BACTERIOLOGICAL STUDIES ON CHRONIC MASTITIS IN BOVINE

PDF created with pdfFactory trial version www.pdffactory.com

http://www.pdffactory.com

الرحمـن الرحیمهللا بسم ا

والبحرأقلـم من شجرةفي االرض ولو أنمــا ﴿

إن اهللا هللامانفدت كلمت أبحر ه من بعدة سبعةیمد

﴾عزيز حكيم

العظیمهللا صدق ا

)٢٧(سورة لقمان االیة



ACKNOWLEDGEMENT

I am greatly indebted to gracious Allah for helping me to carry

out this work.

I would like to express my sincere gratitude and deep

appreciation to Prof. Dr. Mahmoud Essam Hatem Prof. and Head

of Microbiology Department, Faculty of Veterinary Medicine, Cairo

University for his kind supervision, faithful guidance, valuable

advice throughout the course of the present work and for the time

that he has given up to complete the present study.

I would like to introduce my great appreciation to Major

General Dr. Fathy Gaber Awad the consultant of chief stuff of

logistics authority for veterinary services for his kind supervision,

continuous and Valuable encouragement and supplying the facilities

required for my study.

I would like to express my sincere thanks to Brigadier General

Dr. Amin Abd El Fattah Badr Director of The Military Institute of

Health and Epidemiology for his kind help, valuable advice

throughout the course of present study continuous and valuable

encouragement.



List of Contents

Ⅰ. Introduction. ------------------------------------------------ 1

Ⅱ. Review of Literature. ------------------------------------- 4

Ⅱ.1. Etiology of bovine chronic mastitis.----------------------- 4

Ⅱ.2. Pathogenises of chronic Mastitis.-------------------------- 14

Ⅱ.3. Diagnosis of chronic mastitis.------------------------------ 21

Ⅱ.4. Antibiotic sensitivity of different bacterial species causing chronic mastitis 23

Ⅱ.5. Use of antibiotics and vaccines in case of chronic mastitis. 30

Ⅲ. Materials and Methods. ----------------------------------- 34

Ⅲ.1. Material.------------------------------------------------------- 34

Ⅲ.1.1 Animals.------------------------------------------------------- 34

Ⅲ.1.2. Milk samples.------------------------------------------------- 34

Ⅲ.1.3. Materials used for sample collection.--------------------- 35

Ⅲ.1.4. Materials used for direct microscopic examination.------------ 35

Ⅲ.1.5. Media.--------------------------------------------------------- 35

Ⅲ.1.6. Reagents used for biochemical identification.----------- 42

Ⅲ.1.7. Stains.---------------------------------------------------------- 46

Ⅲ.1.8. Materials used for Agar disc diffusion method.---------- 47

Ⅲ.2. Methods.------------------------------------------------------- 50



Ⅲ.2.1. Collection of milk samples.--------------------------------- 50

Ⅲ.2.2. Methods of bacteriological examination.----------------- 50

Ⅲ.2.3. Methods of antibiotic susceptibility testing.------------- 59

Ⅳ. Results. ------------------------------------------------------ 61

V. Discussion.--------------------------------------------------- 84

Ⅵ. Summary.---------------------------------------------------- 100

Ⅶ. Recommendations ------------------------------------------ 103

Ⅷ. Abstract ------------------------------------------------------ 104

Ⅸ References. ---------------------------------------------------

105 X Arabic Summary-------------------------------------------- 127



List of Tables

Table Title Page

1 Concentration of antibacterial discs used in Agar Disc

Diffusion Method (ADD) for antibacterial susceptibility. 49

2 Differentiation between Staphylococcus aureus,

Staphylococcus epidermidis, and Staphylococcus

saprophyticus 54

3 Identification of the isolates of Arcanobacterium

pyogenes. 55

4 Identification of the isolates of

Bacillus sp. 56

5 Differentiation between Escherichia coli and

Klebsiella pneumoniae : 57

6 Identification of the isolates of Pseudomonas

aeruginosa 58

7 Interpretation zones in Agar Disc Diffusion Method

(ADD) for antibacterial Susceptibility. 60

8 The mean of the affected chronic quarters per one cow

in each of the different test locations. 61

9 The prevalence of the isolated organisms in relation to

the total number of examined samples. 62



Table Title Page

10 The prevalence of each isolated organism to the total

number of isolated pathogens. 63

11 The incidence of each of isolated pathogen in different

experiment locations in relation to the total bacterial

isolates. 65

12 The distribution of chronic mastitis according to the

affected quarter involvement of the examined animals. 66

13 The distribution of chronic mastitis according to the

affected quarter involvement for each pathogen. 66

14 Distribution of chronic mastitis within the four udder

quarters according to quarter involvement of the

examined animals in case of affected animals that had no

bacterial growth. 67

15 Antibiogram of Staphylococcus aureus recovered from

cows with chronic bovine mastitis to antimicrobial

agents. 69

16 Antibiogram of Staphylococcus epidermidis recovered

from cows with chronic bovine mastitis to antimicrobial

agents. 71



Table Title Page

17 Antibiogram of Staphylococcus saprophyticus recovered

from cows with chronic bovine mastitis to antimicrobial

agents 73

18 Antibiogram of Streptococcus sp. recovered from cows

with chronic bovine to antimicrobial agents. 75

19 Antibiogram of Bacillus cereus recovered from cows

with chronic bovine mastitis to antimicrobial agents. 77

20 Antibiogram of Escherichia coli recovered from cows

with chronic bovine mastitis to antimicrobial agents. 79

21 Antibiogram of Klebsiella pneumoniae recovered from

cows with chronic bovine mastitis to antimicrobial

agents. 81



List of Graphics

Graph Title Page

1 The prevalence of each isolated organism to the total

number of isolated pathogens. 64 2 Susceptibility pattern of Staphylococcus aureus

recovered from cows with chronic bovine mastitis

against different antibacterials. 70 3 Susceptibility pattern of Staphylococcus epidermidis


against different antibacterials 72 4 Graphic (4): Susceptibility pattern of Staphylococcus

saprophyticus recovered from cows with chronic

bovine mastitis against different antibacterials 74 5 Susceptibility pattern of Streptococcus sp. recovered

from cows with chronic bovine mastitis to different

antibacterials. 76 6 Susceptibility pattern of Bacillus cereus recovered


antibacterials. 78 7 Susceptibility pattern of Escherichia coli recovered


antibacterials. 80



Graph Title Page

8 Susceptibility pattern of Klebsiella pneumoniae


to different antibacterials. 82



List of photographs

photo Title Page

1 Citrate test. 39

2 Methyl Red test 42

3 Voges Proskaur test 43

4 Indole test 44

5 Novobiocin sensitivity test 54

6 Antibiotic sensitivity test to 18 antibacterial agents 68



LIST OF ABBREVIATIONS

API Analytical Profile Index A. pyogenes Arcanobacterium pyogenes A. pyogenes Actinomyces pyogenes

BMEC Bovine mammary epithelial cells BMSC Bulk milk somatic cell count CNS Coagulase-negative Staphylococcus CMT California mastitis tests CP5 Capsular polysaccharide type 5 CLSI Clinical and Laboratory Standards Institute DCT Dry-cow antibiotic therapy

FEMS Federation of European Microbiological Societies IMI Intramammary infection. IP Interpretation

ID Identification IMVIC test Indole, Methyl red, Vogas Proskaur and Citrate tests

MIC Minimum inhibitory concentration PIA Polysaccharide intercellular adhesion SCC Somatic cell count TSI Triple sugar iron

TSST Toxic shock syndrome toxin TSST-1 Toxic shock syndrome toxin-1

VP Voges Proskaur test vs. Versus



INTRODUCTION



INTRODUCTION

1

I. INTRODUCTION

Bovine mastitis is considered to be one of the most important

destructive diseases producing abnormal milk and monetary loses to

dairy industry (Schalm et al., 1971). The economic losses of

mastitis are due to reduced milk production, discarded milk during

treatment, early cow replacement cost, drugs, veterinary service and

labour (Harmon, 1995). Information regarding the prevalence of

mastitis pathogens and costs associated with different bacterial

infection is of interest to the dairy industry (Wilson et al., 1997).

In Egypt mastitis causes great damage through threatening the

dairy animal health. The greatest losses of mastitis are mostly due to

subclinical form in which there are no detectable abnormalities of

the milk. However, the causative microorganism/s act as invisible

potential source for spreading the infection (EL. Kholly et al., 1994).

Most surveys in different countries have shown that up to 50% of

lactating animals, at any time, may suffer from chronic latent

mastitis after suffering from subclinical mastitis (Grunert and

Weight, 1979).

Most infections are caused by various species of streptococci,

staphylococci, and Gram-negative rods, specially lactose-

fermenting organisms of enteric origin, commonly termed



INTRODUCTION

2

coliforms. From an epidemiologic standpoint, the source of infection

may be regarded as contagious or environmental. Except for

Mycoplasma Sp. which may spread from cow to cow through

aerosol transmission and invade the udder subsequent to bacteremia,

contagious pathogens are spread during milking by milker’s hands

or the liners of the milking unit. Species that utilize this mode of

transmission include Staphylococcus aureus, Streptococcus

agalactiae, and Corynebacterium bovis. Most other species are

opportunistic invaders from the cow’s environment, although some

other streptococci and staphylococci may also have a contagious

component. Gangrenous mastitis can also occur, particularly when

subclinical, chronic infections of Staphylococcus aureus, become

severe at times of immunosuppression (MERK, 1998).

Many genera and species of pathogenic bacteria are

incriminated as causal agents of severe mastitis in dairy Friesian

cows .Teat dipping and dry cow therapy have reduced the mastitis

caused by the Staphylococcus aureus and Streptococcus

agalactiae, but failed to prevent mastitis caused by other types of

mastitis causing bacteria (Oz et al., 1985) .



INTRODUCTION

3

The aim of the present study was to:

(A)- Isolation and identification of causative bacterial agent/s of

chronic mastitis in dairy Friesian cows at different dairy farms.

