolete and, where

appropriate, alternatives to antibiotics ought to be considered. There are already

several non-antibiotic approaches to the treatment and prevention of infections

including probiotics, phages and phytomedicines. There is some evidence that

probiotics such as Lactobacillus spp. or Saccharomyces boulardii are useful in the

prevention and treatment of diarrhoea, including Clostridium difficile (a clostridial


57

species that is part of normal flora of colon in infants and sometimes in adult

human beings). The organism causes pseudo membranous entrocolitis in

patients receiving antibiotics associated diarrhea that can be difficult to treat and

recurs frequently. Bacteriophages and their enzymes have received renewed

attention for the control of both staphylococcal and gastrointestinal infections.

Phytomedicines that have been utilized in the treatment of infections includes

artesunate for malaria, tea tree oil for skin infections, honey for wound

infections, gum for Helicobacter pylori gastric ulcers and cranberry juice for

urinary tract infections. Many infections may prove amenable to safe and

effective treatment with non-antibiotics (Carson and Riley, 2003).

The antimicrobial properties of compounds such as phenothiazines, as

well as those of other neurotropic compounds, have only been investigated

sporadically and their application to management of microbial infections has not

been evaluated. Literature suggests that some of those and other membrane

active compounds enhance the activity of conventional antibiotics, eliminate

natural resistance to specific antibiotics (Hendricks et al., 2004) and exhibit strong

activity against multidrug resistant forms of Mycobacterium tuberculosis. Thus

non-antibiotics may have a significant role in the management of certain bacterial

infections (Kristiansen and Amaral, 1997).

Increasing problem of bacterial resistance to common antibiotics might

render non-antibiotics subject to development into antibiotics, and to be utilized

in combination for the treatment of resistant infectious diseases (Bender and


58

Kirstiansen, 1999). Hendricks and his associates (2004) reported that combination

of Thioridazin with erthromycin, oxacillin and ampicillin significantly reduced

the minimum inhibitory concentrations (MICs) of these antibiotics against

Streptococcus pyogenes, S. epidermidis, methicillin resistant S. aureus (MRSA) and

Enterococcus faecalis. A potentiating effect of chlorpromazine on kanamycin,

amikacin and tobramycin against 2 multiresist Escherichia coli strains was recently

reported by Brazilian workers (Coutinho et al., 2008). Published information on a

few selected non-antibiotic antibacterials (chlorpromozine, lidocaine, DMSO,

povidone-iodine etc.) is briefly reviewed in the ensuing subsections.

2.7 CHLORPROMAZINE

Methylene blue, a tricyclic compound is the nucleus of a group of derived

compounds, which together constitute the phenothiazines. Ehrlich (1913) used

methylene blue in dilute concentrations to render motile bacteria and protozoa

immobile. Methylene blue was used to manage urinary tract infections (De Eds et

al., 1939) and parasitic infection of sheep (Harwood, 1946). Charpentier et al.

(1952) developed neuroleptic drug from phenothiazine, called chlorpromazine

(CPZ).

Chlorpromazine and other phenothiazines were found to have activity

against Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium

smegmatis (Levaditi et al.,1951; Bourden, 1961; Molnar et al.,1977; Amaral et al.,

1996; Viveiros et al., 2005; Amaral et al., 2001; Amaral et al., 2004; Amaral et al.,

2007; Martins et al., 2007; Rodrigues et al., 2008) and also against gram negative


59

and gram positive bacteria, (Kristiansen and Mortensen, 1987; Kristiansen and

Amaral, 1997; Kristiansen et al., 2007; Coutinho et al., 2008; Martin et al., 2008;

Bailey et al., 2008), against a wide range of parasitic organisms such as amoeba

(Josefsson et al., 1975; Ockert 1984; Schuster and Mandel, 1984; Schuster and

Visvesvara, 1998), Plasmodia, (Kristiansen and Jepsen, 1988; Satayavivad et

al.,1987; Ohnishi et al.,1989; Tanabe et al., 1989; Basco and LeBras ,1992; Miki et

al.,1992; Oduola et al.,1998; Banerjee et al.,1999; Amaral et al., 2001), Trichomonas

(Dragan et al.,1989), Chlamydomonas. (Schuring et al., 1990), trichinella (Stewart

et al., 1985), trypanosomes (Loiseau et al.,1996; Chan et al., 1998), toxoplasma

(Pezzella et al., 1997), and many other parasites. Although, antibacterial activity

of chlorpromazine including the one against Mycrobactrium tuberculosis has been

known for many decades, this drug has not been considered for the use in the

management of pulmonary tuberculosis due to the severe side effects associated

with its chronic use. In addition, in vitro activity of this compound takes place at

concentration that are well beyond those that can be safely achieved in human

patient (Martins et al., 2008). Ordway et al. (2003) reported that clinical

concentration of thioridazine kill intracellular multidrug-resistant Mycrobactrium

tuberculosis.

Several mechanisms of drug resistance have been discovered. Of these,

active efflux of antifungal, antibacterial and anticancer drugs by broad-specificity

multidrug resistance (MDR) transporters are major obstacles to successful

chemotherapy of infectious diseases and cancer. Kolaczkowski et al. (2003)


60

evaluated the growth inhibitory and MDR modulatory effect of a series of 36

phenothiazines and related compounds, against Saccharomyces cerevisiae strains

exhibiting different levels of expression of MDR transporters Pdr5p, Snq2p and

Yor1p. Several newly synthesized derivates were identified as substrates of

Pdr5p as their growth inhibitory properties were potentiated by deletion of

PDR5. They were synergistic with the antifungal ketoconazole at micromolar

concentrations. The most active phenothiazines contained the amino group at the

end of the alkylene chain substituent. Thioridazine has been shown to reduce

resistance of methicillin-resistance Staphylococcus aureus by inhibiting a reserpine-

sensitive efflux pump (Kristiansen et al., 2006).

2.7.1 Effect of chlorpromazine on susceptible bacteria

The precise mechanism by which chlorpromazine exerts its effect on

susceptible bacteria is not yet known. However, the effect noted from ultra

structural and biomedical studies strongly support the contention that these

agents primarily affect the cell wall of both susceptible and very resistant

bacteria. (Kristiansen and Amaral, 1997) Staphylococcus aureus, an organism that

may be considered fairly susceptible in vitro to chlorpromazine, exhibits a wide

array of changes to its cell wall that are very similar to those produced by β-

lactam antibiotics (Amaral and Lorian, 1991). Chlorpromazine thus appears to

mimic the action of a β-lactam antibiotic on the cell wall of S. aureus and

produces frayed cell walls, thickened cross wall and clusters of bizarre progeny

cells, some of which are devoid of cytoplasmic contents. The action of a β-lactam


61

on the bacterial cell wall is known to involve the inhibition of special membrane

bound enzymes (penicillin binding protein) that are involved in its synthesis and

construction of cell wall (Walter and Heilmeyer, 1965; Gemmel and Lorian, 1991).

Kristiansen and Blom (1981) studied ultrastructure of Staphylococcus aureus

grown on plates containing chlorpromazine (CPZ) in different concentrations up

to a bacteriostatic level (50µg/ml). Cells grown at the concentration of 12.5µg/ml

were indistinguishable from control cells. At 25µg/ml, a number of cells showed

some swelling of the outer layer of the cell wall, but no morphological changes

were found in plasma membrane or in the inner layer of the cell wall. At this

concentration most of the cell divisions were occurring as normally in the

equatorial plane of the cell, but occasionally an asymmetrical cross-wall

formation was seen. The characteristic finding in a number of cells grown at

35µg/ml was some very large and complex mesosome-like structures. They

varied in structure from a regularly striated pattern of dense, 10nm thick lines to

more honeycomb-like structures. The membranes of these structures seemed to

be in continuity with the plasma membrane and sometimes they appeared to be

enclosed within invaginations of the plasma membrane. These cell organelles

were generally observed in dividing bacteria. Asymmetrical cell divisions were

found in about 5%of the sectioned cells.

Kaatz et al. (2003) demonstrated that phenothiazines and thioxathenes

inhibit multidrug efflux pump activity in S. aureus. As per these investigators,

efflux-related multidrug resistance (MDR) is a significant means by which


62

bacteria can evade the effects of selected antimicrobial agents. Genome

sequencing data suggest that S. aureus may possess numerous chromosomally

encoded MDR efflux pumps, most of which have not been characterized.

Inhibition of these pumps, which may restore clinically relevant activity of

antimicrobial agents that are substrates for them, may be an effective alternative

to the search for new antimicrobial agents that are not substrates. The inhibitory

effects of selected phenothiazines and two geometric stereoisomers of the

thiaxanthene flupentixol were studied using strains of S. aureus possessing

unique efflux-related MDR phenotypes. These compounds had some intrinsic

antimicrobial activity and, when combined with common MDR efflux pump

substrates, resulted in additive or synergistic interactions. For S. aureus SA-11

99B, which over expresses the NorA MDR efflux pump, and for two additional

strains of S. aureus having non-NorA-mediated MDR phenotypes, the 50%

inhibitory concentration (IC50) for ethidium efflux for all tested compounds was

between 4 and 15% of their respective MICs. Transport of other substrates was

less susceptible to inhibition; the prochlorperazine IC50 for acriflavine and

pyronin Y efflux by SA-11 99B was more than proton motive force (PMF) of S.

aureus by way of a reduction in the transmembrane potential. These workers

concluded that the mechanism by which phenothiazines and thioxanthenes

inhibit efflux by PMF-dependent pumps is multifactorial and, because of the

unbalanced effect of these compounds on the MICs and the efflux of different


63

substrates, may involve an interaction with the pump itself and, to a lesser

extent, a reduction in the transmembrane potential.

The effects of chlorpromazine on the morphology of Escherichia coli also

mimic the effects of ampicillin, in that both produce significant elongation of the

organism, but not other gram-negative bacteria such as Salmonella (Molnar,

1984; Gemmel and Lorian, 1991). Chlorpromazine binds with calcium binding

protein calmodulin to prevent the influx of calcium into eukaryotic cells. Some

investigators propose similar mechanism with respect to influx and efflux of

potassium into bacterial cells (Kristiansen et al., 1982; Molnar et al., 1992; Ren et

al., 1993; Molnar et al., 1997). Chlorpromazine also increases the permeability of

the cell wall envelope (Amaral and Lorian, 1991; Amaral et al., 1992;

Chattopadhyay et al., 1998; Amaral and Kristiansen, 2000), and binds tightly to

minor groove of eukaryotic and bacterial DNA by intercalation between the base

pairs (deMol and Busker, 1984; de Mol et al., 1986; Portugal and Waring, 1988;

Izbirak, 1989; Kelder et al.,1991; Hagmar, 1992; Lalwani et al.,1995; Kochevar,

1998). Thus, creating the possibility of the inhibition of the replication of the

affected microorganism may take place by this mechanism. Although, this may

be the case for microorganisms that do not have a cell wall. It is doubtful that

these molecules reach beyond the cell envelope of the affected cells. With respect

to bacteria, the morphological response of chlorpromazine-affected cells are

remarkably similar to those produced when such bacteria are exposed to


64

concentrations of beta-lactams below their minimum inhibitory concentration

(MIC), thus suggesting that in some manner they inhibit one or more of the

penicillin binding proteins of the plasma membrane of affected bacteria (Amaral

and Lorian, 1991; Kristiansen and Amaral, 1997). Gram negative bacteria such as

salmonella that are deemed resistant to phenothiazine are susceptible during the

early stages of in vitro growth and the ensuing resistance to phenothiazine is

accompanied by the loss of an outer cell wall protein (Amaral et al., 2000). Thus

the cell wall envelope appears to be a target, one which in all probability is non

specific, because simple washing procedures are effective in eliminating any

phenothizine promoted effects (Amaral et al., 2000).

Attention has been focused on the uptake of chlorpromazine by organs

and tissue (Hu and Curry, 1989; Moriya and Hashimoto, 1999) and the

metabolites produced by them. Human lung tissue of cadavers was found to

contain phenothiazine concentrations upto 300 times higher than those found in

interstitial fluid, (Moriya and Hashimoto, 1999). Studies on the effects of

chlorpromazine on the viability of M. tuberculosis or M. avium which used

purified monocytes of human peripheral blood containing phagocytosed bacteria

demonstrated that the concentration of chlorpromazine that completely killed off

intracellular localized mycobacterium was at least 10 times lower than that

showing any in vitro activity against these same cells (Crowle et al., 1992). Similar

findings have been reported with other infectious agents exposed to

chlorpromazines after phagocytosis and during the time they remain within the


65

human macrophages (Berman and Lee, 1983; Chang and Pechere, 1989). Thus it

seems that the concentrating ability of human pulmonary tissue is reproduced by

human macrophages.

When exposed to light, phenothiazines will yield a variety of derivatives

that have greater activity against bacterial or viral infectious agents than the

parent phenothiazene (Wainwright, 1998; Wainwright et al., 1998). Phenothiazine

derivative, chlorpromazine derived from urine of patients has greater

antimicrobial activity than the parent compound. (Molnar et al., 1991; Csiszar and

Molnar, 1992).

The in vitro antimicrobial activity of phenothiazine has been produced by

concentrations that far exceed those that would be employed for the

management of other conditions like infectious diseases (Williams, 1995).

However, as the in vivo activity is mainly due to high concentration of drug in

monocytes and macrophages and probably neutrophils (Crowle et al., 1992;

Amaral et al., 1996) so the level of drug being used for the treatment of infectious

diseases is much lower than that used for the treatment of psychosis. Therefore,

no significant toxicity would be anticipated, and thus chlorpromazine is a

suitable candidate for mastitis model caused by S. aureus. This is because in

mastitis model the drug is used for short duration so there is little chance for

development of resistance. The use of chlorpromazine in the treatment of

mastitis has not been evaluated thus far.


66

2.8 LIDOCAINE AS ANTIBACTERIAL

That the local anesthetic drugs possess antibacterial properties was first

suggested by (Jonnesco, 1909). Erlich (1961) showed in vitro inhibition of S. aureus

and Candida albicans by 0.5% tetracaine and a decreased recovery of bacteria and

fungi when 1% tetracaine was used at bronchoscopy. Greater concentrations of

lidocaine were required to produce a similar inhibition. Lidociane and tetracaine

(1 ml of 2% solution) prevented the recovery of Mycobacterium tuberculosis from

10 patients with sputum culture that were positive before instillation of the drugs

through a tracheal cathetar (Conte and Laforet, 1962).

In as much as, elucidation of the antibacterial properties of local

anesthetics could have important practical applications, the modes of action of

lidocaine (an amide) and procaine (an ester) were studied with emphasis on

macromolecular biosynthetic pathways. Cumulative percentage of susceptibility

of bacteria to lidocaine has been reported by (Schmidt and Rosenkranz, 1970). Of

1219 clinical bacterial isolates, 80% were inhibited by 2% lidocaine. A total of 28

different species were isolated. Of these, 5 were gram positive and 23 gram

negative. Among the 23 gram negative species, 22 were inhibited by 2 %

lidocaine. Only Pseudomonas aeruginosa was resistant to this drug. Generally, the

gram positive organisms were less sensitive to lidocaine. Fifty three percent of

134 isolates of S. aureus, 68.6% of S. epidermidis, 77.8% Streptococcus viridan, 83.3%

of Enterococcus and 100% of Streptococcus pyogenes were sensitive to 2% lidocaine.


67

In an experiment on the sensitivity of mycobacterium, 20 strains of this

organism derived from human and animals were inhibited by 2% lidocaine.

Exposure of growing culture of E. coli to levels of lidocaine of 0.5% and greater

resulted in loss of viability of the treated bacteria. Lidocaine inhibits the

incorporation of radioactive precursors of DNA, RNA and protein at

concentrations insufficient to inhibit bacterial growth by 50%. There is no distinct

selective inhibition of macromolecular synthesis. In bacterial cultures exposed to

lidocaine, protein synthesis is somewhat more sensitive to inhibition than is

production of DNA or RNA. Since lidocaine does not selectively inhibit the

synthesis of DNA, RNA or protein, the basis of action of this agent may involve

the cell wall or cytoplasmic membrane of bacteria (Jacob et al., 1963; Ryter, 1968;

Smith and Hanawalt, 1967).

Noda et al. (1990) studied the antibacterial activity of lidocaine using

standard colony of S. aureus ATCC 25923, S. epidermidis ATCC 14990, and

Pseudomonas aeruginosa NCTC 10490. According to this study, lidocaine had

bactericidal activity at clinical concentration. At the same clinical concentration,

xylocaine which contained preservative showed a greater antibacterial activity

than the pure lidocaine which contained no preservative.

Feldman et al. (1994) reported that the local anesthetic, lidocaine inhibited

bacterial growth at concentration of 2%, 1.5% and 1%. This effect diminished as

the concentration of drug was reduced. The growth at these concentrations was

compared to the growth observed in agar alone. Fariss et al. (1987) studied the


68

toxicity and anesthetic properties of two anesthetic agents viz. bupivacaine and

lidocaine. These investigators concluded that these anesthetic agents did not

damage tissue defenses or invite infection in experimental animals.

Sakuragi et al. (1996) investigated the rate and potency of the antimicrobial

activity of 2% lidocaine with preservatives and 2% lidocaine without

preservatives on two strains of methicillin resistant S. aureus. The 3-hour

exposure reduced the count by approximately 60%, the 6-hour exposure by 70%

and 24-hour exposure by more than 99%. Antimicrobial activity was observed

shortly after exposure of S. aureus to local anesthetic lidocaine that appeared to

be bactericidal rather than bacteriostatic.

Pina-vaz et al. (2000) evaluated the antifungal activity of lidocaine against

Candida albicans and non-albicans strains. The MIC ranged from 5-40mg/mL. The

inhibitory activity was detected with the fluorescent probe FUN-1 after

incubation for 30 minutes. A very fast fungicidal activity was shown by

50mg/mL of lidocaine. At lower concentration the tested drug had fungistatic

activity, due to yeast metabolic impairment, while at higher concentration it was

fungicidal, due to direct damage to the cytoplasmic membrane.

Aydin et al. (2001) investigated the antimicrobial effect of lidocaine on

isolates of E. coli, S. aureus, P. aeruginosa and Candida albicans. These

microorganisms were exposed to this drug for 0, 30, 60, 120, and 240 min at room

temperature. The inoculum taken from diluted suspensions was reinoculated on

blood agar and incubated for 18-21 hrs at 35°C and then colonies were counted.


69

Lidocaine at 5% and 2% dilutions reduced the viable cells of all microorganisms

tested while lidocaine at 1% dilution reduced only viable organisms of P.

aeruginosa. Lidocaine had more powerful antimicrobial effect than the other two

local anestheltics viz. ropivacaine and bupivacaine.

Parr et al. (1999) characterized the in vitro antibacterial properties of

clinical doses of lidocaine on isolates of a variety of bacterial pathogens including

Enterococcus faecalis, E. coli, P. aeruginosa and S. aureus, as well as a number of

methicillin resistant S. aureus and vancomycin-resistant enterococcal strains.

Time-kill studies were carried out on bacteria exposed to various clinical

concentrations of lidocaine (0, 1, 2 and 4%) with and without epinephrine

(1:100,000). Lidocaine demonstrated a dose dependent inhibition of growth for

all strains of bacteria tested. The greatest sensitivity to lidocaine was shown by

gram-negative organisms; S. aureus being the least sensitive. The addition of

epinephrine had no effect on susceptibility of bacteria to lidocaine.

Thompson et al. (1993) determined the antibacterial activity of lidocaine

buffered with NaHCO3. Rates of killing of six species of bacteria were

determined in the presence of buffered and un-buffered lidocaine. Lidocaine

buffered with NaHCO3 at either 25, 50 or 100mEq/L, affected a decrease of >

99% of all bacteria tested over the 6 hours of the assay. This dramatic rate of

killing was not observed in the unbuffered control. These investigators inferred

that NaHCO3 enhanced the killing effect of lidocaine.


70

Lidocaine treatment of dogs with E. coli septicemia was studied by

(Hardie et al., 1998). Of the 12 dogs with induced E. coli septicemia, six were

treated with lidocaine HCl (6mg/kg/hr). Non treated dogs with septicemia

developed systemic hypotension, decreased cardiac output, increased portal

pressure, increased serum alanine transaminase activity, increased liver

extravascular water, increased liver glycogen depletion and decreased Pa O2,

compared with treated dogs. Lidocaine treatment did not alter the

haemodynamic measurements and resulted in metabolic acidosis and

hypoalbuminemia. Decreased numbers of E. coli were recovered from lidocaine

treated dogs; increased neutrophil sequestration was noticed in the livers but not

in lungs of lidocaine treated dogs.

2.9 POVIDONE-IODINE

A commonly used antimicrobial agent is povidone-iodine, a complex of

iodine, (the bactericidal component) with polyvinylpyrolidone (povidone), a

synthetic neutral polymer and a carrier for iodine. The most common commercial

form is a 10% solution in water yielding 1% available iodine. Povidone-iodine is

available as a surgical scrub or skin cleanser (Mayer and Tsapogas, 1993). The

efficacy of povidone-iodine can be judged in vitro by its ability to kill

microorganisms and in vivo by whether it decreases the severity of infection.

In vivo studies undertaken by Lineaweaver et al. (1985) were based on

rinsing irrigated surgically induced wounds in rats with several solutions,

including saline and 1% povidone-iodine, three times daily. At 4 days


71

postsurgery, tensile strength in wound treated with povidone-iodine was only

21% that of the wounds treated with saline. There were no differences at 8, 12, or

16 days postsurgery, despite continued irrigation. Epitheliazation was found to

be delayed in the wounds treated with povidone-iodine at 4 and 8 days

postsurgery, but not thereafter.

Branemark et al. (1966) used a microwound in the cheek pouch of a

hamster as a model to investigate the effects of povidone-iodine on

microcirculation. Exposure for 60 minutes to 1% povidone-iodine solution

resulted in cessation of blood flow in surface capillaries. Circulation did not

resume within 1 hour. No alteration in blood flow was found with exposure to

1% povidone-iodine solution for 5 minutes nor was any alteration of blood flow

in capillaries covered with epithelial tissue found with exposure to 1% povidone-

iodine solution for 60 minutes. No circulatory changes were seen in cheek

pouches of control hamsters in which the wound were treated with saline.

In a later study, Branemark et al., (1967) examined the effects of

disinfectants on microcirculation in wounds to connective, synovial, and nerve

tissue in mice, hamsters, and rabbits as well as dermal tissue in humans. The

findings based on qualitative evaluation using vital micrography, electron

micrography and vital angiography, showed a very slight reaction in wound

microcirculation when exposed to 1% solution of povidone-iodine. These

researchers commented that other disinfectants produced a much greater

reaction.


72

Hughes-Pepsidero and Levine (1984) exposed the carotid artery and

vagus nerve in wound bed of rabbits. Wounds were kept open until the arteries

were 100% covered with granulation tissue. One group of rabbits received daily

topical applications of saline and a second group of rabbits received daily topical

application of 10% povidone-iodine. The wounds in both groups were treated

identically except for the topical agent that was used. No differences in rate of

healing were found between the groups, nor was any damage to arterial tissue

identified.