(B)- Antibiogram for the bacteria isolated aiming to find the most

suitable treatment for each case.



LITERATURE



LITERATURE

4

Π. LITERATURE

Π. 1. Etiology of bovine chronic mastitis:

Jasper and Dellinger (1975) studied the prevalence of

Escherichia coli infection regarding to the association with the

other infections. The study showed that Escherichia coli

infections were mostly related to the mammary glands that were

not infected with other pathogens. In addition, they reported high

susceptibility of the mammary gland to Escherichia coli

infection particularly after treatment of streptococcal and

staphylococcal infections.

Hill and Shears (1979) examined 28 mastitic cows with

Escherichia coli for persistence and recurrence through 120

days. The study showed that only 5 cases of the 28 had long

recurrent infection.

Inui et al.(1979) applied bacteriological and pathological

studies on the mammary gland of 17 cows with chronic mastitis.

The isolated pathogens were: Staphylococcus aureus (5 cases );

Staphylococcus aureus + Staphylococcus epidermidis (one

case); Staphylococcus aureus, Corynebacterium pyogenes and

Staphylococcus epidermidis (one case); Corynebacterium



LITERATURE

5

pyogenes + Staphylococcus epidermidis (one case);

Corynebacterium pyogenes + Corynebacterium bovis (one case);

Candida krusei, K. pneumoniae, Pseudomonas aeruginosa,

haemolytic streptococci, and Corynebacterium pyogenes each

were isolated from one case; Escherichia coli + Nocardia

asteroides each from 2 cases.

Jain (1979) reported that Streptococcus agalactiae was the

predominant causative microorganism of bovine chronic mastitis before

the antibiotics era.

Smith and Hagstad (1985) studied results of examination of 418

milk samples collected during one year from a herd of 69 cows with

chronic staphylococcal mastitis. The study showed that coagulase-

negative staphylococci and Staphylococcus epidermidis isolates

detected decreased with advancing age, whilst Staphylococcus aureus

infections increased with age. Prevalence of Staphylococcus aureus

infections increased throughout lactation.

Garg and Kapoor (1986) identified pure isolates of Rhodococcus

equi from two cows each with chronic mastitis in one quarter. The two

Rhodococcus equi isolates were of B type colony, did not haemolyse

10% sheep blood agar and produced 'equi factor', catalase and urease.



LITERATURE

6

Wilson et al. (1991) recorded the incidence of different pathogens

in 49 quarters with chronic mastitis. Isolated agents were:

Staphylococcus aureus (18.4%); Staphylococcus sp. (7.3%); no

growth (20.2%). Environmental pathogens streptococci other than

Streptococcus agalactiae, Escherichia coli, Klebsiella sp., Enterobacter sp. and Citrobacter sp.(22.0%). Other pathogens

(Serratia sp., Bacillus sp., diphtheroids, Corynebacterium sp. and

Actinomyces pyogenes), Pseudomonas sp. and Nocardia sp.),

represented 11.9%. Mixed pathogens (2 agents isolated) were 12.8%

and contaminated samples constituted 7.3%.

Samborski et al. (1992) determined the prevalences of pathogenic

bacteria in 114 quarter mastitic milk samples of cows (76 samples were

obtained from subclinical mastitic quarter and 38 samples from clinical

chronic mastitic quarters). Pathogens isolated were Streptococcus

agalactiae (44.7%), Streptococcus uberis (8.8%), Streptococcus

dysgalactiae (5.3%), Staphylococcus aureus (24.5%), Staphylococcus

epidermidis (15.8%) and Escherichia coli (0.9%).

Nemeth et al. (1994) examined and compared the biochemical

properties and certain potential virulence factors of Escherichia coli

isolates that were obtained from bovine mastitis and bovine feces. The

study included 50 isolates of Escherichia coli taken from bovine

mastitic milk, 50 from feces of mastitic cows and 50 from feces of



LITERATURE

7

healthy cows. The study revealed that none of the properties that were

investigated constituted potential virulence factors or markers for the

ability to induce mastitis. They concluded that mastitic Escherichia coli

are simply opportunistic pathogens.

Erer et al. (1996) examined 883 cows at abattoir before slaughter

using California mastitis reagent. The study showed mastitis in 118

cows (13.4%) with 232 mastitic quarters of which 163 chronic mastitic

quarters were identified histologically after slaughter. 184 isolates were

obtained: Staphylococcus aureus from 87 (47.3%), Actinomyces

pyogenes from 30 (16.3%) , Escherichia coli from 15 (8.2%) ,

Candida albicans from 12 (6.5%) , Staphylococcus epidermidis from 8

(4.3%) Klebsiella pneumoniae from 7 (3.8%) , Bacillus subtilis from 4

(2.2%) , Streptococcus dysgalactiae from 4 (2.2%) , Flavobacterium

from 3 (1.6%) , Bacillus cereus from 2 (1.1%) and Proteus mirabilis

from 1 (0.5%).

Gonzalez (1996) identified Bacillus cereus as an uncommon

cause of mastitis. Pure cultures of Bacillus cereus were obtained from

samples taken from first lactation cow with chronic mastitis in two

quarters with frequent flare-ups. Although, few colonies of Bacillus sp.

which were grown on blood agar plates represented contamination.

However, Bacillus sp. was considered as a cause of intramammary



LITERATURE

8

infection in a cow if culture of sequential milk samples was pure with

concomitant high SCC with clinical signs of udder disease.

Owens et al. (1997) compared the results of success of antibiotic

therapy with results of antimicrobial susceptibility testing for bovine

mastitis bacterial pathogens to evaluate the predictability of use of the in

vitro antibacterial susceptibility tests. The study showed that the in vitro

testing was considered to be a predictor of therapy outcome in case of

mastitis caused by Staphylococcus sp., newly acquired Staphylococcus

aureus, Streptococcus uberis, Streptococcus dysgalactiae, and

Streptococcus agalactiae, but was not considered to be a useful

predictor of efficacy for chronic mastitis caused by Staphylococcus

aureus.

Costa et al. (1998) determined the prevalence of environmental

mastitis in dairy herds and identified the main environmental pathogens

of 20,310 quarters of 5,216 cows. The isolated environmental bacteria

were Streptococcus uberis (21.1%), Enterobacteriaceae (8.3%) and

Nocardia sp. (6.6%).

Böhmer et al.(1999) identified the causative organisms of chronic

mastitis in 100 lactating German Black Pied cows. The study showed

that chronic mastitis was a major problem in 14% of cows.

Streptococcus dysgalactiae (3cows), Streptococcus uberis (13),



LITERATURE

9

Enterococcus sp. (1), Staphylococcus aureus (4), and yeasts (4), while

27 cows showed unclear Causes.

Twardona et al. (1999) reported the incidence of chronic mastitis

in 71 Holstein Friesian cows aged 2.8-8 years. Chronic mastitis was

identified in 35 quarters. Staphylococcus epidermidis, Streptococcus

agalactiae and Staphylococcus aureus were isolated from 34.9, 28.1

and 29.1% quarters respectively.

Larsen et al. (2000) examined 625 isolates of Staphylococcus

aureus from bovine mastitis in 9 dairy herds for their diversity and

compared the results with that of isolates from other sites by phage- and

ribotyping. The study included isolates obtained from bovine skin lesions,

asymptomatic human carriers, milking personnel and non-farm-related

human carriers. They aimed to investigate whether colonization of

milkers with Staphylococcus aureus could be a source of infection for

bovine mastitis. The study revealed that certain types predominated in

one or several herds during the study period, whereas the presence of

other types was of a more sporadic nature. Within the individual herds,

there was a close relation between ribo- and phage types of

Staphylococcus aureus isolated from bovine mastitis and bovine skin

lesions. Isolates from milking personnel, however, were not identical to

any of the predominant intramammary strains. These results supported



LITERATURE

10

the hypothesis that the human reservoir of Staphylococcus aureus does

not play any major role as a source of bovine mastitis.

Pitkala et al. (2004) estimated the prevelance of bovine mastitis,

distribution of mastitis pathogens and in vitro antibacterial susceptibility

of different mastitis pathogens in total of 21,661 quarter milk samples

from 3,282 dairy cows. They compared results with a previous survey

done in 1995 and reported decrease in the prevalence of mastitis from

38% to 31% in their study (of bacterial and non bacterial causes). At the

same time they reported a significant increase in the prevelance of

mastitis due to bacterial causes from 21.0% to 33.5% as result to

increase of the prevalence of Corynebacterium bovis mastitis. With a

decrease in Staphylococcus aureus prevalence, the coagulase-negative

staphylococcal mastitis remained the most common bacteria isolated

constituting almost 50% of the reported cases. Staphylococci

showed high penicillin resistance (52.1% for Staphylococcus aureus and

32.0 for coagulase-negative staphylococci).

Armenteros et al. (2002) applied clinical and bacteriological

examinations to determine the clinical and bacteriological status of

3,069 animals. The study revealed 274 mastitic quarters of which

18.02% were chronic mastitis and 3.7% were with atrophy, 3.02%

showed clinical mastitis and 45.1% had subclinical mastitis. The

prevalences of different pathogens were : Staphylococcus aureus



LITERATURE

11

30.5%, Corynebacterium bovis 9.2% and Streptococcus agalactiae

8.3%.

Beytut et al. (2002) performed bacteriological and clinical

examinations for 950 cows before slaughter. They were examined

pathologically after slaughter to determine the incidence of chronic

mastitis and the prevalences of causative bacterial causes. The

study showed that 105 cows (11.05%) with 116 mastitic quarters of

which 47 quarters were with chronic mastitis. The prevalences of

different pathogens were : Staphylococcus aureus (43.75%),

Corynebacterium pyogenes (Arcanobacterium pyogenes) (19.79%),

Streptococcus agalactiae (9.37%), Staphylococcus epidermidis

(8.33%), Escherichia coli (7.29%), Streptococcus dysgalactiae

(4.16%), Bacillus subtilis (3.15%) and Bacillus cereus (2.08%).

Benites et al. (2003) evaluated the microbiological status of

various structures in the mammary glands from naturally infected

dairy cows following slaughter. When all samples were

considered, coagulase-negative Staphylococcus sp. were the

most prevalent (35.7%) followed by coagulase-positive

Staphylococcus (12.2%), Corynebacterium bovis (2.4%),

Prototheca sp. (1.9%), and Streptococcus dysgalactiae (1.5%).



LITERATURE

12

Makovec and Ruegg (2003) examined 83,650 milk samples

submitted for microbiological examination in 2001. Samples

identified as contaminated constituted 9.5%. Samples coded as no

growth were 49.7%. Staphylococcus aureus (9.7%), Streptococcus

agalactiae (3.0%). Coagulase-negative Staphylococcus sp. were

17.5%, environmental Streptococcus sp. were 20.1%, and

Escherichia coli constituted 6.7% of all isolates.

Malinowski et al. (2003) isolated the etiological agents of bovine

mastitis in 3,888 quarter milk samples collected from 972 selected cows

of total 3,288 lactating cows. Cases examined were either with high

bulk milk somatic cell count (more than 400,000 cells) or had received

treatment many times through previous 3-6 months. Chronic mastitis

was noted in 8.1% of the quarters. The prevalences of different

pathogens were: Staphylococcus aureus (34.5%), CAMP-negative

streptococci (21.1%), coagulase-negative staphylococci (15%) and

Streptococcus agalactiae (4.8%).

Zadoks et al. (2003) examined Streptococcus uberis isolates from

infections (n = 84) detected in 70 quarters of 46 cows. Samples were

tested by random amplified polymorphic DNA (RAPD) fingerprinting.

The result showed multiple quarters of a cow were mostly infected by

one strain . The majority of all infections were subclinical, and



LITERATURE

13

infections attributed to predominant strains were more chronic than

infections attributed to other strains.

Zadoks et al. (2005) identified the specific sources of mastitis-

causing Streptococcus uberis strains by investigation of the relation

between the environmental and fecal strains of Streptococcus uberis

and that which were obtained from mastitis samples. Their study

revealed that the environmental and fecal shedding of strains of

Streptococcus uberis play a role in the maintenance of Streptococcus

uberis infection.

Taponen et al. (2006) studied persistence of subclinical and clinical

mastitis caused by coagulase-negative Staphylococcus sp. in cows either

treated with antimicrobials or left untreated and identified the most

common coagulase-negative Staphylococcus sp. causing bovine mastitis

in 133 quarters. The bacteriological diagnosis was based on biochemical

(API) testing. Staphylococcus simulans (43.6%) followed by

Staphylococcus chromogenes (23.3%) were the most common species

isolated from the milk samples. The study revealed that the severity and

persistence of intramammary infection were unaffected by coagulase-

negative Staphylococcus sp. which were involved.



LITERATURE

14

Π. 2 . Pathogenises of chronic Mastitis:

Thorn and Nilson (1962) recorded that Pseudomonas

aeruginosa in mastitic milk samples were shed intermittently in very

small numbers. The organism may disappear spontaneously from

lactation to a next while the Pseudomonas aeruginosa infection still

persists. They recommended that milk samples should be incubated

overnight before culturing on blood agar medium to detect the

organism. The study recorded that the organism showed high resistance

to be overcome by the antibiotic intramammary infusion.

Liu and Mercer (1963) confirmed that the Pseudomonas

aeruginosa may show persistent mastitis in bovine. They recorded that

the Pseudomonas aeruginosa mastitis is characterized by the

intermittent flare-ups with few days or several weeks’ intervals.

Schalm et al. (1964) reported the effect of the inflammatory

reaction on the bovine mastitis causing Escherichia coli in the

mammary gland tissues. The study revealed that the infection induced

an inflammatory reaction that in turn reduced the Escherichia coli

population in the mammary tissue. In a feed-back mechanism, when the

inflammatory reaction subsided the organism began to increase again

giving the infection intermittent manner.



LITERATURE

15

Schalm and Lasminis (1968) studied the recurrent infection in

case of Pseudomonas aeruginosa bovine mastitis. They reported the

intermittent shedding of the Pseudomonas aeruginosa during the

course of infection. The study revealed that in case of confirmed

infection the Pseudomonas aeruginosa isolation in mastitic milk

samples may failed for several times before detection even in case of

milk sample incubation.

Buddle et al. (1987) studied the prevalence of the reinfection of

bovine mammary glands after dry-cow antibiotic therapy (DCT). They

examined quarters for the presence of new or reactivated Staphylococcus

aureus or streptococcal infections. They studied the susceptibility to

reinfection in the following year in relation to the number of quarters

infected prior to DCT. The high susceptibility was recorded in cows with

3–4 quarters that were infected prior to DCT. In contrast, the cows with

1–2 infected quarters showed very low susceptibility to reinfection or

new infection.

Doymaz et al. (1988) evaluated the antigenic effect of chronic

Staphylococcus aureus infection on the humoral immune response of

the bovine mammary gland. They compared results of the infected

quarters with that of the non-infected. They detected no significant

differences that suggested that the antigenic effect of chronic

Staphylococcus aureus infection on the humoral immune response of



LITERATURE

16

the bovine mammary gland is minimal. The results concluded that the

suboptimal stimulation of the mammary immune system may result in

persistent Staphylococcus aureus infection

Matsunaga et al. (1993) examined 58 Staphylococcus aureus

isolates from mastitic milk of cows with chronic, acute and peracute

mastitis to determine and compare their production of virulence

associated factors. Factors studied were toxic shock syndrome toxin-1

(TSST-1), staphylococcal enterotoxins, alpha-haemolysin beta-

haemolysin, delta-haemolysin, DNase, egg-yolk factor, clumping factor

and protein A. These factors were produced at a relatively high

frequency by isolates from chronic mastitis unlike isolates of acute and

peracute mastitis.

Myllys et al. (1997) characterized Staphylococcus aureus isolates

(N=40) from 20 quarters by random amplified polymorphic DNA-PCR

(RAPD-PCR), ribotyping, biotyping. The isolates were taken at the

acute phase of infection then 3 weeks after cessation of therapy. They

compared the paired isolates. The results suggested that the chronic

nature of Staphylococcus aureus infections was due to the persistence

of the original infective strain.

Döpfer et al. (2000) tested seven strains of Escherichia coli

isolated from clinical cases of bovine mastitis and one Salmonella



LITERATURE

17

typhimurium as control strain, for their ability to adhere to and invade

bovine mammary epithelial cells in vitro. Four of them were with

chronic intramammary infections with recurrent episodes of clinical

mastitis and three strains were isolated from single cases of clinical

mastitis. The four strains isolated from chronic recurrent cases invaded

twice as frequently as and three times faster than the strains isolated

from single cases of clinical mastitis

Hensen et al. (2000) investigated the expression of capsular

polysaccharide type 5 (CP5) in situ in both the early and chronic stages

of experimental Staphylococcus aureus mastitis by immunochemical

staining of tissue sections with specific antibodies against CP5. They

recorded that in chronic infection, CP5 - positive Staphylococcus

aureus were located deeply in the interstitial tissue the thing that

probably help the bacteria to withstand the host defense mechanisms.

Tollersrud et al. (2000) examined eighty-six isolates of

Staphylococcus aureus obtained from 81 different cows from dairy

herds with an equal representation of the acute, chronic and subclinical

cases of mastitis. The isolates were characterized biochemically and

with respect to serotype, multilocus enzyme electrophoresis genotypes,

antibiotic sensitivity, and production of enterotoxins A through D and

toxic shock syndrome toxin-1 (TSST-1). No correlation was found

between the factors studied and the clinical classification of mastitis.



LITERATURE

18

Döpfer et al. (2001) examined two strains of Escherichia coli

isolated from the milk of two different cows suffering from persistent

mastitis for their in vitro adhesion to and invasion of three primary

mammary epithelial cell culture. Two of cell cultures derived from

mastitic mammary biopsies of two infected cows while the third was

obtained from non infected quarter of the second cow. They compared

the results with that of the Staphylococcus aureus, Streptococcus

dysgalactiae and Streptococcus uberis. The study indicated that

Escherichia coli invaded the cells less efficiently than Staphylococcus

aureus, about as efficiently as Streptococcus dysgalactiae and more

efficiently than Streptococcus uberis.

Brouillette et al. (2003) used in vitro assays and a mouse model of

mastitis in case of Staphylococcus aureus to demonstrate the

intracellular component of the infection. They as well assessed and

compared the capacities of the parental and fibronectin-binding protein

deficient strains to bind and to invade epithelial cells to determine the

role of fibronectin-binding proteins in the processes of colonization and

internalization. Their study demonstrated that Staphylococcus aureus

was able to cause an intracellular infection and that the elimination of

one adhesion protein delayed, but did not prevent, the infection.



LITERATURE

19

Vasudevan et al. (2003) examined in vitro ability for biofilm

formation and the presence of the ica gene loci responsible for the

biofilm translation in 35 isolates of Staphylococcus aureus from bovine

mastitis. Although only 24 of the 35 Staphylococcus aureus isolates

produced biofilm in vitro, all the 35 isolates were found to possess the

ica locus genes. The presence of the ica locus genes in all

Staphylococcus aureus isolates of bovine mastitis confirms its potential

role as a virulence factor in the pathogenesis of bovine mastitis.

Strandberg et al. (2005) used the quantitative real-time PCR to

study quantitatively the innate immune responses induced by the in

vitro stimulation of bovine primary mammary epithelial cells. They

determined the innate immune responses against each of Gram-negative

lipopolysaccharide and Gram-positive lipoteichoic acid bacterial cell

wall components. The results showed limited cytokine response to

Gram-positive lipoteichoic acid contrary to that of the Gram-negative

lipopolysaccharide. These results may explain why mastitis caused by

Gram-positive bacteria has greater potential for chronic intra-mammary

infection than Gram-negative infection.

Dogan et al. (2006) compared the ability of Escherichia coli strains

associated with persistent bovine mastitis with that associated with

transient mastitis to adhere to, invade, survive and replicate within

cultured mammary epithelial cells. They examined three strains of E. coli



LITERATURE

20

taken from cases of transient bovine mastitis, and three strains associated

with persistent bovine mastitis. They found that E. coli strains associated

with persistent bovine mastitis were better able to invade and replicate

within cultured mammary epithelial cells than transient strains.