Kjolseth et al., (1994) compared the effects of bacitracin 500 µ/g, silver

nitrate 0.5%, silver sulfadiazine 1%, mefenide acetate 8.5% and povidone-iodine

10%, in full thickness wounds in mouse ears. The test substance was applied

once daily to the wounds and covered with a dressing. Wounds treated with

povidone-iodine required more time for epithelization (11.8±0.55 days) than did

controls (7.2±0.7 days) or wounds treated with silver sulfadiazine (7.1±0.3 days)

or mefenide acetate (7.3±0.3 days). The wounds treated with povidone-iodine

showed complete neovascularization in less time (15±0.4 days) than the wounds

treated with other topical agents (range 15.3±0.7 to 18.4±0.56 days). Despite the

cytotoxicity documenteds with in vitro studies, the results of in vivo studies seem

to suggest that povidone-iodine does not interfere with healing, especially if it is

used at concentration of 1% or lower. Povidone-iodine may temporarily decrease

blood flow in the wound bed at higher concentrations, as shown by Brennan and

Leaper (1985), but concentration of 1% or less do not appear to have this effect


73

(Branemark et al., 1966; Branemark et al., 1967). The effect of repeated use of

povidone-iodine on microcirculation has not been investigated. All the above

studies used healthy animal or human subjects with acute, surgically induced

wounds.

Patients have developed systemic iodine toxicity as a result of iodine

absorption from wounds dressed with gauze soaked in povidone-iodine or when

povidone-iodine solution was used as wound irrigant (Cruz et al., 1987; D’Auria

et al., 1990). Patients developed decreased renal function or renal failure

following 10 hours of continuous irrigation of their wounds with povidone-

iodine or 17 days to five weeks of wound dressings with gauze soaked in

povidone-iodine. The four patients described in the above reports aged 50-83

years, had multiple health problems, including pre-existing renal insufficiency

(n=3), diabetes (n=2) and congestive heart failure (n=1). The wounds involved

were pressure ulcers or debrided septic hip wounds. Systemic toxicity does not

appear to be a common occurrence. No toxicity has been reported with

povidone-iodine used as a brief rinse or soak.

In vitro studies, povidone-iodine has been demonstrated to be effective at

killing a broad range of pathogens generally associated with wound infections

(LaRocca et al., 1983). Berkelman et al. (1982) reported that povidone-iodine

solutions diluted to concentrations 0.1% to 5% were more effective in killing

common wound contaminants than was 10% stock solution. Even the 10%

solution was completely effective with 4 minutes of exposure.


74

Van Den Broek et al., (1982) found povidone-iodine to be effective against

S. aureus at concentration of 0.005% or higher, but they questioned whether this

effectiveness would be true in vivo. Lineawear et al. (1985) found povidone-iodine

to be an effective bactericide at a concentration of 0.001% (100 µg/ml). Povidone

is not always effective at killing common bacteria. Anderson (1988) discussed

two reports of povidone-iodine stock solution (10%) contaminated with

Pseudomonas spp. Contamination apparently occurred during production of the

povidone-iodine solution. The bacteria remained viable for several weeks and

were eventually involved in patient infections. Why povidone-iodine failed to

kill these bacteria is not known. These isolated incidents are inconsistent with

other in vitro findings, (Van Den Broek et al., 1982; Lineaweaver et al., 1985;

LaRocca et al., 1983, and Berkelman et al., 1982) and cannot be explained by the

concentration of povidone-iodine solution involved.

There are a small number of in vivo studies in which the ability of

povidone-iodine to control infection in wound was examined. Dire and Welsh

(1991) studied wounds treated in a hospital emergency department. No

differences were found in infection rates between wounds irrigated with

povidone-iodine and those irrigated with normal saline.

Rodeheaver et al. (1982) inoculated experimental wounds in guinea pigs

with 102 to 107 organisms of S. aureus. Ten minutes later, the wounds were

irrigated with either povidone-iodine solution (10%) or normal saline. Four days

after treatment, the authors found no differences between the two groups in the


75

number of viable bacteria present in the wounds or in the number of wounds

with visible purulent exudate. When using povidone-iodine surgical scrub, the

wounds treated with povidone-iodine had higher rate of infection than those

treated with saline. Wounds contaminated with 103 organisms showed 60%

infection when treated with povidone-iodine vs. 0% with saline. Inoculation with

104 organisms produced 90% infection when treated with povidone-iodine vs.

0% with saline. With 105 organisms, the wound treated with povidone-iodine

were 100% infected vs. 15% for saline controls. Edlich et al. (1969) also created

wounds in guinea pigs, which were inoculated with S. aureus. Five minutes later,

the wounds were irrigated with either a povidone-iodine solution (10%) or

saline. After 4 days, wound infection as shown by visible purulent exudate was

lower for the wounds treated with povidone-iodine than for saline treated

wounds. There were no differences in percentage of positive cultures and area of

induration. The findings of this study regarding visible purulent exudate appear

contradictory to the results of study by Rodeheaver et al. (1982).

Kucan et al. (1981) examined infection rates in pressure ulcers under

various treatment conditions. The threshold for infection was defined as 105

bacteria per gram of tissue on biopsy. Wounds were treated with gauze dressing

saturated with either saline or povidone-iodine (10%). Treatment was given for 3

weeks. At the end of this period, 78.6% of the saline treated wounds had bacteria

counts below the infection threshold, compared to 63.6% of the wounds treated

with povidone-iodine. No statistical analysis of this difference was reported. The


76

saline dressings were changed every 4 hours, whereas the povidone-iodine

dressings were changed every 6 hours. No explanation was given for this

difference in procedure. Other researchers have investigated the antibacterial

properties of povidone-iodine for use in surgery. Amstey and Jones (1981) found

povidone-iodine to be no more effective than normal saline for preventing

infection. When used as vaginal irrigant before vaginal hysterectomy. Sindelar

and Mason (1979) investigated superficial infections of surgical wounds. Wounds

irrigated with 10% povidone-iodine solution prior to closure had an overall

infection rate of 2.9%. Whereas wounds irrigated with saline had a 15.1%

infection rate.

Viljanto (1980) found that irrigation of appendectomy wounds with 1%

povidone-iodine before closure resulted in a reduction in wound infection, when

infection before surgery was isolated from the appendix (2.6% infection rate vs.

8.6% for saline controls). If infection had spread beyond the appendix before

surgery, however, there was no difference in infection rates between wound

irrigated with povidone-iodine and saline irrigated controls.

In several of the studies discussed, wounds treated with povidone-iodine

solution were compared with wounds treated with saline to assess impairment

of healing. If povidone-iodine is helpful in promoting wound healing by

decreasing infection rates, healing rates would be expected to be faster in

wounds treated with povidone-iodine. Some researches (Lineaweaver et al., 1985;

Hughes-Papsidero and Levine, 1984; Gruber et al., 1975), however, found no


77

difference in healing rates between wounds treated with povidone-iodine and

saline treated controls. This finding suggests that the antibacterial effects of the

povidone-iodine either did not promote healing or were offset by some other

effect such as cytotoxicity. Although, povidone-iodine clearly is an efficient

bactericide in vitro, the benefits in treating actual wounds appear to be

inconsistent at best. Several studies demonstrated no difference in infection rates

between wound treated with povidone-iodine solution and wound treated with

other topical agents. In the only study involving chronic wounds, povidone-

iodine treatment seemed to be inferior to treatment with saline (Kucan et al.,

1981).

Most in vivo research on the use of povidone-iodine was conducted on

experimentally or surgically induced acute wounds. The applicability of these

findings to chronic wounds typically seen by physical therapists has not been

demonstrated. Apparently, some people assume that chronic wounds, especially

those containing necrotic tissue have a greater risk of infection and ,therefore, a

greater need for treatment to decrease the number of surface bacteria. Moreover,

many patients with chronic wounds generally have compromised health status

and may be less able to produce an effective immune response to bacterial

invasion. These patients, therefore, may need more assistance to prevent

infection than patients with acute wounds and fewer systemic complications.

Conversely, patients with compromised health status may be more susceptible to

the cytotoxic character of povidone-iodine. In the light of findings of Berkelman


78

et al. (1982) that povidone-iodine was more effective when diluted to

concentration of 0.1% to 5%, use of less concentrated solution than the 10% stock

solution may be prudent if povidone-iodine is the treatment of choice.

Rodeheaver (1982) suggests that povidone-iodine surgical scrub may

increase infection rates when used in open wounds. Edlich and associates (1973)

examined the decrease in the effectiveness of antibiotic therapy when the amount

of time that a wound was left open before closure increased. They found that

bacteria become coated with a ‘fibrinous coagulum’ derived from the wound

drainage, which served to protect them from the action of antibiotic. Howell et al.

(1993) suggest that the effectiveness of povidone-iodine in wounds might also be

decreased through this mechanism. This suggestion may explain the finding by

Kucan and colleagues (1981) that povidone-iodine did not reduce bacteria count

in pressure ulcers.

Further investigations with chronic wounds are necessary to establish the

benefit, if any of using povidone-iodine as an adjunct to wound treatment. The

optimal method of application of povidone-iodine has not been clearly

established. Brief contact, such as wound irrigation, especially if followed by a

saline rinse, or use of diluted solutions might minimize the risk of cytotoxicity.

Prolonged contact such as packing the wound with povidone-iodine, however,

might enhance the bactericidal effects. The safety and efficacy of these

alternatives have not been compared in either acute or chronic wounds.


79

The use of povidone-iodine for wound packing requires a particular

scrutiny. This treatment option precludes the use of occlusive or semiocculsive

dressings of the benefits of these ‘moist environment’ require comprehensive

study, however, these dressings have not shown to decrease wound infection

rates (Hutchinson and Lawrence, 1991). Saydak, (1990) reported the result of a

pilot study comprising wound healing rates in pressure ulcers dressed with

either an unsaturated, amorphous hydrogel absorption dressing (Hydra-GranTM)

(moist healing environment) or povidone-iodine solution cleansing followed by

normal saline rinse and dry gauze dressing. Wound treated with absorption

dressing healed more quickly. The percentage of decrease in the length of longest

axis of wound was more than double, and the percentage of decrease in depth

was more than 9 times that of wound treated with povidone-iodine and dry

gauze. No data were reported regarding infection rates. Although, this study did

not isolate the effect of povidone-iodine from that of dry dressings, it suggests

that moist environment dressing may be a viable, possibly safer, alternative to

povidone-iodine.

Wound packing with gauze soaked with povidone-iodine also presents a

small danger of systemic toxicity. Andrews (1994) discusses these concerns in

patients receiving povidone-iodine dressing for prolonged periods. The author

recommends that such patients be observed for symptoms of iodine toxicity,

which include hyperclacemia, metabolic acidosis, cardiovascular instability


80

(bradycardia, hypertension, elevation of hepatic enzymes), central nervous

dysfunction and progressive renal insufficiency.

Povidone-iodine solution appears to be a relatively safe treatment for

small acute wounds. Its safety for treatment of patients with extensive or chronic

wounds has not been adequately investigated. The evidence regarding efficacy of

povidone-iodine solution in treating patients with acute wounds is inconclusive.

There is insufficient evidence to demonstrate effectiveness in treating chronic

wounds.

2.10 DIMETHYLSULPHOXIDE (DMSO)

DMSO is a clear, colourless to straw yellow liquid. It has freezing point of

18.5˚C and boiling point of 189˚C. DMSO traps free radical hydroxide and its

metabolite dimethylsulfide traps free radical oxygen. It appears that these actions

help to explain some the anti-inflamatory qualities of DMSO. It easily penetrates

skin and serves as a carrier agent for drugs and toxin that normally would not

penetrate. DMSO has weak antibacterial activity when used clinically. The anti-

inflammatory/analgesic properties of DMSO have been thoroughly investigated.

The analgesic effect of DMSO is similar to that produced by norcotic analgesics

and is efficacious both for acute and chronic musculoskeletal pain.

One drawback is, it provokes histamine release from mast cell, which

probably contributes to the local vasodilatory effect seen after topical application.

It has also diuretic activity. DMSO also apparently has some anticholinesterase

activity and enhances prostaglandin E, but blocks the synthesis of prostaglandin


81

E2 and PGF2α1H2 and G2. DMSO has been used for enhancement of antibiotic

penetration in mastitis in cattle, where the drug is eliminated quite rapidly. Local

burning effect and garlic or oyster-like breath odour is the most common adverse

effect (Plumb, 1999).

Investigators have reported the following benefits of DMSO therapy:

improved microcirculation, inhibition of inflammatory cells migration,

modulation of cell mediated immune responses and inhibition of fibroblast

proliferation. Inhibition of fibroblast proliferation is important in chronic

conditions. When applied topically DMSO is a potent anti-inflammatory and

analgesic agent. DMSO has a thousand-fold sparing effect on endogenous

cortisone level and favorably influences the endogenous stabilization of

lysosomal membranes by glucocorticoids (Brayton, 1986).

The solvent like DMSO rapidly penetrates tissue membrane with little or

no residual effects and has an affinity for inflamed tissue. This drug was found to

have merit in the treatment of intra-mammary infections by carrying antibiotics

through occluded milk spaces to focci of infection (Ziv, 1967). Very good to

excellent results have been reported by intra mammary infusion of 15-50ml of

90% DMSO + antibiotics / normal saline per quarter in the treatment of sub

clinical or chronic mastitis (Keskintepe et al., 1992; Koller, 1976).

Lorda and Grimeldi (1979) treated 80 mastitic cows during lactation or in

dry period with antibiotic preparations in DMSO, in oil or in water. Treatment

with chloramphenicol / nifuroxazide / prednisolone / DMSO preparations gave


82

a higher percentage of cured quarters than either the water based or oil based

preparations.

In a S. aureus mastitis treatment programme in Brazil, penicillin G (100000

IU), streptomycin (1000 mg) in an aqueous oily or aqueous vehicles with or

without the addition of 20% DMSO was used in three herds. An average of 70.6%

bacteriologically negative glands were obtained using this combination of

antibiotics in the aqueous-oily vehicle and 79.4% in the aqueous vehicle, 120 hour

after single application. The addition of DMSO in formulations had significant

effect only in the herd with chronic mastitis (Figueiredo et al., 1993).

Ballarini (1972) studied the effect of DMSO as vehicle for antibiotics

(Penicillin) and corticosteroid (flumethosone) in the treatment of bovine mastitis.

The DMSO was used as 90% solution. Cases of acute parenchymatous mastitis

due to E. coli in 136 cows were treated by repeated mammary infusions of 0.25 -

0.50 mg flumethasone, associated with an appropriate antibiotic in DMSO.

Favourable results were noticed in 95% of cases. Either during dry period or

during lactation, 151 quarters were similarly treated with flumethasone or

penicillin in DMSO; 90% of the acute cases responded to treatment during

lactation. Of the chronic cases, only 24% responded to treatment during lactation,

while 46% were cured by treatment during dry period. No advantages were

detected from the use of DMSO as vehicle when treating infections localized in

teat canal or milk system.


83

The sensitivity of 132 samples cultured from infected mammary glands

were studied in 3 herds of cattle from the milk basin of Belo Horizonte, Brazil.

The DMSO added to the culture medium in concentrations of 10 and 15% did not

modify the behaviour of the samples which were initially resistant to penicillin

(75%) and streptomycin (38%). However, it increased the sensitivity of samples

that were initially only slightly sensitive and at 6% and 10% concentrations,

DMSO did not inhibit the growth of any of the isolated samples, while at 15% it

inhibited 27.6% and at 20% it inhibited 100% of the samples (Andrade et al.,

1993).

Antibiotic residues in milk was evaluated by Andrade and colleagues

(1993) after using DMSO with antibiotics (Penicillin, 100,000IU, and streptomycin

100mg) in aqueous vehicle only or aqueous vehicle with or without addition of

20% of DMSO. The excretion of residues in milk from treated mammary glands

ceased 120 hr after application. The DMSO caused retention of antibiotic in

mammary parenchyma. The level of antibiotics residue for oily formulations,

with and without DMSO, at 72,96 and 120 hrs were 29.4, 29.4 and 5.9%

respectively and for the aqueous vehicles were 5.9%, 5.9% and 0.0% respectively.

The oily formulations caused retention of antibiotic residues in the milk 68.2%

for longer period than the aqueous formulations 59.4% irrespective of the

addition of DMSO.


84

2.11 OTHER NON-ANTIBIOTIC PREPARATIONS

Researchers (Paape and Bannerman, 2006) at the ARS’ Bovine Functional

Genomics Laboratory, Beltsville, Maryland (USA) have shown that injecting cow

mammary glands with the yeast-derived polysaccharide sugar (Poly-x) reduced

mastitis infections at about one-twelfth the cost of antibiotics. When infused into

non lactating mammary gland, this sugar serves as a kind of biochemical ‘bugle

call’ that mobilizes the animals’ immune system, especially the milk somatic

cells. For their studies, these researchers injected 40 non lactating Holstein cows

with poly-x and 40 with antibiotics. Cows injected with poly-x had a net gain of 5

new infections at the beginning of the next lactation compared to 16 infections in

cows receiving antibiotics. In addition to efficacy superior to the use of

antibiotics, the use of poly-x is avoids the problems of drug residues and this

therapy is much cheaper.

Meaney et al. (1999) evaluated Lacticin 3147, produced by Lactococcus lactis

sub species lactis, for mastitis control in a study conducted jointly at Moorepark

Research Centre and at University College Cork, Irish Republic. Forty five

quarters were selected as untreated controls, 85 were infused with teat seal, and

50 were infused with a teat seal and lacticin formulation. When used in

combination with teat seal, the bacteriocin was effective in preventing clinical

mastitis in dry cows, after they had been exposed to artificial infection with a

mastitic strain of Str. dysgalatiae. The combination of teat seal and lacticin 3147

was also well tolerated within the mammary tissue and produced cellular


85

response levels which were similar to those elicited by a standard intramammary

antibiotic formulation which is used routinely in lactating cows.

Stampfli et al. (1994) experimentally challenged 14 early lactation Holstein

cows by intramammary infusion of 2 teats to compare the virulence of the strains

of Escherichia coli isolated from faeces or from cases of bovine mastitis. The initial

treatment protocol consisted of parenteral antibiotics and fluid therapy with the

onset of high fever, significant systemic signs and local inflammation. Two cows

treated with this protocol developed severe depression, dehydration,

recumbency and were killed. Subsequently the treatment protocol was revised to

commence stripping of the affected quarters plus injections of 30 IU of oxytocin

once the body temperature was a 41˚C. This was repeated at 2 h intervals until

the temperature was ≤39.5˚C. All 12 cows treated with this protocol responded

within 24 h. Bacteria were cleared from all infected quarters within 96 h. The

presence of non-antibiotic inhibitory substances in milk of the infected and non-

infected quarters was evaluated at 12, 24, 36, 48, 72 and 96 h using the Brilliant

Black Reductase Test (RBT), Delvotest P (DTP) and Bacillus stearothermophilus

Disc Assay (BSDA) residue test. False positive results were found in at least one

sample between 12 and 48 h after infection in 4, 5 and 7 cows using BRT, DTP

and BSDA, respectively. It was concluded that non-antibiotic therapy involving

stripping and oxytocin merits further study. In addition, acute coliform mastitis

cases may represent a source of error in routine antibiotic residue testing

programs.


86

Kalorey et al. (1993) used Caliform Mastitis Test in 18 lactating cows,

which were selected for a treatment study. The highest pH value of milk samples

recorded on day 0 was 6.61±0.02 and the lowest, on day 6 of treatment with

trisodium citrate (30 mg/kg orally) and levamisole (10 ml s.c.), was 6.30±0.01. it

was suggested that the decrease in pH achieved with the trisodium citrate was

unfavourable for the growth of bacteria. Cultural examination of quarter milk

samples before treatment revealed Staphylococcus and Streptococcus spp. in 60%

(untreated controls), 66.66% (given trisodium citrate) and 83.33% (given

trisodium citrate and levamisole), while on day 6 after treatment the

corresponding infection rates were 66.66, 46.66 and 33.33%. On day 21 after

treatment, however, 66.66%, 13.33% and 8.33% of these quarters, respectively,

were culturally positive.

Fang et al. (1990) reported that oral administration of a decoction of the

herbs dandelion, honeysuckle flower, Radix isatidis, Radix scutellariae and Radix

angelicae sinensis gave 81.8% clinical and 33.3% bacteriological cure in cows with

mastitis. Bacteriological cure improved to 66.9 when the decoction was combined

with intramammary infusion of the synthetic drug Injecta CD-01. After further

development work, cure rates of 68.5, 911.1% (including 41.7, 71.0 and 71.4

bacteriological cure) were obtained by intramammary infusion of the herbal

preparations Injecta C1, An-Ru and Shuang-Ding respectively these were better

than the rates with penicillin, streptomycin and gentamycin.


87

Prandzheve and Mainkov (1982) infused non-antibiotic hydroxyquinoline

preparation containing 17 mg broxaldine and 83 hydroxyquinoline in 10 g of a

paraffin base. It was used on 52 cows with subclinical and 20 cows with acute

mastitis caused by S. epidermidis, S. aureus, Str. uberis or Str. dysgalactiae. A second

treatment was given 12 or 24 h after the first. Infection was eliminated in the 60%

of the cows with subclinical mastitis, and 15 of the 20 cases of acute mastitis.

Baghaerwal and Shukla (1991) detected the efficacy of cephaloridine in the

treatment of bovine mastitis. They administered the drug at the rate of 10 mg/kg

body weight by IV route at 8 hourly interval to five buffaloes and six cows. On

the second and third day, they infused 250 mg cephaloridine into mastitic

quarters. It was revealed that all animals got cured by third day of treatment

with 99.5% average rate of cure as evidenced by normalization of quarters.

88

CHAPTER 3

MATERIALS AND METHODS

The present study was compartmentalized into two phases viz. phase I

and phase II. During phase I, an in vitro antibacterial activity of 4 non-antibiotics

viz., chlorpromazine (CPZ), lidocaine (Lid), povidone-iodine (PI) and

dimethylsulphoxide (DMSO) was determined against American Type Culture

Collection (ATCC) and field isolates of Staphylococcus aureus, Streptococcus

agalactiae and Escherichia coli. The effect of these non-antibiotics on phagocytosis

by polymorphonuclear leukocytes (PMNLs) was also determined. During phase

II, these non-antibiotics were evaluated alone and in combination with antibiotic

cephradine (Ceph) for the treatment of mastitis in buffalo (Bubalus bubalis).

PHASE I:

Determination of in vitro antibacterial susceptibility, minimum inhibitory

concentrations and effect of non-antibiotics on viability and phagocytic efficiency

of polymorphonuclear leukocytes (PMNLs) were investigated in Phase I.

a. Determination of in vitro antibacterial susceptibility by disc diffusion

susceptibility testing of non-antibiotic antibacterials Principle

When a filter paper disc impregnated with a chemical is placed on agar

medium, the chemical will diffuse from the disc into the agar. Multiple factors

can alter the size of the no-growth-zone around the disc. The solubility of the

CHAPTER 3 MATERIALS AND METHODS

89

drug in question, its molecular size and its antimicrobial potency will determine.