Tamilselvam et al. (2006) defined the ability of Streptococcus

uberis isolates as important bovine chronic mastitis pathogens to persist

intracellularly. Studies were on time-dependent internalization and

survival of S. uberis strains when cultured in bovine mammary

epithelial cells (MAC-T). S. uberis strains and a Staphylococcus

aureus strain used as positive control were cultured. All isolates taken

from cows with clinical mastitis. Results showed that S. uberis could

survive intracellularly up to 120 h without apparent loss of host cells

viability. S. aureus internalized more efficiently and cell death was

observed after 72 h of incubation. Intracellular persistence of S. uberis

may be associated with the spread of the infection to deeper tissues and

development of persistent intramammary infection.



LITERATURE

21

Π. 3 . Diagnosis of chronic mastitis :

Krzywoszynski (1977) performed comparison between results of

bacterial examination of milk samples of cows with chronic mastitis

collected at the first milk jets and residual milk obtained by injection of

40 IU oxytocin after milking. The study showed that Staphylococcus

aureus was isolated from 56.3 and 39.3% of residual and first milk

samples from the same quarters. Study Indicated that residual milk

samples is of greatest value in detecting staphylococcal mastitis.

Hanselmann (1978) carried out bacteriological and cytological

examinations on 672 quarter milk samples from 120 cows. The

frequency of pathogens in chronic mastitis was Streptococcus

agalactiae (15%), other streptococci (7%), Staphylococcus (45%),

Micrococcaceae (31%), others 2%. The study showed that inapparent

infections that excrete pathogens represented 26% of infected quarters.

Barto et al. (1984) determined the prevalence of different

streptococcal clinical mastitis in 309 cows by bacteriological and

cytological investigations. Results showed that chronic mastitis

were 18.9%.

Zecconi et al. (1993) in a Streptococcus agalactiae eradication

programme on 56,130 lactating cows documented that the somatic cell

counts on bulk milk had no close correlation with mastitis prevalence.



LITERATURE

22

Gronlund et al. (2005) evaluated the use of acute phase proteins:

Haptoglobin and serum amyloid A (SAA) as detectors for chronic sub-

clinical mastitis. Their study was performed on 41 cows with composite

somatic cell count (CSCC) above 300,000 cells /mL, and 11 healthy

cows with CSCC below 80,000 cells / mL at least during two months

prior to sampling. Though study revealed that haptoglobin and SAA

concentrations below the detection limit (≥ 0.3 mg/L and ≥ 0.9 mg/L

respectively), were considered as good indicators of healthy udder

quarters, substantial variation in their concentrations in milk was

observed in udder quarters with chronic sub-clinical mastitis.



LITERATURE

23

Π. 4 . Antibiotic sensitivity of different bacterial species

causing chronic mastitis :

Sogaard (1982) applied in vitro antibiotic sensitivity tests on 131

isolates of Escherichia coli recovered from acute and chronic bovine

mastitis by determination of minimum inhibition concentration (MIC)

with eight antibiotics. In milk ampicillin showed the highest bactericidal

activity followed by polymyxin B and colistin. All strains examined

were sensitive to gentamicin.

Bertoldini et al. (1985) tested the susceptibility of 250 isolates of

Staphylococcus aureus and 250 isolates of Streptococcus agalactiae

isolated from chronic bovine mastitis to penicillin, cloxacillin,

ampicillin, tetracycline, oxytetracyciline, thiamphenicol, amiosidine,

erythromycin, neomycin, kanamycin, dihydrostreptomycin, and

rifamycin. Amoxicillin and erythromycin were the most active

antibiotics against penicillin-susceptible strains of Staphylococcus

aureus.

Sikorski (1986) determined the percentage of chronic mastitis

cases that resulted from bovine acute mastitis for 100 cows with 110

mastitic quarters after antibiotic therapy with colistin and ampicillin.

Original pathogens of acute mastitis were Coliforms or E. coli 38.2%,

staphylococci and streptococci each in 15.5%, micrococci and



LITERATURE

24

pseudomonas each in 1.8%, Corynebacterium pyogenes in 2.7%,

yeasts in 0.9% and mixed infections in 0.9%. 22.7% of samples were

negative. The study revealed that acute mastitis had changed into

chronic mastitis in 3.7% of the cases after therapy. He found that 5%

of Escherichia coli or coliforms were resistant to colistin, 62% to

ampicillin, while 25% of staphylococci were resistant to ampicillin.

Binde and Gjul (1988) studied the prevalence of Staphylococcus

aureus in milk samples of chronic mastitic cows in two separate

regions and determined the percentage of penicillin resistance strains in

each case. Penicillin resistant isolates of Staphylococcus aureus were

5.8% and 9.0% in acute mastitis cases. They reported an increase in

penicillin resistant isolates of Staphylococcus aureus in case of chronic

mastitis reaching 12.5% and 22.6%.

Orm et al. (1989) recorded results of laboratory examination of

bovine milk samples for determination of the causative agents of

chronic and subclinical mastitis. The commonest cause of chronic

mastitis were Streptococcus sp. , Streptococcus dysgalactiae and

Streptococcus uberis were predominant. Nearly all streptococci were

sensitive to penicillin.

Saikia et al. (1989) applied in vitro antibiotic sensitivity test on 35

milk samples taken from 35 cows with clinical chronic mastitis. Cases



LITERATURE

25

showed little response to antibiotic therapy. The high sensitivity was

detected with gentamicin (88%) and ampicillin (80%). The least

sensitivity was with oxytetracyciline (40%), penicillin (44%) and

tetracycline (44%) and nitrofurantoin (20%). Each of oxytetracycilline,

penicillin and tetracycline were used previously in conjunction with

streptomycin in treatment of these cows.

Hartmann (1990) performed an antibiogram on 1215 isolates

from cows with acute or chronic mastitis. Pathogens included 304

DNase-positive isolates of Staphylococci, 304 DNase-negative isolates

of Staphylococci, 304 CAMP-negative isolates of Streptococci and 303

Strains of Enterobacteriaceae. The used antibiotics were penicillin,

cloxacillin, neomycin and gentamicin. In addition they used the

combinations of: penicillin+ neomycin, cloxacillin+ gentamicin and

nafcillin+ penicillin+ dihydrostreptomycin. The highest sensitivity of

staphylococci and Enterobacteriaceae was with gentamicin. The

combination of cloxacillin+ gentamicin was fully effective in the Gram-

positive spectrum (against streptococci and staphylococci), as in the

Gram -negative spectrum (against E. coli).

Murdough et al. (1996) determine the alterations over 6-week

period in viability after freezing of isolates of nine bacterial species

Staphylococcus aureus, Staphylococcus hyicus, Staphylococcus

chromogenes, Staphylococcus xylosus, Streptococcus agalactiae,



LITERATURE

26

Streptococcus dysgalactiae, Streptococcus uberis, Corynebacterium

bovis, and Escherichia coli. Minimal inhibition concentration (MIC)

values of 200 isolates of Escherichia coli recovered from clinical

bovine mastitis were determined for ampicillin, cephalexin, ceftazidime,

dihydrostreptomycin, gentamicin, tetracycline, trimethoprim-

sulfadiazine, and ciprofloxacin by an agar dilution method. The freezing

of quarter milk samples for 6 week did not affect viability of any of

these pathogens.

Fang (1996) evaluated the extended effect of certain antibiotics in

case of mastitis-causing Staphylococcus aureus and Escherichia coli.

Fluorometric technique based on the bacterial phosphatase activity was

used. The tested antibiotics were: ampicillin, enrofloxacin, gentamicin

and tetracycline. They recorded no post antibacterial effect of ampicillin

on either bacterial species. Enrofloxacin, gentamicin and tetracycline

exhibited post antibacterial effect of varying effects. Gentamicin and

tetracycline exhibited longer post antibacterial effect than enrofloxacin

on Staphylococcus aureus (4.2–4.8 h vs. 1.4 h). However, longer post

antibacterial effects on Escherichia coli were seen with enrofloxacin

(2.3 h) and gentamicin (1.7 h) than with tetracycline (0.7 h).

Raimundo et al. (1999) performed PCR-coagulase gene typing of

151 Staphylococcus aureus isolates from seven farms. They

distinguished only sex PCR types. PCR type 1 was the predominant



LITERATURE

27

type in five of the seven farms. The study revealed that most isolates

were resistant to penicillin with the exception of only 41 isolates

obtained from one farm.

Costa et al. (2000) determined in vitro susceptibility pattern of

Staphylococcus sp. isolated from mammary parenchyma of slaughtered

dairy cows to 12 different anti-microbials using Kirby and Bauer

standardized diffusion method. A total of 45 isolates of Staphylococcus

sp. [33 coagulase-negative Staphylococcus and 12 Staphylococcus

aureus] were used. 84.44% and 86.66% of the 45 isolates were resistant

to ampicillin and penicillin, respectively. The highest sensitivity was to

cephalothin (84.44%), gentamicin (80%) and to sulphazotrin (77.77%).

Coagulase-negative Staphylococci generally showed higher resistance

(P < 0.05) than Staphylococcus aureus.

DeOliveira et al. (2000) examined 811 strains of Staphylococcus

aureus from cases of chronic mastitis for their minimum inhibitory

concentrations MIC with 16 antibacterials and antibacterial combinations.

The antimicrobial agents tested were penicillin, ampicillin, oxacillin,

cephalothin, ceftiofur, amoxicillin + clavulanate, penicillin + novobiocin,

enrofloxacin, premafloxacin, erythromycin, clindamycin, lincomycin,

pirlimycin, neomycin, lincomycin + neomycin, and sulfamethazine. The

study showed that wide use of different mastitis control programs

decreased the incidence of resistance in Staphylococcus aureus strains.