The size of area of chemical infiltration around the disc. If an organism is placed

on the agar medium suitable for its growth, it will not grow in the area around

the disc, if it is susceptible to that antibacterial agent. This area of no growth

around the disc is known as a ‘zone of inhibition’.

Procedure

a. Organisms

Purified mastitis field isolates of S. aureus (n = 59), Str. agalacliae (n = 65)

and E. coli (n = 78) were used as test organisms, while S. aureus ATCC 25923, Str.

agalactiae ATCC 27956 and E. coli ATCC 25922 were used as control (National

Committee for Clinical Laboratory Standards; NCCLS, 1994b).

b. Medium

Mueller-Hinton agar medium was used, (NCCLS, 1994b). In case of

streptococci, 5% defibrinated sheep blood was added to the medium (EUCAST,

2000).

c. Non-antibiotic antimicrobial test agents

Four non-antibiotic antibacterials viz., chlorpromazine, lidocanine,

povidone-iodine and DMSO, procured from Lahore Pharma Pakistan were used

as antibacterial test agents. Filter paper discs were prepared from Whatman®

filter paper (Cole-Parmer, USA) and impregnated with the following potencies:

1. Chlorpromazine (25, 40, 50, 75 and 100 µg per disc)

2. Lidocaine (10, 20, and 25 mg per disc)


90

3. Povidone-iodine (10 and 20 mg per disc)

4. Dimethylesulphoxide (50 mg per disc)

Cephrasine disc (30µg) was used as a control drug.

d. Stock solutions of non-antibiotic antibacterials

Different dilutions of each non-antibiotic agent and its 10 fold dilutions

were prepared and kept at -20°C until used.

e. Petri plates of culture medium

Mueller-Hinton medium was used (NCCLS, 1994b). Five percent

defibrinated sheep blood was added to the medium for streptococci.

f. Inoculum

The inoculum for each bacterial strain was prepared by taking four or five

pure colonies from an overnight growth using a sterile inoculation loop. These

colonies were emulsified in sterile normal saline. Gentle dilution was performed,

till the turbidity was comparable visually to 0.5 McFarland turbidity standard

with inoculum density approximately 108 cfu/ml.

For each organism, a sterile applicator swab was dipped into its

standardized cell suspension and squeezed gently by rotating the swab against

the inside of the glass tube above the liquid, to remove the excess fluid. The

entire surface of each agar plate was inoculated by streaking the swab in

different directions to ensure a uniform growth. Additional plates for each

organism were incubated by this fashion. The plates were allowed to dry for 5-10

minutes and then kept in incubator for 18 hours at 35 - 37°C.


91

A sterile disposable syringe needle was pierced in the centre of a plain

filter paper disc. The syringe was held upright with disc above. The required

concentration of the non-antibiotic in question was taken by a microdispenser

and discharged gently on the disc till the disc was thoroughly soaked. The disc

was then stamped on the surface of Mueller-Hinton agar medium. Changing the

tips of microdispenser and needles, the process was repeated. All the discs were

placed approximately the same distance from the edge of the plate and from each

other.

The inoculated plates were kept inverted in incubator at 35°C for 18 hours.

The plates were viewed against a black background and illuminated with

reflected light and zones of inhibition were measured by Kirby Bauer ruler. The

diameters of zones of inhibition depend on many factors such as thickness of the

medium base (more than 4-6 mm thick medium in plate reduces the sizes of zone

of inhibition). Similarly, in too thin medium the inoculum may not be

standardized and exaggerated or ill defined zones of inhibition appear.

Humidity and the age of the medium may also affect the zone size. All these

variables were cared for in the procedures.

Since, the sizes of the zones of inhibition in respect of the 4 non antibiotics

do not exist in the available literature, the sizes of zones of inhibition were

interpreted by referring to the zone diameter standards for cephradine and

cephalothin (as class drugs) against the reference strains. The actual zone sizes

have not been standardized as in the Kirby-Bauer method. But a comparision of


92

zones sizes for cephradine provided an approximate effectiveness of the test

drug. Hence the zone sizes have been presented in ranges.

b. Determination of minimum inhibitory concentrations (MICs) of the non-antibiotic anti-bacterials agents by dilution technique

Dilution methods are used to determine the minimum inhibitory

concentrations (MICs) of non-antibiotic agents and are the reference methods for

antimicrobial susceptibility testing against which other methods such as disc

diffusion are calibrated. MICs methods are widely used in the comparative

testing of new agents. In clinical laboratories, they are used to establish the

susceptibility of organisms that give equivocal results in disc tests for tests on

organisms where disc test may be unreliable, and when a more accurate result is

required for clinical management of infections in questions.

In the present study, microorganisms were tested for their ability to

produce visible growth on a series of agar plates at different concentrations of

non-antibiotics with incubation at 37°C for 24 hours. The lowest concentration of

a non-antibiotic agent that inhibited the visible growth of a microorganism was

designed as the MIC of that agent. The agar dilution method, adopted in this

study was based on that mentioned in the report of an international collaborative

study of antimicrobials susceptibility testing, (Ericsson and Sherris, 1971) and

was very similar to those described and recommended in many countries,

including, France (CMI, 1996), Germany, (DIN, 1998), UK, (BSAC 1991) and USA,

(NCCLS, 2000).


93

a. Medium

Mueller-Hinton medium was used (NCCLS, 1996). Five percent

defibrinated sheep blood was added to the medium for the growth of

streptococci.

b. Micro organisms and isolates

Two hundred and two mastitis organisms comparing 59 S. aureus, 65 Str.

agalactiae and 78 E. coli were used to determine their MICs against four non-

antibiotic antibacterials.

c. Antimicrobial agents

Four non-antibiotic antibacterials viz., chlorpromazine, lidocaine, DMSO

and povidone-iodine were used as antimicrobial agents. These agents were

obtained from a commercial source (Lahore Pharma Pakistan), with stated

potency and recommended storage and expiry conditions.

d. Preparation of stock solutions

The non-antibiotic antimicrobials were diluted in sterile distilled water

and the stock solutions thus prepared were kept frozen until required for use.

For preparation of stock solutions and for weight and measures, help was taken

from the following formulae (EUCAST, 2000).

Weight of powder (mg) = (mg/g) powder of Potency

(mg)/Lion concentrat x (mL) solvent of Weight

Alternatively, given a weighed amount of antimicrobial powder, the

volume of diluent needed was calculated from the formula:


94

Volume of solvent (mL) = (mg/L)ion Concentrat

(mg/g) ialantimicrob of potency x (mg) powder of Wt.

The actual concentrations of stock solutions depended upon the method of

preparation working solution.

d. Preparation of working solution

The range of concentrations of antimicrobial depended upon the organism

and antimicrobial agent being tested. The concentrations in stock solutions were

kept at level, 20 time higher as that desired in the final tests in petri dishes. As

one mL of antimicrobial agent was required for 19 ml of molten agar, each 9 cm

petri dish contained a volume of 20 ml. A pattern of conventional scheme of

diluting stock solutions was adopted, as given in Tables 3.1, 3.2 and 3.3. The

dilution series were limited to 3 plates per antimicrobial to avoid cumulative

error inherent in lengthy serial dilutions. So many working solutions were made

to achieve the required minimum inhibitory concentrations in final petri plates.

f. Petri plates preparation

Mueller-Hinton agar medium was prepared according to manufacturer

recommendation. The sterilized molten agar flasks were allowed to cool to 50°C

in water bath. Antimicrobial solution was mixed with molten agar in separate

container, poured into petri plates and then allowed to harden undisturbed.

Drugs free petri plates were included as controls for every dilution.


95

g. Preparation of inoculum

From each isolate, the inoculum was prepared by taking four or five pure

colonies from an overnight growth of the isolate in question. The colonies were

gently diluted. The 0.5 McFarland turbidity standard was used for visual

comparison till the turbidity was equivalent to approximately 108 cfu/ml.

h. Incubation of plates

After inoculation, the plates were incubated at 35°C under aerobic

condition for 18 hours, and read for results. Quality control strains of E. coli

(ATCC 25922), S. aureus (ATCC 25923) and Str. agalactiae (ATCC 27956) were

included.


96

Table 3.1: Stock solutions of chlorpromazine (CPZ), its concentration (μg/mL) and final concentration in petri dishes for determination of MIC.

Stock solutions of CPZ (µg/mL)

Volume of stock

solution (mL)

Volume of distilled

water (mL)

CPZ concentration

obtained (µg/mL)

Final concentrations

of CPZ in medium after

adding to 19 ml agar (µg/mL)

4000 1 0 4000 200

4000 1 1 2000 100

4000 1 3 1000 50

1000 1 1 500 25

1000 1 3 250 12.5

1000 1 7 125 6.25

800 1 0 800 40

800 1 1 400 20

800 1 3 200 10

640 1 0 640 32

640 1 1 320 16

80 1 0 80 4

80 1 1 40 2

80 1 3 20 1


97

Table 3.2: Stock solutions of lidocaine, its concentration (mg/mL) and final concentration in petri plates for the determination of MIC.

Stock solutions of lidocaine

(mg/mL)

Volume of stock

solution (mL)

Volume of distilled

water (mL)

Lidocaine concentration

obtained (mg/mL)

Final concentrations of lidocaine in medium after adding to 19

mL agar (mg/mL)

1000 1 0 1000 50

1000 1 1 500 25

1000 1 3 250 12.5

800 1 0 800 40

800 1 1 400 20

800 1 3 200 10

640 1 0 640 32

640 1 1 320 16

640 1 3 160 8

80 1 0 80 4

80 1 1 40 2

80 1 3 20 1


98

Table 3.3: Stock solutions of povidone-iodine, its concentration (mg/mL) and final concentration in petri plates for the determination of MIC.

Stock solutions of povidone-

iodine (mg/mL)

Volume of stock

solution (mL)

Volume of distilled

water (mL)

Povidone- iodine

concentration obtained (mg/mL)

Final concentration of povidone-

iodine in medium after

adding to 19 ml agar (mg/mL)

400 1 0 400 20

400 1 1 200 10

400 1 3 100 5

100 1 1 50 2.5

100 1 3 25 1.25

100 1 7 12.5 0.625

To get 10% DMSO in petri plates, 2mL DMSO was added to 18mL molten

agar, and to get 20% DMSO in petri plates, 4 mL DMSO was added to 16 mL

molten agar, while to get 30% DMSO, 6 mL DMSO was added to 14 mL molten

agar and so on.


99

C. Effect of non-antibiotics on viability and phagocytic efficiency of polymorphonuclear leukocytes (PMNLs) (Patselas et al., 1989; Hellwell et al., 1997)

Polymorphonuclear leukocytes (PMNLs) were isolated from fresh rabbit

blood onto glass coverslips. These coverslips were then exposed to each non-

antibiotic stock solution or to their 1:10, 1:100 or 1:1000 dilutions, for 30 minutes.

Viability was assayed by trypan blue dye exclusion test and phagocytic

efficiency was assayed by percent capability of PMNLs to ingest opsonized

Candida utilis cells. Testing of each non-antibiotic antibacterial involved blood

from three different rabbits. The test agent and each of its 1:10 dilutions were

evaluated on twelve coverslips four from blood from each rabbit for a total of 24

coverslips per concentration, twelve each for the viability and functionality tests.

During each run, 12 coverslips were used as control samples and were exposed

only to Hank’s balanced salt solution (HBSS). This solution was used as control

in which phagocytosis took place.

The PMNLs were subjected to various stock solutions of four non-

antibiotic antibaterials and their 10 fold dilutions in phosphate buffered saline.

PMNLs + non-antibiotic mixture (either stock solutions or their ten fold

dilutions) were incubated for 1 hour (37ºC, no rocking) followed by rocking for 1

hour (Ziv et al., 1983; Hellwell et al., 1997). The effect of four non-antibiotics on

phagocytic efficiency of PMNLs was calculated as under (Hellwell et al., 1997):


100

Percent effect of non-

antibiotics =

(PE1 of PMNLs at HBSS) – (PE of PMNLs at given conc.) x 100 Phagocytic efficiency of PMNLs at HBSS

The data were analysed using analysis of variance to compare different

means to that of Hank’s Balanced Salt Solution (Steel et al., 1997).

Viability and phagocytic efficiency of PMNLs exposed to test drugs were

compared to controls and results were expressed in percentage.

PHASE II: Determination of in vivo efficacy of non-antibiotic antibacterials alone and in combination with antibiotic in the treatment of bubaline (buffalo) clinical mastitis

Experimental animals and their management:

For this purpose, 270 clinically mastitic quarters of 249 Nili-Ravi lactating

dairy buffaloes were selected. Animals previously treated for mastitis during the

current lactation were not included in the panel of experimental subjects.

Similarly, only those quarters were selected which had contralateral normal

quarters. Animals selected were from those managed at Livestock Experimental

Station, University of Agriculture, Faisalabad, and 6 private dairy farms (Bibi

Jaan Dairy Farm, Aminpur Road, Faisalabad; Councillor Dairy Farm, Satiana

Road, Faisalabad; Raja Dairy Farm, Sammundri Road, Faisalabad; Sheikh Dairy

Farm, Rashidabad, Faisalabad; Chaudhry Dairy Farm, 102 RB, Faisalabad;

Suleman Dairy Farm, Faisalabad). All experimental buffaloes were managed in

‘tie-stall’ cum. loose housing system during the experimental period. These

buffaloes received a diet of concentrate mixture and green fodder. The treatment

PE1 = Phagocytic efficiency


101

trial started in January and culminated in August, 2005. In a cut-and-carry

feeding system, chopped green fodder plus chaffed wheat straw was fed ad lib.

Berseem (Trifolium alexandrinum) and maize (Zea mays) were the main green

fodders fed. Fresh drinking water was made available 3-5 times per day and

animals hosed 1-3 times daily depending upon the weather. The buffaloes were

hand-milked twice a day between 3-5 a.m. and 3-5 p.m. Standard mastitis control

practices (e.g., post-milking antiseptic teat dipping, dry period antibiotic therapy,

segregation or culling of mastitic animals) were not in place at any of the dairy

farms.

Experiment I: Evaluation of non-antibiotics alone in the treatment of bubaline clinical mastitis

Thirty clinically mastitic quarters were treated by intramammary route

with each of the 4 non-antibiotic antibacterials. Table 3.4 gives details concerning

the name of the non-antibiotic antibacterials products and manufacturers,

volume and amount infused into each quarter per day, number of quarters

infused and duration of treatment (days).

Before initiation of treatment, mean milk yield of the quarters affected and

contralateral normal quarters were recorded for all the groups. Similarly, mean

milk yield of the quarters treated and contralateral normal quarters were

recorded over a period of four week after initiation of treatment.


102

Table 3.4: Evaluation of non-antibiotic antibacterials alone in the treatment of bubaline clinical mastitis

Non-Antibiotic antibacterials

Proprietary preparation and

manufacturer

Volume and amount

infused into each quarter

per day

No. of quarters infused

Duration of treatment

(days)

Chlropromazine (CPZ)

Inj. LarjactilTM

2.5% (Aventis Pharma, Pakistan)

2ml (50mg) + 38ml Normal Saline

30 5

Lidocaine (Lid)

XylocaineTM 4% (Barrett Hodgson, Pakistan)

10ml + 30ml Normal Saline

30 5

Povidon-iodine (PI)

PyodineTM Solution 10%, (Brookes Pharmaceutical Lab., Pakistan)

10ml +30ml Normal Saline

30 5

Dimethyl sulphoxide (DMSO)

Dimethyl sulphoxide 99.5% (Sigma-Aldrich, GmbH, Germany)

20ml +20ml Normal Saline

30 5

Immediately before treatment, secretion samples from clinically mastitic

quarters were collected and subjected to microbiological examination as per the

guidelines of the National Mastitis Council Inc., USA (1990). Briefly, teat ends of

the quarters to be treated were scrubbed with cotton gauze soaked in 70% Ethyl

alcohol. Two to three streaks of the secretions were discarded and about 10ml of

milk collected aseptically into sterile milk vials which were placed on crushed ice

immediately after collection for transportation to the Mastitis Research

Laboratory, Department of Clinical Medicine and Surgery, University of


103

Agriculture, Faisalabad. Using a platinum-rhodium inoculation loop, 0.01mL of

milk sample was streaked onto blood agar (containing 5% sheep erythrocytes),

MacConkey’s agar and esculin-blood agar plates. Four milk samples were

cultured on a 100 mm culture plate (by inoculating individual quarter sample on

one quadrant of the plate) and incubated at 37°C for 48 hours. Gram-positive,

catalase-positive, α and β hemolytic coccal isolates presumptively identified as

staphylococci were subjected to tube coagulase test using rabbit plasma and

biotyped using a commercial identification kit (api Staph-Trac, bioMerieux,

France). Gram-positive, catalase-negative, coccal isolates presumptively

identified as streptococci were further grown on Edward’s medium for

confirmation. These isolates were subjected to CAMP-Esculin test and identified

using api 20E Strep kit (bioMerieux, France). Similarly, colonies on MacConkey’s

agar staining negative in Gram staining procedure were presumptively

identified as those of Enterobacteriaceae. These isolates were subjected to citrate,

indole, H2S, and triple sugar iron agar slants and biotyped with api 20E

biochemical kit (bioMerieux, France). Identification Codebooks (bioMerieux,

France) were used for confirmation and assigning 7-digit numerical profiles to

these isolates.

Organisms other than staphylococci, streptococci and members of

Enterobacteriaceae were identified to genus level. Guidelines for significance of

colony numbers of specific organisms isolated in pure cultures or with other


104

colony types as recommended by National Mastitis Council Inc., USA (1987)

were followed (Appendix 1)

Clinically mastitic quarters were infused with the respective non-

antibiotic with the help of plastic part of sterile intravenous infusion catheter No.

20 (Vasocan BraunuleTM Melsungen D-34209) attached to a sterile disposable

syringe. Infusions were given for 5 days. Milk sample were collected again from

the treated quarters on day 14 and day 28 of initiation of treatment (National

Mastitis Council, Inc., 1990) for determination of bacteriological cure rates.

Experiment II: Evaluation of non-antibiotic antibacterials in combination with antibiotic (cephradine) in the treatment of bubaline clinical mastitis

All 4 non-antibiotic antibacterials chlorpromazine (CPZ), lidocaine (Lid),

povidon-iodine (PI), and Dimethylsulphoxide (DMSO) were evaluated in

regimens similar to those given in Table 3.4 except that 500 mg of cephradine

(Inj. VelosefTM; Bristol-Myers Squibb, Pakistan) was additionally added to daily

infusions of all 4 non-antibiotic antibacterials (Table 3.5). Evaluation parameters

and timings of samplings were also the same as those described under

‘Evaluation of non-antibiotic antibacterials alone in the treatment of bubaline

clinical mastitis’ (Table 3.4; Experiment 1).


105

Table 3.5: Evaluation of non-antibiotic antibacterials in combination with antibiotic (cephradine) in the treatment of bubaline clinical mastitis

Non-antibiotic antibacterials

+ antibiotic No. of quarters

infused Duration of treatment

(days) CPZ + Cephradine 30 5

Lid + Cephradine 30 5

DMSO + Cephradine 30 5

PI + Cephradine 30 5

Cephradine alone (control) 30 5

Evaluation parameters included:

1. Surf Field Mastitis Test (Muhammad et al., 1995) and California Mastitis

Test (Schalm et al., 1971) based cure rates.

2. Somatic cell count (Singh and Ludri, 2000; Shailja and Singh, 2002) in pre

and post treatment (day 14 and day 28) quarter milk samples were

performed with a slight modification from original method of Schalm et al.

(1971) as described below

a. 10 µl of milk was spread on a slide having a marked area of 10

mm x 10 mm using a micropipette, and dried at 37ºC in the

incubator.

b. The slides were dipped in xylene for 1-2 minutes to remove fat

globules and dried subsequently


106

c. The slides were then stained with Newman Lampert’s stain for

15 minutes, and dried at room temperature.

d. Stained dried slides were rinsed with tap water to remove

excess of stain from the smear and dried again at room

temperature

e. The poorly stained smears were further stained with blue

aliquot of Diff Quick Stain (Difco Labs., Michigan) for 10-15

seconds, rinsed with tap water and dried.

f. Using oil immersion lens of the microscope, the number of

cells in 50 fields were counted and multiplied by microscopic

factor (MF) to get the number of cells per mL of milk

MF = b x d

100 x 20

d = diameter of the microscopic field

b = number of microscopic fields

The number is then multiplied by 1000.

3. Isolation and identification of pathogens from treated quarters at day 14

and 28 of initiation of treatment (National Mastitis Council, Inc. 1990) for

determination of bacteriological cure rate.

4. Milk production of the quarters in question and clinical cure rate


107

Statistical analysis

The data of somatic cell count was analysed using analysis of variance to

compare mean somatic cell count before and on day 14 and day 28 post-initiation

of treatment. Similarly, mean milk yield loss of the affected quarters was

analysed by analysis of variance to compare the means before and after

treatment (Steel et al., 1997).

108

CHAPTER 4

RESULTS AND DISCUSSION

The present study was conducted in two phases, phase I and phase II.

PHASE I

During this phase, an in vitro antibacterial susceptibility and MIC of 4

non-antibiotic antibacterials viz; chlorpromazine (CPZ), lidocaine (Lid),

povidone-iodine (PI) and dimethylsulphoxide (DMSO) by disc diffusion method

was determined against American Type Culture Collection (ATCC) and field

isolates of Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli. In

addition, the effect of non-antibiotics on viability and phagocytic efficiency of

PMNLs was investigated.

4.1 IN VITRO ANTIBACTERIAL SUSCEPTIBILITY OF FIELD MASTITIS

AND AMERICAN TYPE CULTURE COLLECTION ISOLATES TO NON-ANTIBIOTIC ANTIBACTERIALS Antibacterial susceptibility determined by disc diffusion method against

field mastitis and control (ATCC) isolates has been depicted in Table 4.1. Salient

findings in respect of each of the four non-antibiotic antibacterials tested are as

follows:

CHAPTER 4 RESULTS AND DISCUSSION

109

Chlorpromazine

Filter paper disc harboring 25µg chlorpromazine (CPZ) did not produce

any zone of growth inhibition against field mastitis isolates of S. aureus (Figure

4.1) and E. coli as well as E. coli ATCC 25922 whereas growth of field mastitis

isolates of Str. agalactiae and Str. agalactiae ATCC 27956 was inhibited in circular

zones with radii of 16-18 and 18 mm, respectively.

Fifty µg CPZ disc produced zones of no growth against field S. aureus

isolates and S. aureus ATCC respectively measuring 15-18, and 20 mm. At the

same concentration, CPZ produced zones measuring 18-20, and 22 mm

respectively against field Str. agalactiae and Str. agalactiae ATCC 27956. This disc

potency produced no inhibitory zone against field E. coli and E. coli ATCC 25922.

Chlorpromazine at 75 µg disc dose produced inhibitory zones measuring

15-19mm and 10-19mm against Staphylococcus aureus and Str. agalactiae isolates.

This concentration did not produce any zone of inhibition against E. coli isolates.