LITERATURE

28

Hussain et al. (2002) recorded the antibiotic sensitivity pattern of

Staphylococcus aureus isolates of 21 acute/chronic mastitis samples

taken from 18 cows. Results showed susceptibility to gentamicin and

ciprofloxacin. Moderate susceptibility (75%) was detected with

ofloxacin and cloxacillin. Least sensitivity was reported with

norfloxacin, sparfloxacin and clarithromycin. Complete resistance was

seen with ampicillin, amoxicillin and penicillin.

Lehtolainen et al. (2003) determined the Minimal inhibition

concentration (MIC) values of 200 Escherichia coli isolates from

clinical bovine mastitis by agar dilution method. In vitro antimicrobial

susceptibility of the Escherichia coli isolates was high; only 27%

showed resistance to one or more tested antimicrobial agents. No

gentamicin; ceftazidime; or ciprofloxacin-resistant isolates were

detected. Eleven percent of all isolates were resistant to two or more

antimicrobial agents. Tetracycline was most often associated with

multiresistant patterns. Antimicrobial resistance appeared to pose no

problem in Escherichia coli isolated from mastitic milk of both

countries probably due to the controlled use of antimicrobial agents.

Vintov et al. (2003-a) assessed the antimicrobial resistance of 292

Staphylococcus aureus isolates. The isolates were collected through the

period from 1950 (86 isolates), 1992 (107 isolates), and 2000 (99

isolates). The tested antimicrobial agents were : bacitracin , ceftiofur ,



LITERATURE

29

chloramphenicol , ciprofloxacin , erythromycin, florfenicol , gentamicin

, kanamycin , oxacillin , penicillin , spectinomycin , streptomycin ,

sulphamethoxazole , quinupristin+ dalfopristin , avilamycin ,

trimethoprim , vancomycin , trimethoprim+ sulphamethoxazole ,

tetracycline , tiamulin , and virginiamycin. The prevalences of

antimicrobial resistances remained low through the three periods.

Vintov et al. (2003-b) examined 815 isolates of Staphylococcus

aureus to investigate the diversity of phage types related to their

penicillin resistance. The isolates were tested for their susceptibilities to

different antimicrobial agents. The used antimicrobial agents were:

avilamycin, bacitracin, ceftiofur, chloramphenicol, ciprofloxacin,

erythromycin, florfenicol, gentamicin, kanamycin, oxacillin, penicillin,

spectinomycin, streptomycin, sulphamethoxazole, quinupristin+

dalfopristin, tetracycline, tiamulin, trimethoprim, trimethoprim+

sulphamethoxazol, vancomycin and virginiamycin. The study showed

that a large number of phage types of Staphylococcus aureus can cause

bovine mastitis, but that some types predominate. Penicillin resistance is

widespread among Staphylococcus aureus from bovine mastitis, but

resistance to other antimicrobial agents is limited. An association

between certain phage groups and penicillin resistance was observed

that could suggest that the use of penicillin in the bovine environment

has selected for specific types of Staphylococcus aureus with a high

frequency of resistance.



LITERATURE

30

Π.5. Use of antibiotics and vaccines in case of chronic

mastitis :

Verheijden et al. (1984) tested the sensitivity of 18 isolates of

Staphylococcus sp. using of minimum inhibition concentration (MIC)

method to penicillin-G, cloxacillin and spiramycin. The study together

with the data concerning clinical efficacy trials showed the effectiveness

of spiramycin for treatment of chronic mastitis caused by penicillin-G

resistant Staphylococcus aureus.

Sanchez et al. (1988) concluded that bovine mastitis due to

Staphylococcus aureus may become chronic and show high antibiotic

resistance due to its ability to survive within the mammary gland

macrophages and polymorphonuclear neutrophils. They evaluated the

effectiveness of antibiotics against Staphylococcus aureus surviving

within the bovine mammary gland macrophage. Their study showed an

excellent activity of rifampicin for treatment of the intracellular

Staphylococcus aureus. They explained that this result was due to

potentiation of the rifampicin activity in the intracellular acidic

compartment of the phagolysosome.

Rachid et al. (2000) studied the influence of sub-inhibitory

concentrations of antibiotics on the expression of the ica operon gene

which lead to the synthesis of a polysaccharide intercellular adhesion



LITERATURE

31

(PIA) biofilm in Streptococcus epidermidis. Their study revealed that

subinhibitory concentrations of tetracycline enhanced ica expression.

They recorded no effect on ica expression in case of chloramphenicol,

penicillin, oxacillin, clindamycin, gentamicin and ofloxacin.

Erythromycin showed weak effect.

Diarra et al. (2003) investigated the effects of penicillin G alone or

in combination with bovine lactoferrin on the susceptibility of

Staphylococcus aureus to be phagocytosed by the bovine

polymorphonuclear leukocytes. They used the mouse mastitis model.

Their study clarified that the susceptibility of Staphylococcus aureus to

phagocytosis was decreased in the presence of penicillin G alone. At the

same time lactoferrin alone did not affect phagocytosis. Results

indicated that lactoferrin acts with penicillin G to enhance the

phagocytosis of Staphylococcus aureus by bovine polymorphonuclear

leukocytes and to decrease the invasion of mammary epithelial cells by

this important mastitis pathogen.

Brouillette et al (2004) assessed the ability of Staphylococcus

aureus small-colony variants SCV (isogenic hemB mutant) which have

been implicated in chronic and persistent bovine mastitis, to colonize

mouse mammary glands and persist under antibiotic treatment. They as

well compared the results with that of classical Staphylococcus aureus

Newbould. The study showed that the hemB mutant was as susceptible

as Staphylococcus aureus Newbould to cephapirin, ciprofloxacin,



LITERATURE

32

lysostaphin, oxacillin, rifampicin and vancomycin in vitro. At the same

time, although the hemB mutant has a reduced ability to colonize

mammary glands, the SCV phenotypes were over a 100 times more

persistent when antibiotic was administrated in vivo.

Malouin et al. (2005) assessed the susceptibility of intracellular

Staphylococcus aureus against antimicrobial compounds to be used for

the in vivo treatment of bovine mastitis. They used the hemB deletion

mutant of Staphylococcus aureus Newbould as prototype for small

colony variants intracellular Staphylococcus aureus. Nine compounds

were tested for their minimum inhibitory concentrations (MIC). The

study identified the lead compounds to be useful for treating persistent,

intracellular infections.

Talbot and Lacasse (2005) reported the progress in mastitis

vaccine use. Their study recorded some reports which described

prototype vaccines against Streptococcus uberis, Streptococcus

agalactiae, Escherichia coli and Staphylococcus aureus. Yet, these

vaccines were not in common use especially in case chronic forms of

mastitis. They suggested that these were related to the intracellular

forms of bacteria which were protected from some immune

mechanisms and are often not a reachable target for these vaccines.

Their study revealed a recent strategy by use DNA expression vector

plasmids as vaccines which express virulence-associated antigens in

vivo.



LITERATURE

33

Melchior et al. (2006) studied the epidemiological data concerning

the bacteriological cure following antimicrobials treatment that vary

between 0% and 80% of the recurrent mastitis without any evidence of

a significant loss of activity of the antibiotics used for bovine mastitis.

They concluded that one of the most convincing hypotheses that

explain therapy resistance is the ability of many staphylococci, as well

as other microorganisms, to grow in biofilms in infected tissues, thus

innate resistance is created to almost all therapeutic agents. They as well

reported evidence suggests that antibiotics are not only less effective

against bacterial biofilms, but also may stimulate the biofilm formation.

Smith et al. (2006) evaluated the efficacy of combination of both

vaccination and antibiotic treatment with intramammary pirlimycin in

treatment of Staphylococcus aureus chronic mastitis. The study was

applied on 50 chronically mastitic cows. 20 cows were received 3 doses

of a polyvalent Staphylococcus aureus bacterin and intramammary

pirlimycin treatment. The rest were not received any treatment.

Elimination of Staphylococcus aureus infection in the treated group was

40% while in the non-treated was 9%. Results revealed that combination

of both vaccination and antibiotic treatment can be successful in

eliminating some cases of chronic intramammary Staphylococcus

aureus infections in dairy cattle.



MATERIALS

AND

METHODS



MATERIALS AND METHODS

34

Ⅲ. MATERIALS AND METHODS

Ⅲ.1.Materials:

Ⅲ.1.1. Animals: Seventy-nine machine milked Friesian cows from five dairy

herds located in Nobareia area (Alexandria), Sues, Tal El Kabeer

(Sharkia), Assuit and El-Fayiom during a period of sex months

(February to July, 2006) Animals had chronic mastitis and

showed developing fibrosis. They were treated for mastitis for at

least 2-3 times before they were assigned by their herds’

administrations as unresponsive for treatment. Antibacterials

were banded for all cows for at least one week before collection

of samples. Cows were of different ages (3-5 years), milking

seasons and lactation stages. They showed different patterns of

affected quarter sites.

Ⅲ.1.2. Milk samples: A number of 150 quarter cow milk samples were collected

from 79 cows. Samples were marked for their affected quarter as

follows: anterior right quarter (AR), anterior left quarter (AL),

posterior right quarter (PR) and posterior left quarter (PL). The

samples were labeled with the local farm number of each cow,

the affected quarter and date of sampling. The samples were

collected from the examined animals and immediately transferred




35

in ice tanks to the laboratory within a few hours from sampling

where they were subjected to bacteriological examination. All

samples were taken from quarters which showed clinical signs of

chronic mastitis (fibrosis, firmness, abnormal milk secretions)

and were previously drug-dosed for at least 2-3 times before they

were diagnosed as chronically unresponsive mastitic cases.

Ⅲ.1.3.Materials used for sample collection: - Sterile 50 ml capacity screw capped cups with tight cover.

- Ethyl alcohol (70%).

- Ice box for transportation of milk samples.

Ⅲ.1.4.Materials used for direct microscopic examination: - Clean glass slides.

- Xylol.

- Absolute ethyl alcohol.

- Clean glass covers

- Stains (Ziehl-Neelsen stain – Gram’s stain –Loeffler’s

methylene blue).

Ⅲ.1.5. Media:

Ⅲ.1.5.1.Media used for isolation, identification and

preservation of bacteria through the course of the study:




36

Ⅲ.1.51.1.Blood agar base medium (Oxoid ,1990) :

Nutrient agar plus 5% defibrinated sheep blood freshly

collected apparently healthy animals. The medium was used as

an enriched medium for the isolation of the pathogenic bacteria

as well as the determination of the type of haemolysis.

Peptone 10 g Meat extract 10 g Sodium Chloride 5 g Agar agar 20 g Defibrinated sheep blood 70 mL Distilled water 1 liter pH 7.2 - 7.6 Sterilized by autoclaving 121 oC for 15 minutes and cooled to 50oC before aseptic addition of sterile blood.

Ⅲ .1.5.1.2.MacConkey`s bile salt lactose agar medium

(Cheesbrough,(1984):

It was used as a selective medium for isolation of Enterobacteriaceae

(Cruickshank et al., 1975).

Peptone 20.0 g Lactose 10 g Bile salt 1.5 g Crystal violet 0.001 g Neutral red 10 g Sodium Chloride 5 g Agar agar 15.0 g Distilled water 1 liter pH 7.2 - 7.6 Sterilized by autoclaving 121 oC for 15 minutes




37

Ⅲ.1.5.1.3 Mannitol salt agar medium (Oxoid, 1990):

It was used for the selective isolation of the staphylococci.

Beef extract 1.0 g Peptone 10.0 g Mannitol 10.0 g Sodium chloride 75.0 g Phenol red 0.025 g Agar agar 15.0 g Distilled water 1 liter pH 7.3 - 7.7 Sterilized by autoclaving 121 oC for 15 minutes

Ⅲ.1.5.1. 4. Nutrient agar medium (Oxoid, 1990):

It was used for isolation of the causative organisms aiming

to study the colonial characteristics. It may be used as agar plate

or slant (slope) agar tube to obtain single typical pure colony.

Peptone 10 g Meat extract 10 g Sodium Chloride 5 g Agar agar 20 g Distilled water 1 liter pH 7.2 - 7.6 Sterilized by autoclaving 121 oC for 15 minutes

Ⅲ .1.5.1.5. Semi solid Nutrient agar (soft agar) medium

(0.4%) (Cruickshank et al. (1975):

It was used for preservation of the purified isolates and for

detection of motility 0. 4% agar is added.




38

Peptone 10 g Meat extract 10 g Sodium Chloride 5 g Agar agar 4 g Distilled water 1 liter pH 7.2 - 7.6 Sterilized by autoclaving 121 oC for 15 minutes

Ⅲ1.5.2. Media used for biochemical identification:

Ⅲ.1.5.2.1. Peptone water 1% (Oxoid, 1990):

It was used as the base for the sugar fermentation media

tests and for the detection of indole production by the use of

Kovac`s reagent.

Peptone 10.0 g Sodium chloride 5.0 g Distilled water 1.0 liter pH 7.2 ± 0.2 Sterilized by autoclaving 121 oC for 15 minutes

Ⅲ.1.5.2.2. Glucose phosphate broth (Oxoid, 1990):

It was used in methyl red (MR) and Voges Proskaur (VP)

tests in which the examined isolate was inoculated to be tested by

the specific reagent of each test. It is composed of:

Peptone 7.0 g Glucose 5.0 g Dipotassium hydrogen phosphate 5.0 g Distilled water 1.0 liter pH 7.4 ± 7.6 Sterilized by autoclaving 121 oC for 15 minutes




39

Ⅲ.1.5.2.3. Simmon’s citrate medium (Oxoid, 1990):

It was used for detection of citrate utilization by the

microorganism. It was prepared in slant tube. The suspected

isolate was inoculated on the medium and incubated for 7 days at

37oC. The development of blue coloration indicates citrate

utilization.

Ammonium hydrogen phosphate 0.2 g Magnesium sulphate 0.2 g Sodium chloride 5.0 g Sodium citrate 2.0 g Sodium ammonium phosphate 0.8 g Agar agar 15.0 g Bromo thymol blue 0.08 g Distilled water 1.0 liter ph 6.9 Sterilized by autoclaving 121 oC for 15 minutes

Photo (1) Citrate test. - Bluish color indicate citrate utilization in left tube (positive reaction) - Green color indicates negative result.




40

Ⅲ.1.5.2.4. Sugar fermentation medium (Oxoid, 1990):

It was used for determination of the ability of the isolated

organism to ferment different sugars (produce acid) and the production

of gas. An inverted Durham’s tube is placed in the tube to collect the

produced gas. It contains 1% solution of tested sugar (glucose, lactose,

sucrose, Mannitol, dulcitol, inositol, arabinose, raffinose, trehalose,

sorbitol or inulin). The inoculum of examined isolate is inoculated in

the tube and incubated for 48 hours at 37oC.

Peptone 10.0 g

Beef extract 1.0 g

Sodium chloride 5.0 g

Phenol red 0.018 g

Sugar (tested sugar) 5.0-10.0 g

Distilled water 1.0 liter

pH 6.9 - 7.3

Sterilized by autoclaving 121 oC for 15 minutes




41

Ⅲ.1.5.2.5. Triple sugar iron agar medium “TSI”(Oxoid, 1990):

It was used for the detection of hydrogen sulphide. The

inoculum of suspected isolate is streaked on the medium slant

and stabbed the bottom. The slant is incubated for 48 hours at

37oC. The appearance of yellow color indicates acid formation.

And the black color resulted from hydrogen sulphide that

indicates positive reaction.

Beef extract 3.0 g Yeast extract 3.0 g Peptone 20.0 g Glucose 1.0 g Lactose 10.0 g Sucrose 10.0 g Ferrous sulphate 0.2 g Sodium chloride 5.0 g Sodium thiosulphate 0.3 g Sodium ammonium phosphate 0.8 g Agar agar 20.0 g Distilled water 1.0 liter Phenol red (0.2 % solution) 12.0 ml pH 7.2 ± 0.2 Sterilized by autoclaving 121 oC for 15 minutes




42

Ⅲ.1.6. Reagents used for biochemical identification: The used reagents were proposed according to Cruickshank

et al. (1975) and Finegold and Martin (1982) as follow:

Ⅲ.1.6.1. Methyl red reagent (Finegold and Martin, 1982):

It was added to 5 ml of glucose phosphate broth with the

inoculated isolate after incubation for 48 hours at 37oC. The

appearance of red coloration indicates high hydrogen ion

(positive reaction) while yellow coloration indicates negative

result as seen in Photo (2).

Methyl red 0.1 g Ethanol (absolute) 300.0 ml Distilled water 1 liter

Photo (2) Methyl Red test. - Yellow color indicates negative result tube (Left). - Strong red color indicates positive reaction (Right).




43

Ⅲ.1.6.2. Voges Proskaur (Finegold and Martin, 1982):

It was prepared by addition of 0.6 ml alcoholic to 1 ml of

40% potassium hydrogen solution. The reagent was added to 5

ml of glucose phosphate broth with the inoculated isolate after

incubation for 48 hours at 37oC. The tube was thoroughly shaken

and examined after 15 minutes and one hour. The appearance of

strong red coloration indicates (positive reaction).

alpha-naphthol 0.6 ml 40% potassium hydrogen containing 0.3% creatine 0.2 ml Absolute alcohol 1 liter

Photo (3) Voges Proskaur test. - Clear yellow color of peptone water before addition of alpha-naphthol tube (3). - Yellow color indicates negative result (tube (4). - Strong pinkish red color at the surface of the medium indicates (positive reaction)

tube (1) and (2).




44

Ⅲ.1.6.3. Kovac’s reagent for indole test (Finegold and Martin, 1982):

0.5 ml of the reagent was trickled down the side of a tube

containing 1% peptone water with the inoculated isolate after

incubation for 48 hours at 37oC. The tube was thoroughly shaken

and examined after few minutes. The appearance of rosy colored

ring indicates (positive reaction).

Para dimethyl mine benzaldehyde 0.1 g Iso amyl alcohol 95.0 ml Hydrochloric acid 50.0 ml

photo (4 ) Indole test Pinkish red colored ring at the surface of the medium indicates (positive reaction) .




45

Ⅲ.1.6.4.: Hydrogen peroxide solution 3% for catalase test

(Finegold and Martin, 1982):

1 ml of 3% hydrogen peroxide (30 volume) was added to the isolate

inoculum of 24-hours agar slant culture on a clean slide. Gas bubbles

production indicates the presence of catalase enzyme (produced by both

Gram-positive cocci and Gram-positive bacilli) which is considered

positive reaction. Absence of gas babbles indicates negative results.

Ⅲ.1.6.5. Rabbit plasma for coagulase test (Cruickshank et

al., 1975):

It was used for the detection of the coagulase enzyme

production which agglutinates the citrated rabbit blood plasma.

The reagent was prepared from citrated anticoagulated rabbit’s

blood plasma. The tested inoculum was inoculated to 0.5 ml of

the diluted (1:4) citrated plasma in a sterile agglutination tube.

Then the tube was incubated at 37oC to be examined for 4, 8, 12,

18 and 24 hours for the production of clot.

Ⅲ.1.6.6. Oxidase test discs (Himedia):

It was used for the detection of presence of cytochrome

oxidase enzyme in the examined isolate. The used discs were

containing 1% tetramethyl-p-phenylnedimine dihydrochloride

solution. The discs were streaked with the examined inoculum




46

using glass rod. Reduction of the color of the reagent on the disc

to a deep purple blue color within 60 seconds indicates positive

test. Negative result was judged if the change in color occurs

after 60 seconds or if no change was recorded .

Ⅲ.1.7. Stains:

Ⅲ.1.7.1. Ziehl – Neelsen stain (Cheesbrough, (1984):

For exclusion of tuberculous mastitis all samples were stained.

- Carbol fuchsin (filtered).

- Acid alcohol (1% Hcl in absolute ethanol).

- Methylene blue.

Ⅲ.1.7.2. Gram's stain (Cheesbrough, (1984):

Films were prepared from the mastitic milk as well as

suspected colonies on different media then they were heat-fixed

and stained with Gram stain.