For the control organisms of S. aureus ATCC 25923 and Str. agalactiae ATCC

27956 the inhibition zones were 20mm, and 22mm respectively. Again for control

organism E. coli ATCC 25922 the mentioned concentration produced no zone of

inhibition.

Chlorpromazine at 100 µg produced zones of 20-22, 22-24, and 12-14 mm

respectively against field mastitis isolates of S. aureus, (Figure 4.2) Str. agalactiae

and E. coli. The zones of growth inhibition against control organisms (S. aureus

ATCC 25923, Str. agalactiae ATCC 27956 and E. coli ATCC 25922) measured 22, 24


110

and 14 mm, respectively. In general, filter paper discs of this potency produced

largest zones of growth inhibition amongst all potencies of all 4 non-antibiotic

antibacterials tested in the present study.

Lidocaine

Filter paper disc harboring 10mg lidocaine (Lid) produced no inhibitory

zone against field isolates of S. aureus, Str. agalactiae and the control organism, S.

aureus ATCC 25923 while the Str. agalactiae ATCC 27956 was inhibited in a zone

of 10 mm only. This disc potency of lidocaine prevented the growth of field

isolates of E. coli in zone sizes of 16-18 mm and that of E. coli ATCC 25922 over a

zone size of 20 mm.

Lidocaine at 20 mg per disc produced zones of 8-10 mm, 10-12 mm, and

20-22 mm against the field mastitis isolates of S. aureus, Str. agalactiae and E. coli

(Figure 4.4) respectively. Control organisms (S. aureus ATCC 25923, Str. agalactiae

ATCC 27956, and E. coli ATCC 25922) were inhibited over 12, 16, and 22 mm,

respectively.

At 25 mg per disc, lidocaine produced zones of growth inhibition

measuring 8-11, 13-15, and 22-24 mm against field mastitis isolates of S. aureus,

Str. agalactiae and E. coli (Figure 4.5). This disc potency inhibited growth of

corresponding control organisms (S. aureus ATCC 25923, Str. agalactiae ATCC

27956, and E. coli ATCC 25922) over 14, 17 and 24 mm, respectively. As is evident

from data given in Table 4.1, lidocaine was more effective against field mastitis

isolates of E. coli as well as against E. coli ATCC 25922 than against isolates of Str.


111

agalactiae and S. aureus. In the study of Gajraj et al. (1998), the manufacturer’s

recommended dose of 0.05% lidocaine was not bacteriostatic or bactericidal for

any of the organisms tested, although there were significantly reduced colony

counts for all Gram-positive organisms and two of the Gram-negative organisms.

O.2% was the lowest concentration of lidocaine with bacteriostatic effects. Bazaz

and Salt (1983) concluded that the antibacterial activity of lidocaine was greater

against Gram-positive than against Gram-negative organisms. This is consistent

with the findings of study of Gajraj et al. (1998) but contrary to those of Schmidt

and Rosenkraz (1970) who investigated more than 1200 strains of bacteria and

fungi and showed greater antibacterial activity against Gram-negative

organisms. The findings of the present study with respect to higher activity

against Gram-negative organism (E.coli) concur with findings reported by

Schmidt and Rosenkraz (1970).

Povidone-iodine

Filter paper discs impregnated with 10 mg of povidone-iodine (PI)

produced zones of inhibition of 10-12, 12-14, and 13-15 mm against field isolates

of S. aureus, Str. agalactiae and E. coli. At this concentration, the zones for the

corresponding control (ATCC) organisms were 14, 15, and 16 mm, respectively.

At 20 mg per disc, povidone-iodine produced zones of 12-14, 14-16, and

16-17 mm against field mastitis isolates of S. aureus, Str. agalactiae and E. coli,

respectively. While the corresponding zones of inhibition for control organisms


112

of S. aureus ATCC 25923, Str. agalactiae ATCC 27956, and E. coli ATCC 25922 were

15, 16, and 18 mm, respectively.

Dimethylsulphoxide (DMSO)

Filter paper discs harboring only one potency (50mg) of

dimethylsuphoxide (DMSO) were tested as can be seen in Table 4.1. Of all the 9

potencies of 4 non-antibiotic antibacterials tested, DMSO discs harboring 50mg of

this chemical produced in general the smallest zones of growth inhibition against

field isolates of S. aureus, Str. agalactiae and E. coli as well as against ATCC

isolates of these species.

Cephradine (Control)

Cephradine (the control drug) at 30µg yielded zones of 16-18, 16-20, and

12-14 mm against field isolates S. aureus, Str. agalactiae and E. coli, respectively.

The corresponding zones of inhibition for control organisms were 20, 22 and 14

mm, respectively.


113

Figure 4.1: Inhibitory zones produced by chlorpromazine at 25 and 50 μg per disc against S. aureus isolate.

Figure 4.2: Inhibitory zones against S. aureus expanded as the disc potency

of chlorpromazine was increased to 75 and 100 μg per disc.


114

Figure 4.3: Inhibitory zones against S. aureus increased further as the disc

potency of chlorpromazine was increased to 175 μg per disc

Figure 4.4: Inhibitory zones produced by Lidocaine impregnated discs at

(10mg and 20mg per disc) against E. coli isolate


115

Figure 4.5: Increasing the disc potency of Lidocaine to 25mg per disc further

axpanded the zones of inhibition of this non-antibiotic against E. coli isolate


116

Table 4.1: Zones of inhibition (mm) of the non-antibiotics against field mastitis and control (American Type Culture Collection) isolates

Test agent Disc Potency Mastitis field isolates* Zones of growth

inhibition (radius in mm)

Control organisms Zone of growth

inhibition (radius in mm)

Chlorpromazine (CPZ)

40 µg S. aureus Str. agalactiae E. coli

―― 16 – 18 ――

S. aureus ATCC 25923 Str. agalactiae ATCC 27956 E. coli ATCC 25922

14 18 ――


15-18 18-20 ――


20 22 ――


20-22 22-24 12-14


22 24 14

Lidocaine (Lid)

10 mg S. aureus Str. agalactiae E. coli

―― ――

16-18


―― 10 20


8-10 10-12 20-22


12 16 22


8-11 13-15 22-24


14 17 24

Povidone-iodine (PI)


10-12 12-14 13-15


14 15 16


12-14 14-16 16-17


15 16 18

Dimethylsulphoxide (DMSO) 50 mg

S. aureus Str. agalactiae E. coli

7-8 7-9

8-10


8 9 10

Cephradine (Ceph) 30 µg S. aureus Str. agalactiae E. coli

16-18 16-20 12-14


20 22 14

* S. aureus (n=59) Str. agalactiae (n=65) E. coli (n=78) ―― = No zone of inhibition


117

4.2 MINIMUM INHIBITORY CONCENTRATIONS (MICs) OF NON-ANTIBIOTIC ANTIBACTERIALS AGAINST FIELD MASTITIS AND CONTROL (ATCC) ISOLATES MICs of non-antibiotic antibacterials determined by agar dilution method

against mastitis and ATCC isolates are presented in Table 4.2.

The MIC50 and MIC90 is a way of recording antimicrobials sensitivities

more conviniently. The The MIC50 is minimum inhibitory concentration required

to inhibit the growth of 50% of organisms. Whereas MIC90 is the minimum

inhibitory concentration of antimicrobials required to inhibit the growth of 90%

of organisms under trial. The MIC50 and MIC90 of chlorpromazine were 50μg/mL

and 100μg/mL against mastitis isolates of S. aureus and 40 and 80μg/mL against

Str. agalactiae respectively. While for E. coli isolates, the respective values were

100μg/mL and 200μg/mL.

The values of MIC50 and MIC90 of lidocaine against S. aureus were

25mg/mL and 50mg/mL. The corresponding MICs values against Str. agalactiae

were 20mg/mL and 40mg/mL and against E. coli were 10mg/mL and 20mg/mL,

respectively.

The values of MIC50 and MIC90 of povidone-iodine were 22μg/mL and

44μg/mL against the field mastitis isolates of S. aureus, 16 µg/mL and 32 µg/mL

against the isolates of Str. agalactiae and 10 µg/mL and 20 μg/mL against the

isolates of E. coli. The MIC50 and MIC90 values of DMSO against the isolates of S.

aureus were 40-70% against Str. agalactiae were 40-60% and against E. coli were

30-60% respectively.


118

Table 4.2: Minimum inhibitory concentrations (MICs) of the non-antibiotics determined by agar dilution method

Non-antibitoic anti-

bacterials Mastitis isolates MIC50 MIC90 Range

Cholorpromazine

(CPZ)

S. aureus (n=59)

Str. agalactiae (n=65)

E. coli (n=78)

50 µg/mL

40 µg/mL

100 µg/mL

100 µg/mL

80 µg/mL

200 µg/mL

≤50 >100

≤37.5 >75

100 >200

Lidocaine

(Lid)

S. aureus (n=59)


E. coli (n=78)

25 mg/mL

20 mg/mL

10 mg/mL

50 mg/mL

40 mg/mL

20 mg/mL

≤25 >50

≤20 >40

≤10 >20

Povidone-iodine

(PI)

S. aureus (n=59)


E. coli (n=78)

22 mg/mL

16 mg/mL

10 mg/mL

44 mg/mL

32 mg/mL

20 mg/mL

≤22 >44

≤16 >32

10 >20

Dimethylsulphoxide

(DMSO)

S. aureus (n=59)


E. coli (n=78)

50%

40%

30%

70%

60%

60%

40 >70

40 >60

30 >60

Cephradine

(Ceph)

S. aureus (n=59)


E. coli (n=78)

30 µg/mL

21 µg/mL

36 µg/mL

40 µg/mL

42 µg/mL

48 µg/mL

≤30 >40

≤21 >42

≤36 >48

The results of this study indicate that the in vitro spectrum of activity of

chlorpromazine was closer to that of cephradine, the first generation

cephalosporin. Chlorpromazine at concentration of 50μg produced inhibitory

zones comparable to that of cephradine (30μg) against S. aureus, Str. agalactiae

and E. coli. Both chlorpromazine and cephradine produced bigger inhibitory

zones against Str. agalactiae and S. aureus than E. coli. However, criteria of larger

zones inhibitory may and may not be associated with more antimicrobial activity

of a non-antibiotic agent.


119

The MIC50 and MIC90 of chlorpromazine were 50μg/mL and 100μg/mL

against mastitis isolates of S. aureus and 40µg/mL and 80µg/mL against the

isolates of mastitis of Str. agalactiae respectively (Table 4.2). While for mastitis

isolates of E. coli the respective MICs values were 100μg/mL and 200μg/mL.

These values are in line with MICs values of chlorpromazine against E. coli

reported by Kristiansen (1992). But inconsistent with the MIC value of

chlorpromazine against S. aureus as 25-50μg/mL and Amaral and Lorian (1991)

has given MICs values of chlorpromazine against S. aureus as 10-50μg/mL.

Owing to indiscriminate use of antibiotics, strains of bubaline mastitis might

have become more resistance to antibiotics and non-antibiotic antibacterials.

Lidocaine 10 mg did not produce any inhibitory zone against the field

mastitis isolates of S. aureus and Str. agalactiae but produced a zone of 10 mm

radius against the control organism Str. agalactiae ATCC 27956, and wider zones

16-18 mm against E. coli isolates. The value of MIC50 and MIC90 of lidocaine were

also higher for S. aureus and Str. agalactiae than for E. coli isolates. This indicated

that lidocaine was more effective against gram negative bacilli and least effective

against S. aureus and Str. agalactiae. These findings are in line with those of

previous studies (Schmidt and Rosenkranz, 1970; Parr et al.,1999).

Povidone-iodine produced comparatively smaller zones of inhibition

against S. aureus, Str. agalactiae and E. coli. The values of MIC50 and MIC90 were

22mg/mL and 40 mg/mL against the isolates of S. aureus and 16 mg/mL and 32

mg/mL against the isolates of Str. agalactiae and 10 mg/mL and 20 mg/mL


120

against the isolates of E. coli (Table 4.2) respectively. These values are consistent

with those reported by Lineawear et al. (1985) and are inconsistent with those

given by Van Den Broek et al. (1982) as 50μg/mL against S. aureus.

Povidone-iodine was effective against all the three mastitis isolates.

However, as stated in the ensuing pages its intramammary infusion led to

inflammation after third instillation of the drug into quarters. Teats were painful

and swelling persisted for longer time. Therefore, its internal use may not be

recommended.

Filter paper discs bearing 50mg of DMSO produced the least inhibitory

zones (7-8mm, 7-9mm and 8-10mm) against mastitis isolates of S. aureus, Str.

agalactiae and E. coli. The MICs values were also the highest i.e. 50% to 70% for S.

aureus, 40% to 60% for Str. agalactiae isolates and 30% to 60% for the isolates of E.

coli. This indicates poor antibacterial activity of DMSO against mastitis isolates.

This low antibacterial activity is consistent with that reported previously by

Plumb (1999). While these values are inconsistent with those reported by

Andrade and coworkers (1993), wherein 20% DMSO in the culture medium

inhibited the growth of microorganisms by 100%.


121

4.3 EFFECT OF NON-ANTIBIOTIC ANTIBACTERIALS ON VIABILITY OF POLYMORPHONUCLEAR LEUKOCYTES (PMNLs) Effect of non-antibiotic antibacterials on viability of polymorphonuclear

leukocytes (PMNLs) has been presented in Table 4.3.2 and in Figure 4.3.1. The

details of this table has been depicted in appendices 2-5.

Scrutinizing the effect of chlorpromazine on viability of PMNLs revealed

that only 30.17 ± 5.8% cells remained viable after exposure to 2.5% stock solution

of chlorpromazine. While the cells were 63.83 ± 4.3, 81.50 ± 4.3 and 85.75 ± 2.1%

viable at 1:10, 1:100 and 1:1000 dilutions of stock solution of chlorpromazine,

respectively. Viability at 1:1000 was not-significantly different (P>0.05) than

viability in HBSS.

It can be seen from Table 4.3.2 that 25.33 ± 4.3% cells were viable after

exposure to 2% stock solution of lidocaine. While these cells were 56.83 ± 4.3,

79.75 ± 3.4 and 85.25 ± 2.8% viable at 1:10, 1:100 and 1:1000 dilutions of 2% stock

solution of lidocaine, respectively. Viability at 1:1000 was non-significantally

lower (P > 0.05) than viability in HBSS.

At 10% w/v stock solution of povidone-iodine, PMNLs were not viable.

While the cells were 19.30 ± 3.6, 65.75 ± 5.4 and 83.25 ± 3.8% viable at 1:10, 1:100

and 1:1000 dilutions of 10% stock solution of povidone-iodine. Viability at 1:1000

was not-significantally lower (P > 0.05) than viability in HBSS.

As for the effect of DMSO on the viability of PMNLs, only 16.83 ± 3.3%

cells were viable at 50% v/v solution of this non-antibiotic. Viability of these cells

increased to 40.08 ± 4.2, 78.0 ± 4.5 and 84.08 ± 3.4% at 1:10, 1:100 and 1:1000

dilutions of 50% v/v stock solution of DMSO, respectively. Viability at 1:1000

was not-significantally lower (P > 0.05) than viability in HBSS.


122

Table 4.3.1: Analysis of variance table for viability (%) of polymorphonuclear leukocytes under the influence of different stock solutions of non-antibiotics and their ten fold dilutions.

Source of variation

Degrees of freedom

Sum of squares

Mean squares F-value

Treatment (T)

Concentration (C)

T x C

Error

3

4

12

220

12512.500

169013.775

9850.958

3116.167

4170.833

42253.444

820.913

14.164

294.46**

2983.07**

57.96**

** = Highly significant (P<0.01) Table 4.3.2: Comparison of means for viability (%) of polymorphonuclear

leukocytes under the influence of different stock solutions of non-antibiotic antibacterials and their ten fold dilutions.

Treatment CPZ Lid PI DMSO

Stocck solutions 30.14 ± 5.81 d 25.33 ± 4.31 d 0.00 ± 0.001 d 16.83 ± 0.30 d

1:10 63.83 ± 5.62 c 56.83 ± 4.32 c 19.32 ± 3.60 c 40.08 ± 4.25 c

1:100 81.50± 4.38 b 79.75 ± 3.49 b 65.75 ± 5.46 b 78.00 ± 4.53b

1:1000 85.75 ± 2.18 a 85.25 ± 2.86 a 83.25 ± 3.82 a 84.08 ± 3.40 a

HBSS 87.42 ± 1.93 a 86.25 ± 2.56 a 85.58 ± 2.35 a 86.00 ± 1.41 a

Means sharing similar letters in a column are not significantly different (P>0.05). HBSS = Hank’s Balanced Salt Solution CPZ = Chlorpromazine Lid = Lidocaine PI = Povidone-iodine DMSO = Dimethylsulphoxide


123

0

10

20

30

40

50

60

70

80

90

100

stocksolutions

1:10 1:100 1:1000 HBSS

Viab

ility

(%)

CPZ 2.5% Lidocaine 2%Povidone iodine 10% DMSO 30%

Dilutions of non-antibiotics

Figure 4.3.1: Viability (%) of polymorphonuclear leukocytes under the

influence of different stock solutions of non-antibiotic antibacterials and their ten fold dilutions

CPZ = Chlorpromazine DMSO = Dimethylsulphoxide HBSS = Hank’s Balanced Salt Solution


124

4.4 EFFECT OF NON-ANTIBIOTIC ANTIBACTERIALS ON PHAGOCYTOSIS

The overall percent suppression effects of the four non-antibiotic test

drugs on phagocytosis of PMNLs have been presented in Table 4.4.1, Figure

4.4.1; Table 4.4.2, Table 4.4.3 and appendices 6-8. The reduction in phagocytosis

was sized in percent values extracted from Table 4.4.3 by subtracting the mean

phagocytic efficiency value of a specific concentration from the mean phagocytic

efficiency value in HBSS. Chlorpromazine at its 2.5% stock solution exerted

78.81% inhibitory effects on phagocytic efficiency of PMNLs. While the percent

inhibitory effect on phagocytosis of PMNLs at 1:10, 1:100 and 1:1000 dilutions

were 37.17, 5.20, and 0% respectively. The zero effect of 1:1000 dilution of stock

solution of Chlorpromazine on phagocytosis of PMNLs was same as the effect in

HBBS.

Lidocaine at its 2% stock solution suppressed phagocytic efficiency of

polymorphonuclear leukocytes by 67.29%. The percent reduction in phagocytic

eficiency of PMNLs at 1:10, 1:100 and 1:1000 dilution were 45.80, 6.76 and 0.3%,

respectively.

Povidone-iodine suppressed phagocytic efficiency of PMNLs by 100% at

its 10% stock solution. Whereas the ten fold dilutions of its stock solution i.e.

1:10, 1:100 and 1:1000 exerted a reduction of 78, 10 and 0.87%, respectively.

DMSO 30% exerted a 66% inhibitory effect on phagocytosis of PMNLs,

whereas its 1:10, 1:100 and 1:000 dilutions affected a 24, 0.80 and 0% reduction in

phagocytic efficiency of PMNLs.


125

Table 4.4.1: Suppression of phagocytic efficiency of polymorphonuclear leukocytes by stock solutions of 4 non-antibiotic antibacterials and their ten fold dilutions.


and their stock solutions

Percent suppression

at stock solution strength

Percent suppression

at 1:10 dilution

Percent suppression

at 1:100 dilution

Percent suppression

at 1:1000 dilution

Percent suppression

in HBSS

Chlorpromazine

2.5% w/v 78.810 37.17 5.20 0.00 0

Lidocaine

2 % w/v 67.29 45.80 6.76 0.30 0

Povidone-iodine

10 % w/v 100.00 78.00 10.00 0.87 0

Dimethylsulphoxide

30% v/v 66.00 24.00 0.80 0.00 0

HBSS = Hank’s Balanced Salt solution


126

0

20

40

60

80

100

120

Stocksolutions

1:10 1:100 1:1000 HBSS

% E

ffect

CPZ 2.5% Lidocaine 2%Povidone iodine 10% DMSO 30%

Ten fold dilutions

Figure 4.4.1: Percent inhibitory effect of different stock solutions of non antibiotic antibacterials and their ten fold dilutions on phagocytic efficiency of polymorphonuclear leukocytes

HBSS = Hank’s Balanced Salt Solution CPZ = Chlorpromazine DMSO = Dimethylsulphoxide


127

Table 4.4.2: Analysis of variance table for influence of different stock solutions of non-antibiotic antibacterials and their ten fold dilutions on phagocytic efficiency of polymorphonuclear leukocytes.

Source of variation

Degrees of freedom

Sum of squares

Mean squares F-value

Treatment (T)

Concentration (C)

T x C

Error

3

4

12

220

14.104

140.636

8.190

9.020

4.701

35.159

0.683

0.041

114.67**

857.53**

16.65**

** = Highly significant (P<0.01) Table 4.4.3: Comparison of means for influence of different stock solutions of

non-antibiotic antibacterials and their ten fold dilutions on phagocytic efficiency of polymorphonuclear leukocytes.

Treatment CPZ Lid PI DMSO

Stocck solutions 0.58 ± 1.145 g 0.88 ± 0.075 f 0.00 ± 0.00 h 0.79 ± 0.311 f

1:10 1.70 ± 0.357 d 1.45 ± 0.317 e 0.51 ± 0.067 g 1.78 ± 0.283 d

1:100 2.56 ± 0.220 b 2.37 ± 0.169 b 2.05 ± 0.203 c 2.32 ± 0.102 b

1:1000 2.61 ± 0.255 a 2.66 ± 0.117 a 2.30 ± 0.156 b 2.34 ± 0.091 b

HBSS 2.69 ± 0.270 a 2.66 ± 0.179 a 2.28 ± 0.172 b 2.33 ± 0.097 b

Means sharing similar letters in a row or in a column are statistically non significant (P>0.05). HBSS = Hank’s Balanced Salt Solution CPZ = Chlorpromazine Lid = Lidocaine PI = Povidone-iodine DMSO = Dimethylsulphoxide


128

Phagocytic efficiency of PMNLs (mean±SE) under the influence of stock

solutions of non-antibiotic antibacterials and their 10 fold dilutions have been

presented in Table 4.4.3.

Phagocytosis represents one of the “first line defense” mechanisms against

the pathogens that invade the mammary gland (Schalm et al., 1971).

Polymorphonuclear leukocytes (PMNLs) and macrophages are regarded as the

principal phagocytic cells in lactating and nonlactating mammary glands of cow

(Paape et al., 1979). Rendering the cows neutropenic by administration of anti-

neutrophil serum has been shown to enhance the multiplication of

microorganisms in bovine udder (Schalm et al., 1971). An optimal number and

function of phagocytic cells is essential for preventing as well for rapid clearance

of intramammary infection. This seems especially more important in dairy

buffalo (Bubalus bubalis) than in dairy cow where the number of somatic cell in

milk from uninfected mammary glands is not only low but inflamammatory

response to bacterial invasion is also not as pronoinced as in cattle (Moroni et al.,

2006). One possible biological reason for differences in SCC between dairy

buffalo and dairy cow may be the phagocytic activity of the neutrophils. Sahoo et

al. (1998) reported differences between cow and buffalo in term of concentrations

of hydrolases contents of neutrophils (including lysozyme) and these could affect

the SCC required to reach a given level of phogocytic activity of phogocytes of

the two species.


129

Ideally, microbicidal agents that are not only efficacious, but also lack

toxic effects on host defense mechanisms (most notably phagocytosis) should be

selected for the treatment, prevention and control of mastitis in dairy animals

(Ziv et al., 1983). Phagocytosis and intracellular killing of bacteria by PMNLs are

important physiologic functions mediated through a variety of glycolytic and

oxidative metabolic events (Paape et al., 1979). The most striking metabolic

alteration associated with actual engulfment is a sharp increase in oxygen

consumption due to the increase in glucose metabolism through anaerobic

glycolysis, whereas intracellular killing of ingested bacteria is closely linked with

an oxidative respiratory burst (Sbarra et al., 1977). Antibacterial agents may affect

metabolic processes that are common to microorganisms and to the host. Human

PMNLs incubated with chloramphenicol, rifampin, sodium fusidate, and

tetracyclines, at concentrations similar to in vivo antibiotic levels after treatment

at nomally prescribed dosages, showed markedly depressed migration (Forsgren

and Schmeling, 1977). Incubation with gentamicin caused a similar effect

(Goodhart, 1977). Human PMNLs in the presence of chloramphenicol failed to

show the expected increase in oxygen consumption after particle ingestion, and

the effect was dose-dependent (Kaplan et al., 1969). Preservatives present in

injectable antibiotic products were suspected to decrease phagocytosis

independently of the antibiotic (Goodhart, 1977).

High concentrations of antibiotics are present in the milk during the first

few hours after intramammary injection of antibiotic products intended for the


130

treatment or the prevention of mastitis. The possibility of various antibiotics

producing a defect in phagocytosis has led to the investigation of the effect of

antibacterial drugs, drug products, and vehicles used in intramammary therapy

on in vitro phagocytosis.

Antibiotics and vehicles used in the treatment and prevention of mastitis

were tested in vitro to determine effects on phagocytosis of 32P-labeled S. aureus

by bovine PMNLs isolated from bovine milk (Ziv et al., 1983). Ampicillin,

lincomycin, erythromycin, tetracycline, neomycin, penicillin G, streptomycin,

cephalothin, nitrofurantoin, gentamicin, dicloxacillin, rifampin, trimethoprim,

amikacin, novobiocin-penicillin, polymyxin B, and tiamulin tested at in vitro

concentration of 4000, 2000, and 10µg/ml of incubation mixture. These dilutions

were equivalent to antibiotic values in milk at 0, 6, and 12 hours after injection

into the mammary gland. Vehicles included peanut oil, mineral oil, 3% cabosil in

peanut oil, 2% aluminum monostearate in peanut oil, 25% polyethylene glycol

(PEG) in an aqueous base, 25% PEG in mineral oil, and 25% PEG in peanut oil.

These were tested at dilutions of 1:100, 1:1000, 1:5000, and 1: 10,000.

The percentage of phagocytosis was determined and was compared with

the percentage of phagocytosis in phosphate-buffered saline solution (PBSS).

Antibiotics and vehicles dilutd in 0.0132M PBSS (pH 7.4); combined with

skimmed milk, PMNLs (12.5 x 106), and incubated for 1 hur (37ºC, no rocking)

followed by rocking for 1 hour. A minimum of 2 runs for the antibiotics and 3

runs for the vehicles were performed with all concentrations or dilutions assayed


131

in a single day’s run. Phagocytosis was significantly (P<0.05) reduced at the high

and intermediate concentrations for tiamulin, nitrofurantion, rifampin,

chloramphenicol, amikacin, and at the high concentrations for gentamicin,

tetracycline, and novobiocin-penicillin. The only vehicle that inhibited

phagocytosis significantly was peanut oil without additives at the 1:100 dilution.

Paape et al. (1991) tested in vitro sixty-three drugs, belonging to 10

chemical classes, to determine effect on phagocytosis of 32P-labeled Staphylococcus

aureus by neutrophils isolated from milk. Within each class, the number of

antibiotics tested were: nonsteroidal anti-flamammatory drugs (NSAID; 8),

peptolids (2), aminoglycosides (8), tetracyclines and fusidic acid (4), β-lactam

antibiotics (25), secretolytic agents (2), macrolides (5), polypeptides (2), and

antibacterial quinolones (8). Percentage of phagocytosis was determined after

incubating (2 hours at 37ºC) 12.5 x 106 viable neutrophils, 200 x 106 32P-labeled S.

aureus with antibiotics and 5% skimmed milk. Concentrations of antibiotics

tested were 1000, 500, and 10µg/ml of incubation media. When compared with

nonantibiotic control at the highest drug concentration, the NSAID acetylsalicylic

acid and centrophenoxine increased phagocytosis 23.2 and 8.8%, respectively,

and benzydamine, indomethacin, phenylbutazone, ibuprofen, and

acetaminophen decreased phagocytosis 22.8, 14.2, 9.8, 27.0 and 18.2%

respectively. The peptolids novobiocin and pristiamycin decreased phagocytosis

24.5, and 22.0%, respectively. The aminoglycosides tobramycin, amikacin, and

gentamicin decreased phagocytosis 21.1, 15.4, and 9.2%, respectively. For the


132

tetracyclines and fusidic acid, minocycline and doxycycline decreased

phagocytosis 39.8 and 54.2%, respectively. The β-lactam antibiotics carfecillin,

cephapirin sodium, and cephacetrile sodium decreased phagocytosis 11.2, 12.8,

and 23.8%, respectively. The secretolytic agent, bromhexin, increased

phagocytosis 10.8%. these data indicate that the potential for enhanced

phagocytosis exist through use of some NSAID, and for depressed phagocytosis

through use of aminoglycosides, peptolids, tetracyclines, and β-lactams, as ell as

certain other NSAID.

Heretofore, the effects of nonantibiotic antibacterials on the viability of

PMNLs and their phagocytic abilities have not been investigated.

Influence of antibiotics on viability and phagocytic efficiency of PMNLs

have been studied by many investigators; (Paape et al., 1991; Nickerson et al.,

1985; Ziv et al., 1983; and Dulin et al., 1984). The present study is the first report

dealing with the effect of non-antibiotic antibacterials on the viability and

phagocytic efficiency of PMNLs.

It can be seen from Table 4.4.3 that mean phagocytic efficiency value of

chlorpromazine 2.5% and its 1:10, 1:100 dilution were significantly lower (P<0.05)

than that of control. Whereas at 1:1000 dilution mean phagocytic efficiency value

(2.61 ± 0.25) was not significantly lower (P > 0.05) than control (i.e. HBSS).

It indicates that under the influence of 2.5% chlorpromazine and its 1:10

and 1:100 dilutions, the mean phagocytic efficiency figures were significantly

lower (P < 0.05) as compared to phagocytic efficiency figures of PMNLs in HBSS.


133

This mean these three concentration put significantly reducing effect on

phagocytosis of PMNLs.

Chlorpromazine at 1:1000 dilution put same not significant effect (P>0.05)

on phagocytosis as HBSS, which served as control. This means that 1:1000

dilution of chlorpromazine has same effect on phagocytosis as that exerted by

HBSS.

It can be seen from Table 4.4.3 that mean phagocytic efficiency value of

PMNLs in lidocaine 4% and its 1:10, and 1:100 dilution were significantly lower

(P < 0.05) than that in control. Contrarily, at 1:1000 dilutions the mean phagocytic

efficiency value (2.66 ± 0.11) was non-significantly lower (P > 0.01) than that in

control (HBSS).

This indicates 1st three concentrations were detrimental to both viability

and functionality of PMNLs. The highest dilution of lidocaine (1:1000) was safe

for the cells functions and viability.

As can be seen in Table 4.3.3, povidone-iodine at 10% concentration was

100% toxic to the viability of PMNLs, as no live cell survived at this

concentration. Next two concentrations (1:10 and 1:100) of povidone-iodine were

also associated with a significant reduction in viability as compared to the value

in HBSS. This means that these two dilutions were not favourable for the

viability of PMNLs. However, viability in 1:1000 dilution of 10% povidone-iodine

was near to the viability (P ≥ 0.05) recorded in HBSS (control).


134

It is clear that phagocytosis was zero percent in 10% solution of povidone-

iodine. It was concluded that this concentration was 100% toxic to PMNLs cells.

Even in 1:100, dilution phagocytic efficiency of PMNLs was significantly lower (P

< 0.05) as compared to phagocytosis in HBSS. But at 1:1000 dilution of povidone-

iodine phagocytic efficiency was non-significantly (P>0.05) different from that

recorded in HBSS.

The Federal Agency for Food and Drug Administration (FDA), USA has

approved povidone-iodine for use in nonprescription first-aid antiseptic

products. Use of the term first-aid implies that povidone-iodine can be used for

short-term treatment (approximately 1 week). Results from both superficial and

deeper wounds with contralateral control indicates that there were no

statistically significant differences in mean healing times between any of the

treatment groups and controls. In addition, these pathological and histological

studies did not indicate any deleterious effect of povidone-iodine on wound

healing. However, in in vitro studies, the researchers have examined the effect of

povidone-iodine on several of the cellular component of healing mechanisms.

Van Den Broek et al. (1982) found povidone-iodine solution at concentrations

greater than 0.05% to be toxic to granulocytes; monocytes showed some effects of

toxicity at concentrations above 0.05% and complete toxicity at concentrations

above 1%. Tatnall et al. (1987) found concentrations of povidone-iodine greater

than 0.004% to be 100% toxic to keratinocytes.


135

Lineaweaver et al. (1985) identified 0.05% as safe concentration of

povidone-iodine for fibroblasts; higher concentrations (including the 10%

concentration that is commonly used in clinical practice) were 100% cytotoxic.

These studies show that in vitro povidone-iodine unless it is diluted to a far lower

concentration than that commonly used in clinical settings, is toxic to all of the

cells types that are essential to the healing process.

As can be seen in Table 4.3.3, the values of mean viability in DMSO 30%

and its 1:10 and 1:100 dilution were significantly lower (P < 0.05) than the

viability value in HBSS. However, viability at 1:1000 concentration of DMSO 30%

was not significantaly lower (P > 0.05) than the value in HBSS.

Plymorphonuclear leukocytes (PMNLs) in stock solution of DMSO (30%)

and its 1:10 dilution had significantly lower phagocytic efficiency as compared to

that in HBSS. Contrarily, at concentrations of 1:100 and 1:1000 phagocytic

efficiency values were not significantly different (P > 0.05) from that in HBSS. It

was concluded that at these concentrations of DMSO did not reduce phagocytic

efficiency of PMNLs.

PHASE II

In this phase the in vivo antibacterial effects of the four non-antibiotics

either alone or in combination with cephradine were studied by instilling these

drugs into buffalo quarters affected with mastitis. Treatment effect on mastitis

cure was studied on the basis of the following parameters:


136

1) Mastitis screening tests (SFMT and CMT) based cure rates

2) Quarter somatic cell count.

3) Bacteriological cures on day 14 and day 28 post initiation of

intramammary treatment.

4) Effect of treatment on the milk yield of the treated quarters.

4.5 MASTITIS SCREENING TESTS (SFMT AND CMT) BASED CURE RATES IN QUARTERS TREATED WITH NON-ANTIBIOTIC ANTIBACTERIALS ALONE AND IN COMBINATION WITH CEPHRADINE

Using Surf Field Mastitis Test (SFMT), testing of quarters treated with

intramammary infusions of non-antibiotic antibacterials alone on day 14 post

initiation of treatment, the highest cure rate 60% was achieved with

chlorpromazine followed by povidone-iodine 46.6%, lidocaine 43.3% and

dimethylsulphoxide 10 %, respectively (Table 4.5.1). Barring PI, all non-antibiotic

antibacterials tested in vivo in the present study exhibited a synergy with

cephradine (Ceph). Thus day-14 post initiation of treatment, SFMT based cure

rates in quarters treated with chlorpromazine + cephradine, lidocaine +

cephradine, povidone-iodine + cephradine, and dimethylsulphoxide +

cephradine were 66.6, 66.6, 50 and 66.6 percent, respectively. Surf Field Mastitis

Test based quarter cure rate on day 28 post initiation of treatment with non-

antibiotic antibacterials alone as well as their combinations with Cephradine

were similar to those recorded on day 14 Table 4.5.2.

As can be seen in Tables 4.5.3, 4.5.4 and 4.5.5, California Mastitis Test

based percent quarter cure rates on day 14 and day 28 in non-antibiotic

antibacterials treatment groups G1 thru G4 and in groups treated with non-


137

antibiotic antibacterials + cephradine (G6 thru G9) were almost similar to those

recorded with Surf Field Mastitis Test on the corresponding time points. This

close proximity of percent quarter cure rates detected with these two animals-

side screening tests attests their comparable efficiency of mastitis detection

reported previously (Muhammad et al., 1995; Fazal-ur-Rehman, 1995).

Table 4.5.1 Surf Field Mastitis Test (SFMT) based percent quarters cure rates in various non-antibiotics antibacterials/antibiotic groups on day 14 post initiation of treatment.

Non-antibiotic/antibiotic Groups

SFMT Reaction Proportion of cured quarters to total treated

quarters

Percent cure rate - T + ++ +++

Chlorpromazine (G1) (CPZ) 18 5 6 1 0 18/30 60

Lidocaine (G2 ) (Lid) 13 7 8 2 0 13/30 43.3

Povidone-iodine (G3) (PI) 14 7 7 2 0 14/30 46.6

Dimethylsulphoxide (G4) (DMSO) 3 4 15 4 4 3/30 10

Cephradine (G5) (Ceph) 19 8 3 0 0 19/30 63.3

CPZ + Ceph (G6) 20 6 2 2 0 20/30 66.6

Lid + Ceph (G7) 20 6 2 2 0 20/30 66.6

PI + Ceph (G8) 15 7 6 2 0 15/30 50

DMSO + Ceph (G9) 20 5 4 1 0 20/30 66.6

SFMT Surf Field Mastitis Test - Normal (Negative) T Trace + Weak Positive ++ Distinct positive +++ Strong positive All treated quarters (n = 30) were clinical and reacted strongly (+++) on the day of first intramammary infusion.


138

Table 4.5.2 Surf Field Mastitis Test (SFMT) based percent quarter cure rates in various non-antibiotic antibacterials/antibiotic groups on day 28 post initiation of treatment.


SFMT Reaction Proportion of cured quarters to total treated

quarters



Lidocaine (G2 ) (Lid) 14 6 8 2 0 14/30 46.6




CPZ + Ceph (G6) 20 7 2 1 0 20/30 66.6

Lid + Ceph (G7) 20 6 2 2 0 20/30 66.6

PI + Ceph (G8) 16 6 5 3 0 16/30 53.3

DMSO + Ceph (G9) 20 5 4 1 0 20/30 66.6

SFMT Surf Field Mastitis Test - Normal (Negative) T Trace + Weak Positive ++ Distinct positive +++ Strong positive All treated quarters (n = 30) were clinical and reacted strongly (+++) on the day of first intramammary infusion


139

Table 4.5.3 California Mastitis Test (CMT) based percent quarter cure rates in various non-antibiotic antibacterials/antibiotic groups on day 14 post initiation of treatment.


CMT Reaction Proportion of cured quarters to total treated

quarters



Lidocaine (G2 ) (Lid) 13 7 8 2 0 13/30 43.3


Dimethylsulphoxide (G4) (DMSO) 2 4 16 4 4 2/30 6.6


CPZ + Ceph (G6) 20 4 4 2 0 20/30 66.6

Lid + Ceph (G7) 20 5 5 0 0 20/30 66.6

PI + Ceph (G8) 15 7 6 2 0 15/30 50

DMSO + Ceph (G9) 20 4 5 1 0 20/30 66.6

CMT California Mastitis Test - Normal (Negative) T Trace + Weak Positive ++ Distinct positive +++ Strong positive All treated quarters (n = 30) were clinical and reacted strongly (+++) on the day of first intramammary infusion


140

Table 4.5.4 California Mastitis Test (CMT) based percent quarter cure rates in various non-antibiotic antibacterials alone and in combination with cephradine on day 14 and day 28 post initiation of treatment.


CMT Reaction Proportion of cured quarters to total treated

quarters


Chlorpromazine (G1) (CPZ) 19 3 7 1 0 19/30 63.3

Lidocaine (G2 ) (Lid) 14 8 6 2 0 14/30 46.6

Povidone-iodine (G3) (PI) 15 7 7 1 0 15/30 10



CPZ + Ceph (G6) 20 5 3 2 0 20/30 66.6

Lid + Ceph (G7) 20 6 2 2 0 20/30 66.6

PI + Ceph (G8) 16 8 6 2 0 16/30 53.3

DMSO + Ceph (G9) 20 4 5 1 0 20/30 66.6

CMT California Mastitis Test - Normal (Negative) T Trace + Weak Positive ++ Distinct positive +++ Strong positive All treated quarters (n = 30) were clinical and reacted strongly (+++) on the day of first intramammary infusion


141

Table 4.5.5 Surf Field Mastitis Test and California Mastitis Test based percent cure rates of non-antibiotic antibacterials alone and in combination with cephradine on day 14 and day 28 post initiation of treatment.

Non-antibiotic/ antibacterials Groups

SFMT on day 14

%

SFMT on day 28

%

CMT on day 14

%

CMT on day 28

% Chlorpromazine (G1) (CPZ)

60 60 60 63.3

Lidocaine (G2 ) (Lid)

43.3 46.66 43.3 46.6

Povidone-iodine (G3) (PI)

46.6 46.6 46.6 50

Dimethylsulphoxide (G4) (DMSO)

10 10 6.6 10

Cephradine (G5) (Ceph)

63.3 63.3 63.3 63.3

CPZ + Ceph (G6) 66.6 66.6 66.6 66.6

Lid + Ceph (G7) 66.6 66.6 66.6 66.6

PI + Ceph (G8) 50 53.3 50 53.3

DMSO + Ceph (G9) 66.6 66.6 66.6 66.6

SFMT = Surf Field Mastitis Test CMT = California Mastitis Test All quarters (n = 30) were clinical and reacted strongly (+++) on the day of first intramammary infusion


142

4.6 EFFECT OF THE INFUSIONS (N = 4) OF NON-ANTIBIOTIC ANTIBACTERIALS ALONE AND IN COMBINATION WITH CEPHRADINE ON QUARTER SOMATIC CELL COUNT (QSCC) Table 4.6.1 and Table 4.6.2 respectively depict the quarter somatic cell

counts (Mean ± SD), at day 14 and day 28 post infusions of non-antibiotic

antibacterials singly and in combination with cephradine. The same has been

stylized in Figure 4.5.1. Appendices 9-18 give the details of there values. As can

be seen in Table 4.6.1, of the 4 non-antibiotic antibacterials infused singly,

chlorpromazine affected the highest percent reduction (61.48%) in quarter

somatic cell count on day 14 post initiation of treatment followed by lidocaine

(51.9%) and povidone-iodine (47.52%). Contrarily, quarters infused with DMSO

were found to have an increment of 9.51% in somatic cell count on day 14 post

initiation of treatment as compared to the pretreatment count. On day-28 post

initiation of infusions, quarters treated with chlorpromazine, lidocaine, and

povidone-iodine were observed to have only marginally different reductions in

somatic cell count (62.5, 47.93 and 53.76%, respectively) from those recorded on

day 14 post initiation of treatment with these non-antibiotic antibacterials.

Quarters treated with DMSO, however, registered 11.39% further increment in

somatic cell count over the value recorded on day 14 post initiation of treatment.

Thus, the quarter somatic cell count in DMSO treated group (G4) was 20.09%

higher than the values recorded on the first day of infusion.

Infusions of chlorpromazine, lidocaine, povidone-iodine and DMSO in

combinations with cephradine, respectively were found to have affected 66.57,


143

67.30, 53.58 and 65.20% reductions in quarter somatic cells counts on day 14 post

initiation of treatment (Table 4.6.1). The corresponding percent reductions in

quarter somatic cell counts two weeks later (i.e. day 28) were only marginally

different from those recorded on day 14 (Table 4.6.2).

Statistical analysis revealed that chlorpromazine, lidocaine and povidone-

iodine when used singly affected a significant decrease (P < 0.05) in quarter

somatic cell counts on day 14 post initiation of treatment (Table 4.6.4; Figure

4.5.1). The values recorded at day 28 differed non significantly (P > 0.05) from

those recorded at day 14. Quarters treated with DMSO on the other hand had

significantly (P < 0.05) higher somatic cell counts both on day 14 and day 28 post

initiation of treatment than those recorded on the day of first infusion of this

non-antibiotic antibacterials. Quarter somatic cell counts recorded on day 14 as

well day 28 post initiation of treatment with chlorpromazine + cephradine,

lidocaine + cephradine, and povidone-iodine + cephradine were not-significantly

(P > 0.05) lower than those recorded on these two sampling time points with

uncomplemented use of the 3 non-antibiotic antibacterials. The quarter somatic

cell counts on day 14 and day 28 post infusion of DMSO with cephradine were

sampling time points in quarters treated with a combination of significantly (P <

0.05) lower than somatic cell counts of quarters treated with DMSO alone. This

indicates a favourable effect of cephradine + DMSO combination in mastitis

therapy.


144

As a consequence of injury to the udder tissue, the blood leukocytes,

(popularly known as milk somatic cells) especially neutrophils, migrate through

the intercellular spaces of the capillary and the mammary alveolar wall into the

milk in an attempt to remove the invading pathogens. The number of milk

somatic cells, therefore, increases tremendously in the wake of invasion by the

microorganism (depending upon the severity of the injury induced by the

mastitis pathogens). Worldwide milk somatic cell count is the most widely used

criteria to determine and monitor the udder health status as well as for

estimating the milk yield losses. Normal somatic cell count in buffalo milk from

non infected quarters reportedly varies from 50,000 to 375,000 per ml (Dhakal et

al., 1992; Silva and Silva, 1994; Singh and Ludri, 2001; Moroni et al., 2006).

Differences between cow and buffalo milk in terms of total somatic count as well

as differential counts were documented by Sri Lankan workers (Silva and Silva,

1994). As per these workers, milk somatic cell count in buffalo is lower than that

in cow and neutrophil is the predominant cell type in buffalo milk from

uninfected quarters whereas, in cow the macrophage is considered to be the

predominant cell type. Even though intramammary infusions of chlorpromazine,

lidocaine and povidone-iodine when given singly or in combination with

cephradine affected a statistically significant reduction in quarter somatic cell

count, the number of somatic cells at both day 14 and day 28 did not decease to

the levels considered normal for buffalo (Dhakal et al., 1992; Silva and Silva, 1994;

Singh and Ludri, 2001; Moroni et al., 2006). It may be due to persistency of


145

inflammatory response for several weeks after the offending microorganism has

been eliminated. At the least 40% of the quarter treated with non-antibiotic

antibacterials were not cured in terms of elimination of the bacterial infections.

These non responder quarters might have also contributed considerably to high

somatic cell count observed in all treatment groups even at day 28 post initiation

of treatment.