-Crystal violet

-Lugols iodine.

- 90% ethyl alcohol.

- Diluted carbol fuchsin.




47

Ⅲ.1.7.3. Loeffler's methylene blue (Cheesbrough, (1984):

It was used for the examination of milk samples obtained

from chronic mastitic cows that were suspected to be infected

with streptococcus agalactiae.

- Methylene blue. - Ethanol (absolute). - Potassium hydroxide.

Ⅲ.1.8. Materials used for agar disc diffusion method

(determinations of antibiogram of the isolates):

Ⅲ.1.8.1. Media used for antibiotic sensitivity:

Ⅲ.1.8.1.1. Mueller Hinton agar (Finegold and Martin, 1982):

This medium was used for disk diffusion test. It produce

large and clear zone of inhibition when sensitive organism meet

active antibiotic.

Beef infusion 300.0 g

Bio-case 17.5 g

Starch 1.5 g

Agar 17 g

Distilled water 1 liter

ph 7.4

Sterilized by autoclaving 121 oC for 15 minutes




48

Ⅲ.1.8.1.2 Nutrient broth (Finegold and Martin, 1982):

It was used in standardization of the inoculum dilution of

bacterial isolates before the inoculation on the Mueller Hinton

agar plates.

Peptone 10 g Meat extract 10 g Sodium Chloride 5 g Distilled water 1 liter ph 7.4 Sterilized by autoclaving 121 oC for 15 minutes

Ⅲ.1.8.2. McFarland 0.5 nephlometer turbidity tube:

The standard McFarland 0.5 nephlometer tube [0.05ml of

1% barium chloride hydrate (Bacl2-2H2O) to 9.95 ml of 1%

sulfuric acid] was used to standardize the dilution of the

inoculum in the Muller Hinton broth according to Finegold and

Martin (1982).

Ⅲ.1.8.3. Antibiotic discs:

Eighteen antibiotic discs supplied from Oxoid were used in

this investigation to detect the susceptibility of the obtained

isolates according to the method described by Finegold and

Martin (1982) The content of each disc is given in Table (1):




49

Table (1): Concentration of antibacterial discs used in Agar Disc

Diffusion Method (ADD) for antibacterial susceptibility.

Conc. µg disc Symbol CompanyAntibacterial Agent NO

10 AML Oxoid Amoxicillin 1 30 AMC Oxoid Amoxicillin & clavulanic acid 2 10 AMP Oxoid Ampicillin 3 30 FOX Oxoid Cefoxitin 4 30 C L Oxoid Cephalexin 5 30 C Oxoid Chloramphenicol 6 30 CTX Oxoid Cefotaxim 7 5 CIP Oxoid Ciprofloxacin 8 30 DO Oxoid Doxycycline 9 15 E Oxoid Erythromycin 10 10 CN Oxoid Gentamicin 11 30 K Oxoid Kanamycin 12 30 N Oxoid Neomycin 13 10 NOR Oxoid Norfloxacin 14 5 RA Oxoid Rifampin 15 10 S Oxoid Streptomycin 16 5 TE Oxoid Tetracycline 17

10 units P Oxoid Penicillin----------------------- 18

Adapted from CLSI (Clinical and Laboratory Standards

Institute, 2005).




50

Щ .2.Methods :

Щ.2.1.Collection of milk samples (Blood and Handerson,

1986) : All samples were obtained from clinically chronic mastitic

cows that showed a degree of developing fibrosis. The cow ID,

quarter ID and the date of collection were recorded. All udder s,

teats and hands of the milkers were washed perfectly with soap

and water. Just before collection of samples, 70% ethyl alcohol

was used to disinfect teats, teat orifices. The first two or three jets

of milk from each quarter were discarded. The next 20-25 mL

were collected separately in a clean sterile pre-labeled screw-

capped cups. Each quarter sample was labeled to show the local

farm-cow ID number, quarter ID number and a study serial

number. Samples were kept chilled to 4°C in ice container and

were delivered to the laboratory within few hours.

Щ .2.2. Methods of bacteriological examination

(Quinn et al. , 2002):

Щ .2.2.1. Direct microscopical examination:

Щ .2. 2.1.1. Defattening of milk samples: Milk samples were centrifuged at 3000 r.p.m. for 20

minutes. Smears were prepared from the deposit of samples.

The heat fixed milk smears were defattined by xylol for 2




51

minutes then fixed by ethyl alcohol 95% followed by watching

with water, films were stained using different stains.

Щ .2.2.1.2. Staining:

Щ.2.2.1.2.1. Ziehl – Neelsen stain:

It was used for exclusion of tuberculous mastitis. The

staining was according to Cheesbrough (1984). All milk

samples stained and examined were T.B. microorganisms free.

Щ.2.2.1.2.2. Loeffler’s methylene blue:

All milk samples obtained from chronic mastitic cows were

stained with methylene blue according to Cheesbrough (1984).

Щ.2.2.2. Cultivation on ordinary and selective media:

Bacteriological culturing was carried out as follow:

1- Milk samples were incubated for 18-24 hours at 37oC before

culturing on the ordinary and selective media (Seadawy,

2004).

2- Using sterile platinum loop, approximately 0.01–0.03 ml of

each milk sample were streaked onto each of nutrient agar

medium, blood agar and MacConkey agar media.

3- The plates were incubated at 37 °C, and examined after 24 and

48 h. When slow growing or unusual bacteria were




52

suspected, longer incubation periods were used. If growth

did not appear within 3 days, plates were considered

negative.

4- Plates of solid media were examined macroscopically for

growth, colonial morphology and any change of the media

such as haemolysis and change in color of the medium or

colonies.

5- Single pure culture of each bacterium was obtained by sub -

culturing from single typical colony and transfer onto an

agar slant.

6- Inoculated slants were incubated at 37°C for 24 hours.

7- Single pure colony was picked up and stained with Gram’s

stain, examined microscopically under oil immersion lens.

8- Stained films were examined and their characteristics were

recorded (Gram’s stain reaction; the aggregation, shape and

arrangement of the isolated organism.

9- Another identical colony was picked up and stabbed onto semi

solid agar for preservation.

Щ .2. 2.3 Gram's stain:

Films were prepared from mastitic milk as well as from

suspected colonies on different media, heat fixed and stained

with Gram stain according to Cheesbrough (1984).




53

Щ.2. 2. 4. Methods of biochemical identification of:

Pure cultures of the isolates were identified biochemically

according to Koneman et al. (1992) and Quinn et al. (2002) as

follow:

Щ.2. 2. 4.1. Identification of Gram positive cocci:

They were identified by their characteristic morphology,

mannitole salt tolerance, cell aggregation, type of haemolysis,

catalase production, coagulase production, and novobiocin

sensitivity into staphylococci and streptococci. Furthermore

staphylococci were differentiated into Staphylococcus aureus,

Staphylococcus epidermidis, and Staphylococcus saprophyticus

while the streptococci were classified into hemolytic streptococci

and non hemolytic streptococci as follow:

Щ .2. 2. 4.1. 1. Identification of Staphylococcus sp:

The suspected colonies were picked up and prepared in pure

culture for biochemical and cultural characteristics according to

Collee et al. (1996) and Quinn et al. (2002) by use of the following

tests: catalase tests, coagulase tests using tube method, haemolysis

pattern, pigmentation, Mannitole and maltose fermentation test, salt

tolerance using mannitole salt agar, novobiocin sensitivity as shown

in photo (5). Tests and characteristics used for Differentiation




54

between the isolated mastitis Staphylococcus pathogens are seen in

Table (2):

Table (2); Differentiation between Staphylococcus aureus,

Staphylococcus epidermidis, and Staphylococcus saprophyticus

tests Staphylococcus aureus

Staphylococcus epidermidis

Staphylococcus saprophyticus

Coagulase test (tube test) + - - Haemolysis + -W -

Colony pigmentation +W - d Mannitol fermentation test + - -

Mannitol salt agar W - d Novobiocin sensitivity S S R

D: 11%-89% of the colonies give positive S: sensitive R: resistant

+W: positive to weak reaction -W: weak to negative reaction W: weak reaction.

+: over 90% of strains are positive - : over 90% of strains are negative

Photo (5) Novobiocin sensitivity test to differentiate between Staphylococcus epidermidis (sensitive) (at the right) and Staphylococcus saprophyticus (resistant) (at the left)




55

Щ.2. 2. 4.1.2. Identification of Streptococcus sp.:

Streptococcus sp. were identified into hemolytic streptococci

and non hemolytic streptococci according to Collee et al. (1996)

and Quinn et al. (2002) by their characteristics morphology and

catalase negative reaction. Then they were subjected for further

identification by their haemolysis pattern on blood agar, CAMP

test and sugar fermentation test .

Щ.2. 2. 4.2. Identification of Arcanobacterium pyogenes:

Isolates of Arcanobacterium pyogenes were identified

according to their cultural and morphological examination.

Then they were confirmed by the catalase tests, glucose,

mannitol, lactose and oxidase test according to Quinn et al.

(2002) as shown in Table(3):

Table (3): identification of the isolates of Arcanobacterium pyogenes:

Features and tests Arcanobacterium pyogenes Shape Gram positive Pleomorphic (cocci,

coccoid, club ,rod ) Cell aggregations Chinese litter arrangement Spore formation Non spore forming Haemolysis on blood agar β- haemolysis Catalase - Glucose + Mannitol + Lactose + Oxidase test -




56

Щ .2. 2.4.3. Identification of Gram positive bacilli (Bacillus

sp.):

Isolates of Gram positive bacilli (Bacillus sp.) were

identified according to Quinn et al. (2002) by their colonial

characteristics, Gram’s stain, endospore formation, catalase test

and haemolysis pattern, as shown in Table (4):

Table ( 4 ): identification of the isolates of (Bacillus sp.):

Features and tests Bacillus sp.