146

Table 4.6.1: Effect of intramammary infusions (n = 4) of non-antibiotic antibacterials alone and in combination with cephradine on quarter somatic cells count (QSCC; X 105/mL of milk) on day 14 post initiation of treatment.

Non-antibiotic antibacterials alone and in combination with

cephradine

Group designation *

Pre-treatment QSCC

(_X 1 ± SD)

QSCC on day 14 post initiation of

treatment

(_X 2 ± SD)

Difference of means

(_X 1 -

_X 2 )

Percent reduction (↓)/ increment (↑) in QSCC on day 14 post-initiation of

treatment

Chlorpromazine (CPZ) G1 14.80 ± 9.39 5.70 ± 4.73 9.10 61.48 ↓

Lidocaine (Lid) G2 14.52 ± 9.35 6.97 ±7.07 7.55 51.9 ↓

Povidone-iodine (PI) G3 15.55 ± 8.32 8.167 ± 7.32 7.39 47.52 ↓

Dimethylsulphoxide (DMSO) G4 17.02 ± 10.27 18.64± 11.51 -1.62 9.51 ↑

Cephradine (Control) G5 17.33 ± 7.50 6.02± 4.91 11.31 65.26 ↓

CPZ + Cephradine G6 14.51 ± 7.83 4.85 ± 4.57 9.66 66.57 ↓

Lid+ Cephradine G7 14.53 ± 9.08 4.75 ± 3.90 9.78 67.30 ↓

PI + Cephradine G8 13.42 ± 7.64 6.23 ± 5.75 7.19 53.58 ↓

DMSO + Cephradine G9 15.32 ± 8.99 5.33 ± 4.83 9.99 65.20 ↓

QSCC = Quarter Somatic Cell Count CPZ = Chlorpromazine PI = Povidone-iodine Lid = Lidocaine DMSO = Dimethylsulphoxide * No. of quarters in each treatment group = 30


147

Table 4.6.2: Effect of intramammary infusions (n = 4) of non-antibiotic anti-bacterials alone and in combination with cephradine on quarter somatic cells count (QSCC; X 105/mL of milk) on day 28 post initiation of treatment.

Non-antibiotics alone and in combination with cephradine

Group designation *

Pre-treatment

QSCC

(_X 1 ± SD)

QSCC on day 28 post initiation of

treatment

(_X 2 ± SD)

Differences of mean

(_X 1 -

_X 2 )


treatment Chlorpromazine (CPZ) G1 14.80 ± 9.39 5.55 ± 5.12 9.25 62.50 ↓ Lidocaine (Lid) G2 14.52 ± 9.35 7.56 ± 8.22 6.96 47.93 ↓ Povidone-iodine (PI) G3 15.55 ± 8.32 7.19 ± 6.52 8.36 53.76 ↓

Dimethylsulphoxide (DMSO) G4 17.02 ± 10.27 20.44 ±12.60 -3.42 20.09 ↑

Cephradine (Control) G5 17.33 ± 7.50 6.21 ± 5.56 11.12 64.16 ↓ CPZ + Cephradine G6 14.51 ± 7.83 4.80± 4.52 9.71 66.91 ↓ Lid+ Cephradine G7 14.53 ± 9.08 4.15 ± 3.33 10.38 71.43 ↓ PI + Cephradine G8 13.42 ± 7.64 5.24 ± 5.10 8.18 60.95 ↓ DMSO + Cephradine G9 15.32 ± 8.99 5.07 ±4.69 10.25 66.90 ↓

QSCC = Quarter Somatic Cell Count CPZ = Chlorpromazine PI = Povidone-iodine Lid = Lidocaine DMSO = Dimethylsulphoxide

* No. of quarters in each treatment group = 30


148

Table 4.6.3: Effect (% decrease or increase) of infusions (n = 4) of non-antibiotic antibacterials alone (groups G1 thru G4) and in combinations with cephradine (groups G6 thru G9) on quarter somatic cells count (QSCC) recorded on day 14 and day 28 post-initiation of treatment

Non-antibiotic antibacterials alone and in combination with cephradine

Group designation *


treatment


treatment Chlorpromazine (CPZ) G1 61.48 ↓ 62.50 ↓

Lidocaine (Lid) G2 51.9 ↓ 47.93 ↓

Povidone-iodine (PI) G3 47.52 ↓ 53.76 ↓

Dimethylsulphoxide (DMSO) G4 9.51 ↑ 20.09 ↑

Cephradine (Control) G5 65.26 ↓ 64.16 ↓

CPZ + Cephradine G6 66.57 ↓ 66.91 ↓

Lid+ Cephradine G7 67.30 ↓ 71.43 ↓

PI + Cephradine G8 53.58 ↓ 60.95 ↓

DMSO + Cephradine G9 65.20 ↓ 66.90 ↓



149

Table 4.6.4: Effect of infusions (n = 4) of non-antibiotic antibacterials alone (groups G1 thru G4) and in combination with cephradine (Groups 6 thru 9) on quarter somatic cells count (QSCC x 105) recorded on day 14 and day 28 post-initiation of treatment

Non-antibiotic antibacterials alone

and in combination with cephradine

Group designation *

Pre-treatment QSCC

QSCC on day 14 post-initiation of treatment

QSCC on day 28 post-initiation of

treatment Chlorpromazine (CPZ) G1 14.80±14.48bc 5.70±2.98d 5.55±2.93d Lidocaine (Lid) G2 14.52±9.35bc 6.97±7.07d 7.56±8.22d Povidone-iodine (PI) G3 14.55±8.32bc 8.16±7.32d 7.19±6.50d Dimethylsulphoxide (DMSO) G4 17.02±10.27abc 18.64±11.51ab 20.44±12.06a

Cephradine (Control) G5 17.33±7.48abc 6.02±4.80d 6.21±4.43d CPZ + Cephradine G6 14.52±7.83bc 4.85±4.69d 4.80±4.52d Lid+ Cephradine G7 14.54±9.08bc 4.75±3.90d 4.15±3.33d PI + Cephradine G8 13.42±7.64c 6.23±5.75d 5.24±5.10d DMSO + Cephradine G9 15.32±8.99bc 5.33±4.82d 5.07±4.69d Means sharing similar letters in a row or in a column are statistically non-significant (P>0.05).



150

0

5

10

15

20

25

G1 G2 G3 G4 G5 G6 G7 G8 G9

WSC

Cx1

05

Pre-treatmentPost-treatment on day 14Post-treatment on day 28

Groups

Figure 4.5.1: Effect of infusions (n = 4) non-antibiotic antibacterials alone and

in combination with cephradine in reducing QSCC (means ± SE) after treatment

G1 = Chlorpromazine G2 = Lidocaine G3 = Povidone-iodine G4 = Dimethylsulphoxide G5 = Cephradine (Control drug) G6 = Chlorpromazine + Cephradine G7 = Lidocaine + Cephradine G8 = Povidone-iodine + cephradine G9 = Dimethylsulphoxide + Cephradine QSCC = Quarter somatic cell counts


151

4.7 BACTERIOLOGICAL CURE RATES OF INFUSIONS (n = 4) OF NON-ANTIBIOTIC ANTIBACTERIALS SINGLY AND IN COMBINATIONS WITH CEPHRADINE AT DAY 14 AND 28 POST INITIATION OF TREATMENT

Table 4.7 depicts the bacteriological cure rates (% quarters cured out of 30)

of infusions of 4 non-antibiotic antibacterials used in a dissociated regimen (i.e.

non-antibiotic antibacterials alone) and in an associated regimen (i.e.

combination of non-antibiotic antibacterials and cephradine). As can be seen in

this table, of the 4 non-antibiotic antiobacterials infused singly, chlorpromazine

affected the highest bacteriological cure rate (53.3%), followed by povidone-

iodine (43.3%), lidocaine (33.3%) and DMSO (6.6%) on day 14 post initiation of

treatment. All non-antibiotic antibacterials but DMSO had higher bacteriological

cure rates (chlorpromazine = 56.6%; povidone-iodine = 46.6%; lidocaine = 40%)

on day 28 post initiation of treatment than the corresponding values on day 14.

In the case of DMSO, the bacteriological cure rates on day 28 post initiation of

treatment was the same (i.e. 6.6%) as that on day 14.

Daily intramammary infusions of the control drug (cephradine) for 4 days

cured 53.3 and 56.6 % of 30 quarters on day 14 and day 28 post initiation of

treatment, respectively. The associated (i.e. combined) use of chlorpromazine +

cephradine, lidocaine + cephradine, povidone-iodine + cephradine, and DMSO +

cephradine, respectively cured 56.6, 60, 50 and 56.6% of 30 quarters treated with

each of these non-antibiotic antibacterial + cephradine combination on day 14

post initiation of treatment. The corresponding percent cure rates on day 28 post


152

initiation of these combination treatments were 60, 60, 50, 56.6%, respectively.

Thus whereas, combination of chlorpromazine, lidocaine and povidone-iodine

with cephradine exhibited a synergy in terms of bacteriological cure rate,

combination of DMSO with cephradine was bereft of this desirable outcome. A

very low bacteriological cure rate of DMSO (6.6% quarters at both day 14 and

day 28 post initiation of treatment) can be ascribed to a weak antibacterial

activity of this solvent chemical (Brayton, 1986; Plumb, 1999). DMSO, however, is

a very potent anti-inflammatory agent. It rapidly penetrates tissue membranes

with little or no residual effects and has an affinity for inflamed tissues. This

drug was found to have merit in the treatment of intra-mammary infections by

carrying antibiotics through occluded milk spaces to foci of infection (Ziv, 1967).

Very good to excellent results have been reported by intramammary infusion of

15-50 ml of 90% DMSO + antibiotics / normal saline per quarter in the treatment

of subclinical or chronic mastitis (Keskintepe et al., 1992; Koller, 1976).

Lorda and Grimeldi (1979) treated 80 mastitic cows during lactation or in

dry period with antibiotic preparations in DMSO, in oil or in water. Treatment

with chloramphenicol / nifuroxazide / prednisolone / DMSO, preparation gave

a higher percentage of cured quarters than either the water based or oil based

preparations.

In a S. aureus mastitis treatment programme in Brazil, penicillin G (100000

IU), streptomycin (1000 mg) in an aqueous-oily or aqueous vehicles with or

without the addition of 20% DMSO was used in three herds. An average of 70.6%


153

bacteriologically negative glands were obtained using this combination of

antibiotics in the aqueous-oily vehicle and 79.4% in the aqueous vehicle 120

hours after single application. The addition of DMSO in formulations had

significant effect only in the herd with chronic mastitis (Figueiredo et al., 1993).

Ballarini (1972) studied the effect of DMSO as vehicle for antibiotic

(penicillin) and corticosteroid (flumethasone) in the treatment of bovine mastitis.

DMSO was used as a 90% solution. Cases of acute parenchymatous mastitis due

to E. coli in 136 cows were treated by repeated intramammary infusions of 0.25-

0.50 mg flumethasone, associated with an appropriate antibiotic in DMSO.

Favourable results were noticed in 95% of cases. Either during dry period or

during lactation, 151 quarters were similarly treated with flumethasone or

penicillin in DMSO; 90% of the acute cases responded to treatment during

lactation. Of the chronic cases, only 24% responded to treatment during lactation,

while 46% were cured by treatment during dry period. No advantages were

detected from the use of DMSO as vehicle when treating infections localized in

teat canal or milk system.

The sensitivity of 132 samples (isolates) cultured from infected mammary

glands was studied in 3 herds of cattle from the milk basin of Belo Horizonte,

Brazil. DMSO added to the culture medium in concentrations of 10 and 15% did

not modify the behavior of the isolates which were initially resistant to penicillin

(75%) and streptomycin (38%). However, it increased the sensitivity of isolates

that were initially only slightly sensitive and at 6% and 10% concentrations,


154

DMSO did not inhibit the growth of any of the isolate, while at 15% it inhibited

27.6% and at 20% it inhibited 100% of the isolates (Andrade et al., 1993).

A reasonably high (53.3%) bacteriological cure rate obtained by

intramammary infusions of chlorpromazine when used singly may be attributed

to a high antibacterial activity of this phenothiazine (Levaditi et al., 1951;

Bourden, 1961; Molnar et al., 1977; Kristiansen and Mortensen, 1987; Amaral et

al., 1996; Kristiansen and Amaral, 1997; Amaral et al., 2001).

A special type of synergy is produced by the combination of conventional

antibiotics and non-antibiotic antibacterials (e.g. β-lactam antibiotics and

phenothiazines). Strains of S. aureus that are resistant to methacillin (MIC 100

mg/L) became sensitive to concentrations of methacillin as low as 6.2 mg/L

when they were cultured in medium containing chlorpromazine to which the

organism was resistant. Other chlorpromazine related compounds can reduce

the MIC even further to 1.6 mg/L. The same effect was seen when penicillin

resistant corynebacterium isolates were cultured in the presence of low

concentration of chlorpromazine and penicillin (Kristiansen et al., 1993).

The studies cited above have primarily been in vitro. However, there are a

number of reports that describe the antimicrobial activity of phenothiazine in

human and animals. Children with recurrent pylonephritis produced by

Escherichia coli reportedly had significantly fewer relapses, when antibiotic (e.g.

gentamicin) was accompanied by subclinical doses of chlorpromazine (Molnar et

al., 1990). This has been attributed to an enhancement in the activity of


155

gentamicin as a consequence of increased permeability of the antibiotic into E.

coli as the MIC is reduced as much as four folds by traces of the chlorpromazine

(Molnar et al., 1990). Soon after chlorpromazine was widely used, psychotic

patients presenting with cavitary infections of Mycobacterium tuberculosis were

cleared of this infection (Kaminska, 1967). The MIC of chlorpromazine for

various strains of mycobacteria were 10μg/ml and 15 μg/ml, the bactericidal

concentration of chlorpromazine for mycobacterium was shown to be 100-300

μg/ml (Molnar et al., 1997).

Higher bacteriological cure rates of chlorpromazine and cephradine

combination may be attributed to synergy between these two microbicidal

agents. The present study is the first ever report on the use of chlorpromazine

(singly and in combination with cephradine) in the treatment of mastitis.


156

Table 4.7: Bacteriological cure rates of infusions (n = 4) of non-antibiotic antibacterials singly and in combinations with cephradine on day 14 and day 28 post-initiation of treatment.

Non-antibiotic antibacterials alone and in combination

with cephradine

Group designation

*

Pretreatment bacteriologically +ve quarters (%)

Bacteriological cure rate (%)

on day 14 post initiation of

treatment

Bacteriological cure rate (%)

on day 28 post-initiation of treatment

Chlorpromazine (CPZ) G1 27/30 = 90 16/30=53.3 17/30=56.6

Lidocaine (Lid) G2 25/30=83.3 10/30= 33.3 12/30=40

Povidone-iodine (PI) G3 28/30 =93.3 13/30=43.3 14/30=46.6

Dimethylsulphoxide (DMSO) G4 26/30=86.6 2/30=6.6% 2/30=6.6

Cephradine (Control) G5 28/30= 93.3 16/30=53.3 17/30=56.6

CPZ + Cephradine G6 27/30 = 90 17/30=56.6 18/30=60

Lid+ Cephradine G7 28/30 =93.3 18/30=60 18/30=60

PI + Cephradine G8 26/30=86.6 15/30=50 15/30=50

DMSO + Cephradine G9

27/30 = 90

17/30=56.6

17/30=56.6

*No. of quarters in each treatment group = 30


157

4.8 EFFECT OF INFUSIONS (n = 4) OF NON-ANTIBIOTIC ANTIBACTERIALS ALONE AND IN COMBINATION WITH CEPHRADINE ON MILK YIELD OF THE BUFFALO QUARTERS AFFECTED WITH CLINICAL MASTITIS OVER A PERIOD OF 4 WEEKS POST INITIATION OF TREATMENT

The overall effect of infusions of non-antibiotic antibacterials on milk yield

of the buffaloes quarters affected with clinical mastitis has been depicted in Table

4.8.1. Prioritization of different treatments on the basis of mean milk yield loss

before and at day 28 post-initiation of treatment has been given in table 4.8.2. The

same has been stylized in Figure 4.8.

The pre-treatment milk yield losses in quarters treated with CPZ, Lid, PI

and DMSO alone were 31.69, 30.06, 33.08 and 24.80%, respectively. The

corresponding values on day 28 post-initiation of treatment were 16.31, 22.53,

27.4 and 34.55%. Thus the highest percent net recovery of milk yield was

observed in CPZ group (15.38) followed by Lid (7.53) and PI (5.68) group.

Contrarily, in DMSO group, the milk yield loss increased further instead of

decline. In other words, a significant (p < 0.05) reduction (table 3) in losses of

milk yield were observed in CPZ group as compared to other treatment groups.

This may be due to strong antibacterial effect of CPZ (Kristiansen et al., 1990;

William, 1995 and Amaral et al., 1996; Martins et al., 2008). On the other hand, the

quarters treated with DMSO registered a further significant (p < 0.05) increase in

loss of milk yield. This may be attributed to inability of dimethylsulphoxide to

control intramammary bacterial infections as this chemical possesses only a weak


158

antibacterial activity (Plumb, 1999). However, it is inconsistent with the previous

findings of Andrade et al. (1993). The net recovery of milk yield in quarters

treated with povidone-iodine was very poor. It may be due to its irritability on

udder tissue. Udder irritation produced by antimastitic preparations is one of the

main criterions in the evaluation experiments (Neumeister, 1971; Muhammad et

al. 1990). More irritant the drug, the lesser it is desirable for intramammary

infusion (Uvrov, 1971). In the present study, although all four non-antibiotic

antibacterials with and without cephradine were administered by intramammary

infusion, no attempt was made to study their comparative irritability in the form

of increases in milk somatic cell counts. This is one of the shortcomings of the

present study which should be addressed in any similar future investigation on

the use of non-antibiotic antibacterials in mastitis treatment.

Looking at combination regimens (table 4.8), the pre-treatment milk yield

losses in quarters treated with CPZ, Lid, PI and DMSO all in combination with

cephradine were 29.19, 34.75, 31.42 and 33.10%, respectively. The corresponding

values on day 28 post-initiation of treatment were 11.85, 13.47, 19.01 and 15.38%

with a resultant net recovery of milk yields of 17.25, 21.28, 12.41 and 17.72%.

Thus the highest net recovery of milk yield on day 28 post-initiation of treatment

was 21.28% for Lid + Ceph group followed by DMSO + Ceph, CPZ + Ceph and

PI + Ceph groups. We may say that the quarters receiving combination

treatments the most important reduction (p < 0.05) in the loss of milk yield was

observed in Lid + Ceph. group (table 4.8.2) as compared to other treatment


159

groups. This may be due to strong antibacterial effect of Lid against Gram

negative bacteria and that of Cephradine against Gram positive bacteria

(Schmidt and Rosenkranz, 1970).

In sum, among the 4 non-antibiotic antibacterials tested alone,

Chlopromazine (CPZ) showed a relative more promising recuperative effect on

the milk yield of clinically mastitic quarters of dairy buffaloes.

Dimethylsulphoxide (DMSO) when infused alone aggravated the milk loss of

clinically mastitic quarters. Adjuncting Cephradine with each of the 4 non-

antibiotic antibacterials showed that the lidocaine-cephradine combination had

the highest effect (p< 0.05) on the net recovery of milk yield loss at day 28 post

initiation of treatment.

The present study was the first one on the use of Chlorpromazine and

Lidocaine in the treatment of mastitis. In view of a small number of quarters

(n=30) on which each of the 4 non-antibiotic antibacterials were tested, a larger

field trials involving a larger number of mastitis affected animals and quarters is

clearly warranted.


160

Table 4.8.1: Effect of non-antibiotic antibacterials infusions (n = 4) alone and in combination with cephradine on milk yield (Loss of milk in liters/quarter/day) in buffaloes affected with clinical mastitis over a period of 4 weeks post initiation of treatment

Non-antibiotic

antibacterials alone and in combination

with cephradine

Milk yield of Mastitic quarters before and after

treatment

Milk yield of Contralateral

Normal quarters

% Reduction in milk yield loss of affected quarters compared to that of contralateral

normal quarters

Net Recovery of milk yield

(%) of treated quarters on day

28

CPZ Pre-treatment 0.97 ± 0.10 Post-treatment 1.18 ± 0.13

1.42 ± 0.11 1.41 ± 0.11

0.45 ± 0.05 0.23 ± 0.07

31.69% 16.31%

15.38

Lid Pre-treatment 1.0 ± 0.09 Post-treatment 1.10 ± 0.07

1.43 ± 0.11 1.42 ± 0.09

0.43 ± 0.12 0.32 ± 0.02

30.06% 22.53%

7.53

PI Pre-treatment 0.89 ± 0.11 Post-treatment 0.98 ± 0.17

1.33 ± 0.12 1.35 ± 0.11

0.44 ± 0.08 0.37 ± 0.11

33.08% 27.4%

5.68

DMSO Pre-treatment 1.03 ± 0.07 Post-treatment 0.89 ± 0.08

1.37 ± 0.07 1.36 ± 0.07

0.34 ± 0.09 0.47 ± 0.08

24.8% 34.55%

-9.75

Ceph (Control) Pre-treatment 0.95 ± 0.09 Post treatment 1.16 ± 0.13

1.45 ± 0.09 1.46 ± 0.08

0.50 ± 0.08 0.30± 0.09

34.48% 20.54%

13.94

CPZ + Ceph Pre-treatment 0.95 ± 0.12 Post-treatment 1.19 ± 0.11

1.34 ± 0.10 1.35 ± 0.10

0.39 ± 0.06 0.16 ± 0.09

29.10% 11.85%

17.25

Lid+ Ceph Pre-treatment 0.92 ± 0.11 Post-treatment 1.22 ± 0.14

1.41 ± 0.10 1.41 ± 0.10

0.49 ± 0.10 0.19 ± 0.08

34.75% 13.47%

21.28

Pi + Ceph Pre-treatment 0.96± 0.26 Post-treatment 1.15 ± 0.11

1.40 ± 0.11 1.42 ± 0.11

0.44 ± 0.07 0.27 ± 0.07

31.42% 19.01%

12.41

DMSO + Ceph Pre-treatment 0.97 ± 0.19 Post-treatment 1.21 ± 0.14

1.45 ± 0.10 1.43 ± 0.09

0.48 ± 0.10 0.22 ± 0.12

33.10% 15.38%

17.72

CPZ = Chlorpromazine HCl Lid = Lidocaine HCl DMSO = Dimethylsulphoxide PI = Povidone-iodine * No. of quarters in each treatment group = 30


161

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

G1 G2 G3 G4 G5 G6 G7 G8 G9

Groups

Yiel

d de

pres

sion

Before treatment After treatment

Figure 4.8: Mean effects of infusions (n = 4) of non-antibiotic antibacterials

alone and in combination with cephradine on milk yield of the buffalo quarters affected with clinical mastitis over a period of 4 weeks post initiation of treatment.

G1 = Chlorpromazine HCl (CPZ) G2 = Lidocaine HCl (Lid) G3 = Povidone-Iodine (PI) G4 = Dimethylsulphoxide (DMSO) G5 = Cephradine (Ceph.) (control) G6 = CPZ + Ceph. G7 = Lid. + Ceph. G8 = PI + Ceph. G9 = DMSO + Ceph.


162

Table 4.8.2: Prioritization of different treatments on the basis of mean milk yield loss (mean ± SE) of buffalo mastitic quarters


alone/ and in combination with cephradine

Pre-treatment milk yield loss (L/quarter/day)

Post-treatment milk yield loss

(L/quarter/day) CPZ 0.45 ± 0.05 abc 0.23± 0.07 hi

Lid 0.43 ± 0.12 c 0.32 ± 0.02 ef

PI 0.44 ± 0.08 bc 0.37 ± 0.11 de

DMSO 0.34 ± 0.09 def 0.47 ± 0.08 abc

Cephradine (Control) 0.50 ± 0.08 a 0.30 ± 0.09 fb

CPZ + Cephradine 0.39 ± 0.06 d 0.16 ± 0.09 j

Lid+ Cephradine 0.49 ± 0.10 ab 0.19 ± 0.08 ij

PI + Cephradine 0.44 ± 0.07 bc 0.27 ± 0.07 gh

DMSO + Cephradine 0.48 ± 0.10 ab 0.22 ± 0.12 hi

Means sharing similar letters in a column or row are statistically non-significant (P > 0.05) CPZ = Chlorpromazine HCl Lid = Lidocaine HCl PI = Povidone - Iodine DMSO = Dimethylsulphoxide

163

CHAPTER 5

SUMMARY

The use of antibiotics in the treatment of mastitis in dairy animals has

been a common practice since 1940s. However, development of antibiotic

resistance, poor cure rates as well as the concern for antibiotic residues in milk

and meat of treated animals have spurred interest in the investigation and use of

some non-antibiotic antibacterials chemicals and non-antibiotic approaches in the

treatment of this disease. The aims of the present study were to determine the in

vitro and in vivo effect of 4 non-antibiotic compounds viz. chlorpromazine (CPZ),

lidocaine (Lid), povidone-iodine (PI), and dimethylsulphoxide (DMSO) in the

treatment of bubaline (dairy buffalo) clinical mastitis and to evaluate their effect

on cell viability and phagocytic efficiency of polymorphonuclear leukocytes.

The study was conducted in two phases, Phase I and Phase II. In Phase-I,

an in vitro antibacterial activity of 4 non-antibiotics (CPZ = 25, 40, 50, 75 and

100µg per disc; Lid = 10, 20, and 25 mg per disc; PI = 10, and 20mg per disc and

DMSO = 50mg per disc) against field mastitis isolates of S. aureus (n = 59), Str.

agalactiae (n = 65) and E.coli (n = 78) and American Type Culture Collection

isolates (S. aureus ATCC 25923, Str. agalactiae ATCC 27956 and E.coli ATCC

25922) was determined by disc difussion method as per the guidelines of

National Committee for Clincial Laboratory Standards. Cephradine (30 µg per

CHAPTER 5 SUMMARY

164

disc) was used as the control drug. Minimum inhibitory concentration (MIC) of 4

non-antibiotics against field mastitis and ATCC isolates was determined by an

agar dilution method. Effect of 4 non-antibiotics on in vitro viability of

polymorphonuclear leukocytes (PMNLs) of rabbits was assayed by trypan blue

dye exclusion test. Their effect on in vitro phagocytic efficiency was assayed in

terms of percent capability of PMNLs to ingest opsonized Candida utilis cells.

Phase II involved evaluation of non-antibiotics alone and in combination

with cephradine in the treatment of clinical bubaline mastitis. A total of 270

clinically mastitic quarters of 249 Nili-Ravi buffaloes were infused with either

non-antibiotic antibacterial alone (CPZ = 50mg + 38ml normal saline; Lid = 10ml

(4%) + 30ml normal saline; PI = 10ml (10%) + 30ml normal saline; DMSO 99.5% =

20ml + 20ml normal saline) or in combination with cephradine (Ceph) (500mg)

for 5 days. The response to these treatments was monitored in terms of Surf Field

Mastitis Test (SFMT) and California Mastitis Test (CMT) based cure rates, effect

of treatment on quarter somatic cell counts (SCC), and bacteriological cure rates

at day 14 and day 28 post initiation of treatment. Effect of infusions of non-

antibiotic antibacterials alone and in combination with cephradine on quarter

milk yield was determined at day 28 post initiation of treatment.

Disc potencies of all 4 non-antibiotic antibacterials and their zones of

growth inhibition were related in a linear fashion. The zones sizes were larger

against ATCC isolates than against field mastitis isolates. In general, filter paper

disc harboring 100µg of CPZ produced the largest zones of growth inhibition

CHAPTER 5 SUMMARY

165

among all potencies of all 4 non-antibiotic antibacterials tested. The lowest disc

potency (25 µg) of CPZ did not produce any zone of growth inhibition against

field mastitis isolates of S. aureus, E.coli as well as E. coli ATCC 25922 whereas

growths of field mastitis isolates of Str. agalactiae and Str. agalactiae ATCC 27956

were inhibited in circular zones with radii of 16-18 and 18 mm, respectively. As

the potency of CPZ increased from 25 to 100 µg per disc, so did the zones of

growth inhibition of field mastitis and ATCC isolates. Ten mg disc of Lid was not

inhibitory for field mastitis isolates of S. aureus, and Str. agalactiae whereas this

potency produced zones of inhibition measuring 16 to 18mm against field

mastitis E. coli isolates. The other two potencies of Lid (20 and 25mg per disc)

were inhibitory over zones ranging from 8 to 24mm and 12 to 24mm for field

mastitis isolates and ATCC isolates. Two disc potencies of PI (10 and 20mg per

disc) tested inhibited growth of field mastitis isolates over zones ranging from 10

to 17mm, the corresponding zones of inhibition against ATCC isolates being 14

to 18mm. DMSO discs harboring 50mg of this chemical produced in general the

smallest zones of growth inhibition against field isolates (7-10 mm) of S. aureus,

Str. agalactiae and E. coli as well as against ATCC isolates (8-10mm) of these

species. The zone sizes of Ceph. (30 µg per disc) ranged from 12 to 20mm against

field mastitis isolates and 14 to 22mm against ATCC isolates. MIC50 of CPZ

against S. aureus, Str. agalactiae and E. coli were 50, 40, and 100 µg/mL

respectively. The corresponding values for Lid were 25, 20, and 10mg/mL. MIC50

of PI against these organisms were 22, 16, and 10 mg/mL. DMSO had higher

CHAPTER 5 SUMMARY

166

MIC50 values against S. aureus (50%), than against Str. agalactiae (40%) and E. coli

(30%). The control drug (Ceph) had MIC50 values of 30, 21, and 36 µg/mL,

respectively against S. aureus, Str. agalactiae and E. coli. MIC90 of CPZ against S.

aureus, Str. agalactiae and E. coli were 100, 80, and 200 µg/mL respectively. The

corresponding values for Lid were 50, 40, and 20mg/mL. MIC90 of PI against

these organisms were 44, 32, and 20 µg/mL. DMSO had higher MIC90 values

against S. aureus (70%) than against Str. agalactiae and E. coli (60% each). The

control drug (Ceph) had MIC90 values of 40, 42, and 48 µg/mL, respectively

against S. aureus, Str. agalactiae and E. coli.

Viability of PMNLs exposed to stock solutions of CPZ (2.5%), Lid (2%), PI

(10%), and DMSO (30%) were the lowest and significantly different (P < 0.05)

from their 10 to 1000 fold dilutions of stock solutions. The viability of 1000 fold

dilutions of these non-antibiotic antibacterials were non significantly different

from viability in Hank’s Balanced Salt Solution (HBSS) (P > 0.05). The

suppression of phagocytosis in 1: 1000 dilution of stock solution of CPZ and

DMSO was 0%, being the same as in HBSS. A 1: 1000 dilution of Lid and PI

affected slightly higher suppression of phagocytosis (0.30 and 0.87%) compared

with suppression in HBSS (0%).

Surf Field Mastitis Test (SFMT) based cure rate on day 14 post initiation of

treatment was the highest (60%) in quarters treated with CPZ followed by those

treated with PI (46.6%), Lid (43.3%), and DMSO (10%). Barring PI, all non-

antibiotic antibacterials tested in vivo exhibited a synergy with cephradine

CHAPTER 5 SUMMARY

167

(Ceph). Thus on day 14 post initiation of treatment, SFMT based cure rates in

quarters treated with CPZ + Ceph, Lid + Ceph, PI + Ceph, and DMSO + Ceph

were 66.6, 66.6, 50 and 66.6 percent, respectively. Surf Field Mastitis Test based

quarter cure rates on day 28 post initiation of treatment with non-antibiotic

antibacterials alone as well as their combinations with Ceph were similar to those

recorded on day 14. California Mastitis Test (CMT) based percent quarter cure

rates on day 14 and day 28 in non-antibiotic antibacterials treatment groups and

in groups treated with non-antibiotic antibacterials + Ceph were almost similar

to those recorded with SFMT on the corresponding time points.

A comparison of all treatments showed a non-significant difference (P >

0.05) in quarter SCC (OSCC) at pre-treatment stage. Quarter somatic cell counts

on day 14 and day 28 post initiation of treatment were significantly higher (P <

0.05) in DMSO treated groups than those of all other treatments which in turn

were non-significantly different (P > 0.05) from each other. QSCC at day 14 and

day 28 post initiation of treatment showed a significant decrease in all treatment

groups compared with pre-treatments levels with the exception of DMSO whose

pre and post treatment values differed non-significantly (p > 0.05).

Bacteriological cure rates on day 14 was the highest (53.3%) in quarters

treated with intramammary infusions of CPZ followed by those infused with PI

(43.3%), Lid (33.3%) and DMSO (6.6%). The corresponding cure rates at day 28

were only marginally different (56.6, 46.6, 40, and 6.6% respectively). The control

drug (Ceph) affected 53.3, and 56.6% bacteriological cure on day 14 and day 28,

CHAPTER 5 SUMMARY

168

respectively. The percent bacteriological cure rates obtained with CPZ + Ceph,

Lid + Ceph, PI +Ceph, and DMSO + Ceph combination on day 14 were 56.6, 60,

50, and 56.6%, the corresponding values on day 28 being 60, 60, 50, and 56.6%.

Among the 4 non-antibiotic antibacterials tested alone, Chlorpromazine

(CPZ) showed a relatively more significant (p<0.05) recuperative effect on the

milk yield of clinically mastitic quarters of dairy buffaloes. Whereas

Dimethylsulphoxide (DMSO) when infused alone, further aggravated (p<0.05)

the milk yield loss, indicating negative effect on milk yield improvement.

Adjuncting Cephradine with each of the non-antibiotic antibacterials, the

Lidocaine-cephradine group had the highest effect (p<0.05) on net recovery of

milk yield on day 28 postinitiation of treatment.

Based on the in vitro and in vivo results of the present study, it can be

tentatively concluded that chlorpromazine (CPZ) was the most effective of all

four non-antibiotic antibacterials tested. The residues of this drug in the milk of

treated animals were not investigated. Additional work is required before the

treatment of bubaline mastitis with this low cost non-antibiotic antibacterials can

be recommended independently or as an adjunct to antibacterial therapy.

169

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APPENDICES

i

Appendix 1: Guidelines for significance of colony numbers of specific micoorganisms isolated in pure or with other colony types (based on 0.01ml quarter milk sample streaked on blood agar)

Total number colonies One Several More than 10

Culture Pure Pure Mixed

two types

Mixed several types

Pure Mixed

two types

Mixed several Types

Str. Agalactiae 4* 4 4 4 4 4 4

Group G streptococci 4 4 4 4 4 4 4

Streptococcal species 2 3 2 2 4 3 1

S. aureus 3 4 4 4 4 4 4

Staphylococcal species 1 2 2 2 4 2 1

E. coli, Klebsiella Enterobacter, Serratia 2 3 2 2 4 2 1

Pasteurella 4 4 4 4 4 4 4

Pseudomonas 2 3 2 2 4 4 2

Yeast, Mold & Other Fungi 2 3 1 1 4 2 1

Nocardia 2 3 2 2 4 3 3

Prototheca 2 3 3 2 4 3 3

Corynebactrium bovis 1 2 2 2 4 3 3

Corynebactrium pyogenes 2 3 3 3 4 3 3

C. ulcerans 2 4 3 2 4 4 3

Proteus 2 3 1 1 4 2 1

Source: (National Mastitis Council, Inc., 1987) *Degree of confidence in diagnosing an infection:

1- not significant 3 – probable significance 2 – questionable significance 4 – highly significant

ii

Appendix 2: Viability (%) of polymorphonuclear leukocytes (PMNLs) under the influence of Chlorpromazine (CPZ) 2.5% and its ten fold dilutions

Coverslip# 2.5% w/v 1:10 1:100 1:1000 HBSS

1 25 59 75 87 85

2 39 71 83 88 89

3 31 65 85 86 88

4 28 63 79 80 90

5 36 60 86 87 84

6 29 65 84 88 86

7 20 72 86 85 87

8 29 68 74 86 90

9 35 57 87 86 88

10 37 70 81 85 87

11 30 61 80 87 89

12 23 55 78 84 86

x ±SD 30.16±5.8 63.83±5.6 81.5±4.19 85.75±2.17 87.41±1.92

% 30.16 63.8 81.5 85.75 87.41

CPZ = Chlorpromazine HBSS =Hank’s Balanced Salt Solution

iii

Appendix 3: Viability (%) of polymorphonuclear leukocytes (PMNLs) under the influence of Lidocaine (Lid) 2% and its ten fold dilutions.

Coverslip# 2 % w/v 1:10 1:100 1:1000 HBSS

1 20 54 76 88 90

2 19 51 83 87 86

3 30 56 80 87 84

4 25 59 77 86 91

5 29 61 84 80 88

6 33 63 85 86 87

7 21 58 78 84 82

8 28 50 81 83 87

9 26 62 79 81 84

10 27 53 73 88 86

11 24 60 82 89 85

12 22 55 79 84 85

x ±SD 25.3±4.7 56.83±4.14 79.75±3.3 85.25±2.8 86.5±2.6

% 25.3 56.83 79.75 85.25 86.5

PMNLs = Polymorphonuclear leukocytes HBSS = Hank’s Balanced Salt Solution

iv

Appendix 4: Viability (%) of polymorphonuclear leukocytes (PMNLs) under the influence of Povidone-iodine (PI) 10% and its ten fold dilutions.


1 0 17 58 83 89

2 0 21 66 76 85

3 0 20 63 86 80

4 0 18 57 84 87

5 0 25 61 87 86

6 0 19 64 89 84

7 0 23 72 85 87

8 0 17 65 79 85

9 0 24 71 83 85

10 0 15 70 84 84

11 0 13 68 78 88

12 0 20 74 85 87

x ±SD 0 19.3±3.6 65.75±5.4 83.25±3.3 85.58.±2.2

% 0 19.3 65.7 83.2 85.5

PMNLs = Polymorphonuclear leukocytes HBSS = Hank’s Balanced Salt Solution

v

Appendix 5: Viability (%) of polymorphonuclear leukocytes (PMNLs) under the influence of dimethylsulphoxide (DMSO) 30% and its ten fold dilutions.

Coverslip# 30 % v/v 1:10 1:100 1:1000 HBSS

1 15 35 76 79 88

2 19 49 82 83 87

3 14 40 83 86 85

4 13 41 72 84 86

5 14 35 85 87 86

6 21 45 79 85 87

7 17 37 70 78 85

8 13 43 75 87 88

9 17 36 78 80 84

10 23 38 81 88 87

11 20 42 75 87 85

12 16 40 80 85 84

x ±SD 16.75±3 40.08±4.25 78.0±4.3 84.08±3.3 86.0±1.41

% 16.75 40.08 78.0 84.08 86.0

PMNLs = Polymorphonuclear leukocytes HBSS = Hank’s Balanced Salt Solution DMSO = DimethylSulphoxide

vi

Appendix 6: Effect of Chlorpromazine (CPZ) 2.5% and its ten fold dilutions on phagocytic efficiency of polymorphonuclear leukocytes (PMNLs).

Coverslip# 2.5 % w/v 1:10 1:100 1:1000 HBSS

1 0.74±0.40 1.69±1.6 2.35±1.3 2.41±1.1 2.28±1.5

2 0.71±0.30 1.84±1.7 2.28±0.8 2.30±1.0 3.25±1.6

3 0.67±0.8 2.10±1.6 2.78±1.5 2.89±0.7 296±1.7

4 0.60±0.41 2.17±0.5 2.89±1.6 2.81±1.3 2.73±1.5

5 0.49±0.53 2.03±1.0 2.76±1.1 2.79±0.9 2.61±1.7

6 0.35±0.15 2.06±0.8 2.59±1.4 2.64±1.2 2.70±1.5

7 0.40±0.6 1.70±1.4 2.28±1.5 2.30±1.4 2.47±1.5

8 0.50±1.4 1.35±1.5 2.49±1.4 2.47±1.0 2.50±1.6

9 0.75±0.50 1.67±6.1 2.43±1.6 2.46±1.2 2.69±1.7

10 0.63±0.39 1.20±4.7 2.65±1.2 2.77±1.5 2.45±1.6

11 0.70±1.1 1.15±5.0 2.37±1.5 2.38±1.3 3.00±1.7

12 0.39±0.37 1.40±0.4 2.82±1.0 3.07±1.8 2.69±2.5

x ±SD 0.57±0.1 1.69±0.3 2.55±0.21 2.69±0.29 2.69±0.25

% 78.81 37.7 5.20 0 0

CPZ = Cholorpromazine

vii

Appendix 7: Effect of Lidocaine (Lid) 2% and its ten fold dilutions on phagocytic efficiency of polymorphonuclear leukocytes (PMNLs).


1 0.85±1.1 1.25±1.1 2.16±1.4 2.44±1.0 2.85±1.3

2 0.79±1.2 1.21±1.0 2.43±1.6 2.62±1.3 2.75±1.2

3 0.80±1.1 1.29±1.3 2.40±1.0 2.68±1.3 2.97±0.9

4 0.98±1.3 1.00±1.4 2.36±1.4 2.50±1.1 2.48±1.2

5 0.95±1.2 1.40±1.1 2.17±1.5 2.69±1.2 2.67±1.4

6 0.78±1.3 1.39±1.2 2.45±1.3 2.65±1.3 2.41±1.2

7 0.90±1.0 1.20±1.3 2.16±1.1 2.56±1.4 2.50±1.3

8 0.91±1.2 1.82±1.5 2.20±1.2 2.68±1.1 2.75±1.4

9 0.83±1.1 2.07±1.2 2.58±1.4 2.80±1.4 2.72±1.5

10 0.98±1.4 1.88±1.4 2.67±1.3 2.86±1.2 2.80±1.6

11 0.81±1.2 1.47±1.4 2.35±1.3 2.70±1.4 2.42±1.2

12 0.94±1.3 1.40±1.2 2.48±1.2 2.69±1.3 2.61±1.3

x ±SD 0.87±.07 1.44±0.40 2.36±0.16 2.65±0.11 2.66±0.17

% 67.29 45.8 6.76 0.30 0

viii

Appendix 8: Effect of Povidone-iodine (PI) 10% and its ten fold dilutions on phagocytic efficiency of polymorphonuclear leukocytes (PMNLs).


1 0 0.51±8.6 2.02±1.13 2.05±1.20 2.66±1.70

2 0 0.40±5.7 1.53±1.20 2.24±1.26 2.15±1.00

3 0 0.61±8.4 2.10±1.50 2.21±1.21 2.21±1.12

4 0 0.43±6.0 2.21±1.10 2.30±1.30 2.32±1.30

5 0 0.49±7.2 2.07±1.20 2.31±1.13 2.20±1.18

6 0 0.56±6.1 2.27±1.31 2.29±1.40 2.10±1.31

7 0 0.62±8.1 2.23±1.30 2.24±1.21 2.07±1.21

8 0 0.49±5.5 2.05±1.10 2.73±1.63 2.50±1.34

9 0 0.50±7.5 1.82±1.20 2.36±1.11 2.40±1.40

10 0 0.54±8.0 2.12±1.20 2.28±1.40 2.36±1.20

11 0 0.51±7.8 2.17±1.31 2.32±0.91 2.22±1.42

12 0 0.44±5.9 2.03±1.21 2.26±1.20 2.22±1.41

_X ±SD 0 0.50±0.06 2.05±0.3 2.26±0.08 2.28±0.17

% 100 78 10 0.87 0

ix

Appendix 9: Quarter SCC (X 105/ml of milk) of group G1 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of Chlorpromazine (CPZ) 50mg (2ml + 8ml normal saline) in dairy buffaloes.

Quarter No. Pre-treatment Count

Post-treatment count on day 14


1 31.69 ± 2.02 10.80 ± 1.69 10.30 ± 2.11 2 18.71 ± 01.92 8.51 ± 0.12 7.90 ± 0.08 3 15.96 ± 1.12 7.70 ± 0.17 7.16 ± 0.13 4 8.97 ± 2.40 4.15 ± 006 4.40 ± 0.021 5 7.59 ± 0.78 2.61 ± 0.25 2.12 ± 0.09 6 8.93 ± 0.67 3.10 ± 0.11 2.83 ± 0.032 7 11.50 ± 0.19 2.82 ± 0.07 2.16 ± 0.06 8 7.95 ± 0.08 2.56 ± 0.04 2.00 ± 0.03 9 9.32 ± 0.85 3.47 ± 0.08 3.02 ± 00.7 10 10.81 ± 1.10 3.11 ± 0.32 2.60 ± 0.09 11 7.16 ± 0.72 2.70 ± 0.17 2.50 ± 0.04 12 7.88 ± 0.91 2.21 ± 0.06 2.03 ± 0.04 13 9.12 ± 1.02 6.00 ± 0.46 5.10 ± 0.07 14 20.09 ± 4.11 7.80 ± 0.09 7.21 ± 0.08 15 7.91 ± 0.77 2.28 ± 0.05 2.05 ± 0.05 16 9.50 ± 0.54 3.20 ± 0.04 2.60 ± 0.02 17 11.66 ± 1.7 4.49 ± 0.62 4.20 ± 0.18 18 87.0 ± 0.44 4.61 ± 0.14 4.17 ± 0.12 19 17.84 ± 2.1 12.54 ± 1.10 12.07 ± 1.0 20 8.10 ± 0.45 2.84 ± 0.60 2.41 ± 0.02 21 7.79 ± 1.2 2.40 ± 0.07 2.05 ± 0.06 22 16.91 ± 2.0 8.11 ± 0.73 7.62 ± 0.09 23 10.15 ± 1.01 4.20 ± 0.08 3.86 ± 0.05 24 7.90 ± 0.81 2.48 ± 0.06 2.10 ± 0.02 25 8.82 ± 0.32 2.73 ± 0.34 2.17 ± 0.06 26 17.11 ± 1.20 9.50 ± 0.05 9.10 ± 0.02 27 12.0 ± 1.3 5.66 ± 0.41 5.21 ± 0.71 28 8.40 ± 1.02 2.87 ± 0.07 2.27 ± 0.01 29 15.73 ± 2.0 7.21 ± 0.92 6.86 ± 0.09 30 21.10 ± 2.41 10.85 ± 0.51 10.20 ± 1.2 _X ± SD 14.80 ± 14.48 5.70 ± 2.98 5.55 ± 2.93

x

Appendix 10: Quarter SCC (X 105/ml of milk) of group G2 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of Lidocaine (Lid) 200mg (10ml + 30ml normal saline) in dairy buffaloes.




1 50.57 ± 2.81 35.13 ± 01.70 34.60 ± 01.02 2 23.10 ± 1.12 14.21 ± 0.94 13.96 ± 0.18 3 10.00 ± 0.92 4.59 ± 0.67 4.70 ± 0.09 4 16.47 ± 0.76 5.54 ± 0.13 4.12 ± 0.11 5 32.10 ± 0.91 19.35 ± 0.14 18.09 ± 0.16 6 9.17 ± 0.07 2.94 ± 0.03 2.41 ± 0.02 7 9.82 ± 0.10 5.27 ± 0.06 5.79 ± 0.15 8 13.45 ± 0.21 4.56 ± 0.17 4.70 ± 0.1 9 21.77 ± 0.19 10.81 ± 0.47 10.05 ± 0.23 10 8.91 ± 0.06 2.45 ± 0.04 2.11 ± 0.08 11 10.31 ± 0.10 4.90 ± 0.12 4.30 ± 0.14 12 15.06 ± 0.13 6.21 ± 0.19 5.57 ± 0.09 13 9.34 ± 0.05 2.74 ± 0.06 2.21 ± 0.15 14 7.89 ± 0.05 2.10 ± 0.02 2.09 ± 0.07 15 9.50 ± 0.12 3.10 ± 0.16 2.77 ± 0.03 16 16.11 ± 0.09 7.51 ± 0.10 7.06 ± 0.06 17 7.91 ± 0.07 2.33 ± 0.03 2.04 ± 0.01 18 10.20 ± 0.11 5.42 ± 0.09 5.14 ± 0.12 19 7.95 ± 0.06 2.55 ± 0.03 2.15 ± 0.10 20 9.35 ± 0.04 2.69 ± 0.02 2.09 ± 0.07 21 8.29 ± 0.03 2.27 ± 0.09 2.05 ± 0.02 22 8.76 ± 0.12 2.70 ± 0.06 2.21 ± 0.04 23 11.45 ± 0.09 4.94 ± 0.13 4.10 ± 0.06 24 9.49 ± 0.13 3.15 ± 0.04 2.50 ± 0.02 25 31.10 ± 01.06 19.41 ± 0.17 18.78 ± 0.19 26 17.39 ± 0.94 10.24 ± 0.06 9.35 ± 1.00 27 9.47 ± 0.10 3.61 ± 0.12 2.71 ± 0.03 28 8.81 ± 0.12 2.57 ± 0.11 2.03 ± 0.08 29 22.45 ± 0.18 13.10 ± 0.03 12.79 ± 0.02 30 9.56 ± 0.10 2.50 ± 0.06 2.05 ± 0.05 _X ± SD 14.52 ± 9.35 6.97 ± 7.07 7.56 ± 8.22

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Appendix 11: Quarter SCC (X 105/ml of milk) of group G3 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of Pivodine-iodine (PI) 10% (10ml + 30ml normal saline) in dairy buffaloes.




1 40.17 ± 4.01 34.10 ± 3.48 30.51 ± 4.12 2 27.92 ± 3.90 19.36 ± 2.71 14.23 ± 2.46 3 11.35 ± 1.23 3.10 ± 0.92 2.90 ± 0.77 4 13.49 ± 1.20 7.93 ± 0.72 4.18 ± 0.94 5 7.66 ± 0.32 2.49 ± 0.02 2.11 ± 0.09 6 22.90 ± 2.13 10.74 ± 2.00 09.90 ± 1.74 7 9.40 ± 1.21 3.52 ± 0.76 3.05 ± 0.32 8 8.53 ± 1.01 2.71 ± 0.09 2.14 ± 0.06 9 17.944 ± 2.01 10.41 ± 1.10 10.60 ± 1.03 10 10.21 ± 1.41 3.53 ± 0.92 3.72 ± 0.74 11 8.12 ± 0.70 2.27 ± 0.06 2.09 ± 0.044 12 12.90 ± 1.00 6.22 ± 0.15 5.46 ± 0.75 13 9.34 ± 0.93 2.50 ± 0.16 2.90 ± 0.09 14 17.61 ± 2.03 10.27 ± 1.02 09.90 ± 0.10 15 22.13 ± 2.40 13.75 ± 2.10 12.95 ± 1.00 16 13.56 ± 1.09 5.47 ± 1.40 5.11 ± 0.41 17 8.47 ± 0.92 2.15 ± 0.08 1.80 ± 0.032 18 7.70 ± 0.31 2.11 ± 0.04 1.73 ± 0.03 19 34.16 ± 4.20 21.30 ± 2.50 20.78 ± 2.1 20 25.05 ± 2.30 15.8 ± 1.90 14.64 ± 2.09 21 12.33 ± 1.60 5.73 ± 0.10 5.10 ± 0.91 22 9.13 ± 1.50 2.85 ± 0.04 2.16 ± 0.08 23 11.86 ± 0.95 3.09 ± 0.02 4.26 ± 0.22 24 7.37 ± 0.06 2.15 ± 0.07 1.80 ± 0.04 25 16.90 ± 2.10 9.20 ± 0.05 08.76 ± 0.18 26 8.14 ± 1.20 2.41 ± 0.02 2.05 ± 0.03 27 10.45 ± 1.09 3.25 ± 0.07 2.47 ± 0.12 28 25.90 ± 2.70 16.96 ± 1.50 12.11 ± 1.50 29 14.35 ± 1.02 7.20 ± 1.03 6.42 ± 0.96 30 21.51 ± 2.11 12.42 ± 1.93 10.92 ± 2.11 _X ± SD 14.55 ± 8.32 8.16 ± 7.32 7.19 ± 6.50

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Appendix 12: Quarter SCC (X 105/ml of milk) of group G4 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of dimethylsuphoxide (DMSO) (20ml + 20ml normal saline) in dairy buffaloes.




1 25.13 ± 2.40 27.01 ± 1.90 28.95 ± 1.21 2 16.47 ± 1.26 18.20 ± 1.32 25.39 ± 2.10 3 9.76 ± 0.92 10.99 ± 1.21 12.27 ± 1.02 4 18.97 ± 0.09 26.10 ± 0.03 27.15 ± 2.13 5 9.361 ± 1.25 10.28 ± 1.02 11.92 ± 1.09 6 10.02 ± 1.06 12.91 ± 1.22 13.40 ± 2.01 7 22.00 ± 0.85 27.21 ± 0.61 30.49 ± 2.92 8 11.36 ± 1.72 16.58 ± 0.03 17.80 ± 7.97 9 8.20 ± 0.16 12.75 ± 0.14 16.23 ± 0.21 10 7.96 ± 0.12 2.21 ± 0.12 2.69 ± 0.17 11 9.87 ± 0.15 10.10 ± 0.16 11.73 ± 0.13 12 15.25 ± 0.11 17.02 ± 0.19 19.30 ± 1.07 13 14.10 ± 0.13 15.30 ± 0.20 16.23 ± 0.14 14 8.12 ± 0.09 9.45 ± 0.17 10.09 ± 0.08 15 30.20 ± 0.36 37.12 ± 1.10 40.21 ± 01.13 16 21.27 ± 0.71 22.82 ± 0.57 24.42 ± 0.66 17 11.32 ± 0.42 12.89 ± 0.16 13.25 ± 0.90 18 10.84 ± 0.17 12.07 ± 0.21 13.19 ± 0.18 19 7.20 ± 0.13 2.70 ± 0.11 3.06 ± 0.16 20 13.57 ± 0.16 14.60 ± 0.20 15.11 ± 0.18 21 8.46 ± 0.08 2.86 ± 0.05 3.17 ± 0.07 22 12.79 ± 0.70 14.96 ± 0.26 15.51 ± 0.37 23 16.09 ± 0.13 17.80 ± 0.64 20.72 ± 0.12 24 28.17 ± 0.90 31.11 ± 0.19 36.21 ± 0.18 25 41.61 ± 0.24 43.30 ± 0.18 44.4 ± 0.06 26 17.36 ± 0.11 18.32 ± 0.26 20.93 ± 0.11 27 9.15 ± 0.05 10.22 ± 0.15 11.90 ± 0.09 28 24.1 ± 0.61 26.93 ± 0.60 28.69 ± 0.21 29 19.27 ± 0.53 21.53 ± 0.24 23.21 ± 0.29 30 52.67 ± 0.70 54.10 ± 0.27 55.86 ± 0.42 _X ± SD 17.02 ± 10.27 18.64 ± 11.51 20.44 ± 12.06

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Appendix 13: Quarter SCC (X 105/ml of milk) of group G5 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of Cephradine (Ceph) 500mg (in 40ml normal saline) in dairy buffaloes.




1 40.08 ± 2.15 19.73 ± 1.76 17.16 ± 0.90 2 25.12 ± 1.27 10.20 ± 0.42 10.90 ± 0.51 3 10.36 ± 0.16 2.15 ± 0.09 2.21 ± 0.18 4 9.42 ± 0.30 2.25 ± 0.91 2.11 ± 0.23 5 17.64 ± 0.6 3.27 ± 0.21 3.40 ± 0.37 6 16.57 ± 1.02 3.11 ± 0.66 3.00 ± 0.30 7 30.21 ± 0.64 15.09 ± 0.39 14.17 ± 0.45 8 9.80 ± 0.27 2.70 ± 0.61 2.35 ± 0.52 9 21.00 ± 2.10 9.44 ± 0.87 8.20 ± 0.06 10 15.04 ± 1.02 3.01 ± 0.21 3.10 ± 0.13 11 23.97 ± 0.64 9.15 ± 0.11 8.90 ± 0.91 12 14.61 ± 0.96 2.80 ± 0.39 2.05 ± 0.21 13 29.11 ± 2.05 15.27 ± 0.61 14.23 ± 1.06 14 16.10 ± 0.41 3.70 ± 0.75 3.16 ± 0.93 15 12.13 ± 0.63 2.15 ± 0.21 2.75 ± 0.11 16 23.50 ± 1.04 11.37 ± 0.08 10.60 ± 0.60 17 8.93 ± 0.32 2.09 ± 0.94 2.00 ± 0.59 18 14.64 ± 0.92 2.30 ± 0.12 2.05 ± 0.93 19 18.20 ± 1.00 7.76 ± 0.63 6.48 ± 0.47 20 9.75 ± 0.42 2.30 ± 0..09 2.15 ± 0.81 21 13.66 ± 0.70 2.80 ± 0.76 3.05 ± 0.98 22 19.05 ± 0.23 7.17 ± 0.81 7.08 ± 1.22 23 10.78 ± 0.60 2.78 ± 0.90 2.10 ± 0.79 24 11.31 ± 0.91 2.81 ± 0.32 2.60 ± 0.84 25 25.56 ± 1.00 10.21 ± 0.43 9.70 ± 0.17 26 15.78 ± 1.02 3.05 ± 0.12 4.12 ± 0.92 27 9.65 ± 0.96 2.45 ± 0.80 2.06 ± 0.61 28 8.92 ± 0.73 2.10 ± 0.91 2.03 ± 0.21 29 20.17 ± 1.04 7.18 ± 0.69 7.10 ± 0.74 30 24.11 ± 1.30 12.10 ± 0.29 11.76 ± 0.69 _X ± SD 17.33 ± 7.48 6.02 ± 4.80 6.21 ± 4.43

xiv

Appendix 14: Quarter SCC (X 105/ml of milk) of group G6 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of Chlorpromazine (50mg = 2mL) + Cephradine (500mg) in 38ml normal saline in dairy buffaloes.




1 15.90 ± 0.93 6.64 ± 0.74 6.10 ± 0.31 2 43.21 ± 01.24 22.80 ± 0.34 20.00 ± 0.61 3 12.59 ± 0.23 3.10 ± 0.09 2.80 ± 0.12 4 7.70 ± 0.05 1.61 ± 0.03 2.05 ± 0.67 5 9.83 ± 0.12 2.00 ± 0.07 2.15 ± 0.06 6 12.11 ± 0.06 3.16 ± 0.03 3.10 ± 0.65 7 8.19 ± 0.15 2.21 ± 0.11 2.03 ± 0.14 8 20.06 ± 01.0 7.09 ± 0.07 7.00 ± 0.06 9 11.29 ± 0.02 3.04 ± 0.14 2.61 ± 0.13 10 9.75 ± 0.17 2.23 ± 0.04 2.00 ± 0.06 11 10.63 ± 0.88 2.05 ± 0.79 2.02 ± 0.18 12 18.62 ± 0.72 7.34 ± 0.32 7.16 ± 0.11 13 13.21 ± 0.26 3.17 ± 0.13 3.09 ± 0.27 14 9.20 ± 0.34 2.27 ± 0.24 2.14 ± 0.21 15 8.79 ± 0.09 2.00 ± 0.14 2.09 ± 0.18 16 10.21 ± 0.28 3.34 ± 0.32 2.70 ± 0.29 17 14.71 ± 0.30 2.10 ± 0.16 2.06 ± 0.22 18 31.10 ± 1.50 7.58 ± 0.53 15.16 ± 0.36 19 8.74 ± 0.64 2.19 ± 0.18 2.03 ± 0.09 20 9.90 ± 0.31 2.03 ± 0.36 2.26 ± 0.10 21 11.35 ± 0.21 2.28 ± 0.27 2.01 ± 0.29 22 8.14 ± 0.17 2.09 ± 0.42 2.07 ± 0.44 23 15.90 ± 0.36 3.17 ± 0.46 4.20 ± 0.52 24 22.11 ± 0.73 10.47 ± 0.75 9.10 ± 0.12 25 7.80 ± 0.06 2.00 ± 0.13 2.12 ± 0.11 26 10.66 ± 0.637 2.39 ± 0.12 2.76 ± 0.36 27 9.97 ± 0.51 2.21 ± 0.38 2.45 ± 0.20 28 20.11 ± 0.37 8.06 ± 0.12 8.10 ± 0.16 29 16.30 ± 0.54 6.09 ± 0.82 5.40 ± 0.32 30 27.38 ± 01.10 16.21 ± 0.92 15.24 ± 0.59 _X ± SD 14.52 ± 7.83 4.85 ± 4.69 4.80 ± 4.52

xv

Appendix 15: Quarter SCC (X 105/ml of milk) of group G7 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of Lidocaine (4% = 10ml) + Cephradine (500mg) in 30ml normal saline in dairy buffaloes.




1 10.09± 07 3.64 ± 0.23 2.50 ± 0.19 2 36.10 ± 1.50 16.70 ± 0.18 14.96 ± 0.36 3 9.28 ± 0.12 2.31 ± 0.09 2.00 ± 0.20 4 8.26 ± 0.09 2.29 ± 0.11 2.11 ± 0.16 5 7.64 ± 0.07 2.21 ± 0.06 1.70 ± 0.08 6 9.10 ± 0.17 2.69 ± 0.13 2.15 ± 0.10 7 28.05 ± 1.00 08.55 ± 0.37 9.47 ± 0.51 8 14.17 ± 0.92 3.60 ± 0.14 4.25 ± 0.47 9 8.01 ± 0.16 2.24 ± 0.11 1.81 ± 0.09 10 10.10 ± 0.38 3.15 ± 0.08 2.50 ± 0.12 11 7.47 ± 0.21 2.12 ± 0.06 1.78 ± 0.05 12 9.56 ± 0.18 2.72 ± 0.022 2.10 ± 0.14 13 12.29 ± 0.27 4.66 ± 0.75 3.45 ± 0.91 14 29.00 ± 0.91 13.81 ± 0.29 11.39 ± 0.83 15 8.19 ± 0.15 2.10 ± 0.12 1.90 ± 0.14 16 10.15 ± 0.067 3.15 ± 0.36 2.43 ± 0.32 17 13.91 ± 0.71 2.25 ± 0.12 3.21 ± 0.51 18 8.14 ± 0.17 2.10 ± 0.41 1.96 ± 0.39 19 7.81 ± 0.06 2.00 ± 0.13 1.78 ± 0.11 20 15.20 ± 0.56 6.31 ± 0.67 5.50 ± 0.72 21 9.18 ± 0.63 2.90 ± 0.09 2.41 ± 0.03 22 11.29 ± 0.62 3.51 ± 0.17 2.70 ± 0.21 23 8.74 ± 0.64 2.19 ± 0.12 2.11 ± 24 24 28.11 ± 0.90 08.41 ± 0.95 06.21 ± 0.53 25 24.14 ± 0.61 7.56 ± 0.39 6.24 ± 0.71 26 7.30 ± 0.08 2.26 ± 0.13 1.50 ± 0.21 27 9.14 ± 0.25 2.07 ± 0.54 2.45 ± 0.69 28 10.22 ± 0.42 2.70 ± 0.27 2.31 ± 0.90 29 38.13 ± 0.72 13.21 ± 0.64 10.19 ± 0.85 30 27.29 ± .49 9.35 ± 0.80 7.53 ± 0.32 _X ± SD 14.52 ± 9.08 4.75 ± 3.90 4.15 ± 3.335

xvi

Appendix 16: Quarter SCC (X 105/ml of milk) of group G8 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of Povidone-iodine 10%(10ml) + Cephradine (500mg) in 30ml normal saline in dairy buffaloes.




1 32.27 ± 1.09 15.20 ± 0.67 13.55 ± 0.39 2 10.18 ± 0.20 3.39 ± 0.31 2.69 ± 0.23 3 8.51 ± 0.11 2.79 ± 0.26 2.25 ± 0.12 4 19.27 ± 0.21 12.85 ± 0.16 12.48 ± 0.41 5 8.10 ± 0.28 2.52 ± 0.23 2.10 ± 0.08 6 7.80 ± 0.10 2.15 ± 0.12 1.75 ± 0.02 7 10.12 ± 0.14 3.05 ± 0.66 2.58 ± 0.21 8 9.02 ± 0.21 2.64 ± 0.19 2.10 ± 0.06 9 7.60 ± 020 2.41 ± 0.16 1.77 ± 0.09 10 11.12 ± 0.43 3.15 ± 0.35 2.51 ± 0.32 11 18.31 ± 0.15 12.76 ± 0.18 12.05 ± 0.91 12 16.35 ± 0.70 7.31 ± 0.84 5.80 ± 0.32 13 7.80 ± 0.63 2.10 ± 0.91 1.68 ± 0.24 14 9.75 ± 0.31 2.87 ± 0.24 2.20 ± 0.61 15 12.06 ± 0.32 5.22 ± 0.67 4.11 ± 0.22 16 8.50 ± 0.72 2.66 ± 0.09 2.10 ± 0.02 17 10.37 ± 0.16 4.15 ± 0.12 4.73 ± 0.6 18 7.28 ± 0.07 2.50 ± 0.04 2.02 ± 0.08 19 9.54 ± 0.03 2.87 ± 0.06 2.24 ± 0.11 20 21.06 ± 1.02 10.69 ± 0.15 8.72 ± 0.24 21 13.20 ± 0.62 5.80 ± 0.14 4.10 ± 0.21 22 7.20 ± 0.09 2.41 ± 0.07 1.91 ± 0.13 23 14.51 ± 0.35 6.72 ± 0.11 4.50 ± 0.32 24 19.10 ± 0.41 11.16 ± 0.24 8.40 ± 0.21 25 8.31 ± 0.18 2.82 ± 0.05 2.37 ± 0.16 26 9.11 ± 0.25 2.95 ± 0.06 2.21 ± 0.11 27 12.36 ± 0.16 5.35 ± 0.31 4.32 ± 0.24 28 10.20 ± 0.73 4.31 ± 0.52 3.80 ± 0.24 29 22.65 ± 0.74 13.0 ± 0.06 11.31 ± 0.09 30 41.02 ± 0.30 29.10 ± 01.16 25.09 ± 0.21 _X ± SD 13.42 ± 7.64 6.23 ± 5.75 5.24 ± 5.10

xvii

Appendix 17: Quarter SCC (X 105/ml of milk) of group G9 before treatment and at day 14 and day 28 post initiation of treatment with intramammary infusions (n = 5) of dimethylsulphoxide (20ml) + Cephradine (500mg) in 20ml normal saline in dairy buffaloes.




1 28.37 ± 1.02 8.12 ± 0.92 7.83 ± 0.32 2 10.20 ± 0.23 2.45 ± 0.31 2.51 ± 0.16 3 29.96 ± 1.06 9.73 ± 0.95 9.67 ± 0.24 4 8.35 ± 0.12 2.70 ± 0.26 2.35 ± 0.13 5 7.76 ± 0.10 1.50 ± 0.16 1.72 ± 0.19 6 11.03 ± 0.41 3.25 ± 0.305 2.68 ± 0.32 7 7.61 ± 0.23 2.01 ± 0.11 1.71 ± 0.15 8 9.25 ± 0.10 2.81 ± 0.32 2.42 ± 0.23 9 27.31 ± 01.02 7.30 ± 0.69 7.43 ± 0.59 10 10.09 ± 0.14 3.06 ± 0.71 2.50 ± 0.21 11 9.02 ± 0.22 2.55 ± 0.19 2.11 ± 0.06 12 25.10 ± 1.00 10.21 ± 0.95 11.16 ± 0.35 13 7.71 ± 0.63 2.10 ± 0.80 1.41 ± 0.21 14 9.46 ± 0.32 2.72 ± 0.24 2.25 ± 0.33 15 12.10 ± 0.67 3.61 ± 0.05 3.25 ± 0.29 16 8.51 ± 0.72 2.20 ± 0.17 2.08 ± 0.05 17 14.70 ± 0.30 6.10 ± 0.16 6.41 ± 0.22 18 31.05 ± 1.51 14.31 ± 0.58 14.60 ± 0.37 19 8.73 ± 0.64 2.11 ± 0.21 2.00 ± 0.06 20 9.92 ± 0.31 2.18 ± 0.27 2.10 ± 0.24 21 11.35 ± 0.21 3.21 ± 0.06 2.29 ± 0.10 22 10.12 ± 0.61 2.20 ± 0.31 2.17 ± 0.29 23 8.70 ± 0.25 2.37 ± 0.09 2.05 ± 0.12 24 13.21 ± 0.90 4.05 ± 0.11 3.06 ± 0.41 25 15.90 ± 0.84 6.20 ± 0.92 6.50 ± 0.49 26 9.26 ± 0.34 2.21 ± 0.21 2.15 ± 0.35 27 26.12 ± 1.06 9.10 ± 0.57 9.35 ± 0.58 28 11.36 ± 0.73 3.15 ± 0.28 2.70 ± 0.19 29 27.10 ± 1.12 14.33 ± 0.26 12.65 ± 0.85 30 40.27 ± 2.10 23.05 ± 1.08 21.20 ± 0.37 _X ± SD 15.32 ± 8.99 5.24 ± 4.82 5.07 ± 4.69

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