Shape Gram positive bacilli

Spore formation +

Haemolysis on blood agar β- haemolysis

Catalase test +




57

Щ .2. 2. 4.4. Identification of Enterobacteriaceae:

Isolates of Gram negative bacilli (Escherichia coli and

Klebsiella pneumoniae) were identified according to Quinn et

al.(2002) by their colonial growth on the MacConkeys bile salt

lactose agar medium, Gram’s stain, oxidase test, sugar fermentation

test, and the IMViC test which included indole test, methyl red

test, Voges Proskaur test and citrate utilization test as shown in

Table (5):

Table(5): Differentiation between Escherichia coli and Klebsiella

pneumoniae :

Features and tests Escherichia coli Klebsiella pneumoniae

Shape Gram negative bacilli Gram negative bacilli

Spore formation non spore former non spore former

Haemolysis on blood agar Some strains show haemolysis -

Catalase test + +

Oxidase test - -

Indole test, + -

Methyl red test + -

Voges Proskaur test - +

Citrate utilization test - +




58

Щ .2. 2. 4.5. Identification of Pseudomonas aeruginosa:

Isolates of Pseudomonas aeruginosa were identified

according to Kerig and Holt (1984) and Quinn et al. (2002) by

use of catalase test, oxidase test, pigment production, sugar

fermentation of glucose, sucrose and lactose, haemolysis on

blood agar and growth on MacConkey agar as shown in the

following Table (6):

Table ( 6 ): identification of the isolates of Pseudomonas

aeruginosa:

tests Pseudomonas aeruginosa

Shape Gram negative bacilli

Spore formation non spore former

Catalase test +

Oxidase test +

Glucose fermentation test +

Lactose fermentation test -

Sucrose fermentation test -

Diffuse pigment production +

Colony odor grape like odor

Growth on the MacConkey’s agar +




59

Щ .2.3. Methods of antibiotic susceptibility testing:

For antibiotic susceptibility testing, pure isolates were

suspended in nutrient broth and the suspension was adjusted to a

turbidity equivalent to 0.5 McFarland standards. The antibiotic

susceptibility test was carried out by the agar disc diffusion

technique (ADD) according to Bauer et al. (1966) and Quinn et

al. (2002).

Sterile cotton swab was dipped into the bacterial suspension of

each isolate. Then it was streaked on a Muller-Hinton agar plate.

Eighteen selected antibacterial discs were placed on the inoculated plate

for each isolate. The plates were inverted and incubated at 35°C - 37°C

for 16-24 hours. The isolates were categorized as susceptible,

intermediate and resistant based on the interpretive criteria developed by

CLSI (2005).

The tested antibiotics were amoxicillin, amoxicillin &

clavulanic acid, ampicillin, cefoxitin, cephalexin, chloramphenicol,

cefotaxim, ciprofloxacin, doxycycline, erythromycin, gentamicin,

kanamycin, neomycin, norfloxacin, rifampin, streptomycin,

tetracycline and penicillin.

The concentration of each antibiotic and interpretative diameter was reported in Table (7).




60

Table (7): Interpretative zone diameters of the antibiotic discs

used in Agar Disc Diffusion Method (ADD) for antibacterial

Susceptibility.

Interpretation Antibacterial Agent NO I P

S ≥ I R ≤ ≥20 19 ≤18 Amoxicillin 1

≥18 14-17 ≤13 Amoxicillin & clavulanic acid 2

≥17 14-16 ≤13 Ampicillin 3

≥18 15-17 ≤14 Cefoxitin 4

≥18 15-17 ≤14 Cephalexin 5

≥18 13-17 ≤12 chloramphenicol 6

≥23 15-22 ≤14 Cefotaxim 7

≥21 16-20 ≤15 Ciprofloxacin 8

≥16 13-15 ≤12 Doxycycline 9

>23 14-22 ≤13 Erythromycin 10

>15 13-14 ≤12 Gentamicin 11

>17 14-15 ≤13 Kanamycin 12

≥17 13-16 ≤12 Neomycin 13

≥17 13-16 ≤12 Norfloxacin 14

≥20 17-19 ≤16 Rifampin 15

≥15 12-14 ≤11 Streptomycin 16

≥19 15-18 ≤14 Tetracycline 17

≥29

≥22

12-21

≤28

≤11

Penicillin-------------------------

Organisms other than Staph-

18

IP: interpretation S: sensitive R: resistant I: intermediate Adapted from CLSI (Clinical and Laboratory Standards Institute, 2005).



RESULTS



RESULTS

61

IV. RESULTS:

Table (8): The mean of the affected chronic quarters per one

cow in each of the different test locations.

Location Number of

cows Quarter milk

samples Affected

quarter/ cow

Alexandria - farm 21 37 1.76

Sues -farm 30 60 2

Sharkia-farm 23 47 2

Assuit farm 3 3 1

Fayiom -farm 2 3 0.66

Total number of animals 79 150 1.88 - Samples were collected from chronic cases only.



RESULTS

62

Table (10): The prevalence of the isolated organisms in

relation to the total number of examined samples:

Isolated microorganism Number of isolates

% of the isolate to the total number of samples tested

1- Staphylococcus aureus 23 15.3%

2- Staphylococcus epidermidis 25 16.7%

3- Staphylococcus saprophyticus 19 12.7%

4- Streptococcus sp. 3 2 %

5- Bacillus cereus 24 16 %

6- Arcanobacterium pyogenes 1 0.7%

7- Escherichia coli 25 16.7%

8- Klebsiella pneumoniae 2 1.3 %

9- Pseudomonas aeruginosa 1 0.7 %

Number of isolates to total number of samples = 123 82%

10- Samples with no bacterial growth 27 18%

Total number of Quarter milk samples 150 100.10%

%: was calculated according to the total number of the examined

quarter milk samples.

All milk samples stained and examined were T.B. microorganisms

free.



RESULTS

63

Table (10): The prevalence of each isolated organism to the

total number of isolated pathogens.

Isolated microorganism Number of isolates

% of the isolate to the total number of isolated pathogens

1- Staphylococcus aureus 23 18.7%

2- Staphylococcus epidermidis 25 20.3%

3- Staphylococcus saprophyticus 19 15.4%

4- Streptococcus sp. 3 2.4%

5- Bacillus cereus 24 19.5%

6- Arcanobacterium pyogenes 1 0.8%

7- Escherichia coli 25 20.3%

8- Klebsiella pneumoniae 2 1.6%

9- Pseudomonas aeruginosa 1 0.8%

Total number of bacterial isolates 123 100 %

% : was calculated according to the total number of the

recovered bacterial isolates.



RESULTS

64

Graphic(1): The prevalence of each isolated organism to the

total number of isolated pathogens .

Stap

hylo

cocc

us a

ureu

s

Stap

hylo

cocc

us e

pide

rmid

is

Stap

hylo

cocc

us sa

prop

hytic

us

Stre

ptoc

occu

s sp

.

Bac

illu

s ce

reus

Arca

noba

cter

ium

pyo

gene

s

Esc

heri

chia

col

i

Kle

bsie

lla

pne

umon

iae

Pseu

dom

onas

aer

ugin

osa

0.00%

5.00%

10.00%

15.00%

20.00%

25.00%

1 2 3 4 5 6 7 8 9

Series1



RESULTS

65

Table (11): The incidence of each of isolated pathogen in

different experiment locations in relation to the total

bacterial isolates.

Isolated

microorganism

Nobareia- farm (Alexandria) Suis -farm Sharkia-farm Assuit Fayiom -farm

No % No % No % No % No %

Staphylococcus aureus 7 20 7 15.56 7 18.9 1 33.3 1 33.3

Staphylococcus epidermidis 8 22.85 8 17.78 8 21.6 1 33.3 0 0

Staphylococcus saprophyticus 6 17.15 6 13.33 7 18.9 0 0 0 0

Streptococcus sp. 1 2.86 2 4.44 0 0 0 0 0 0

Bacillus cereus 4 11.4 12 26.67 7 18.9 0 0 1 33.3

Arcanobacterium pyogenes 1 2.86 0 0 0 0 0 0 0 0

Escherichia coli 6 17.15 10 22.22 8 21.6 1 3..33 0 0

Pseudomonas aeruginosa 1 2.86 0 0 0 0 0 0 0 0

Klebsiella pneumoniae 1 2.86 0 0 0 0 0 0 1 33.3

Total number of bacterial isolates 35 100 45 100 37 100 3 100 3 100

% was calculated according to the total number of isolated

pathogens to total isolates in each farm.



RESULTS

66

Table (12): The distribution of chronic mastitis according to the affected quarter involvement of the examined animals.

Affected animals

Quarter involvement

One quarter Two quarters Three quarters All

No % No % No % No %

79 40 50.9% 19 24% 8 10.1% 12 15.2% * No. of the animals mentioned refers to the animals that have affected udder pattern with one, two, three or four affected udder quarter/s, even if the animal have different isolated pathogen for each quarter. Table (13): The distribution of chronic mastitis according to

the affected quarter involvement for each pathogen.

The pathogen

Affected animals

Quarter involvement One quarter Two quartersThree quarters All

No % No % No % No %

Staphylococcus aureus 21 20 95.25% - 0 % 1 4.75 % - 0 % Staphylococcus epidermidis 21 18 85.75% 2 9.5 % 1 4.75 % - Staphylococcus saprophyticus 15 12 80.0 % 2 13.33% 1 6.66 % 0 0 % Streptococcus sp. 3 3 100 % 0 0 % 0 0 % 0 0 % Bacillus cereus 3 3 100% 0 0% 0 0% 0 0% Arcanobacterium pyogenes 1 1 100% 0 0% 0 0% 0 0% Escherichia coli 3 3 100% 0 0% 0 0% 0 0% Klebsiella pneumoniae 2 2 100% 0 0% 0 0% 0 0% Pseudomonas aeruginosa 1 1 100 0 0% 0 0% 0 0%

*No. of the animals mentioned refers to the animals that have affected udder

pattern with one, two, three or four affected udder quarter/s with one isolated

pathogen from each of the mentioned affected quarters.



RESULTS

67

Table (14): Distribution of chronic mastitis within the four

udder quarters according to quarter involvement of the

examined animals in case of affected animals that had no

bacterial growth .

Affected animals

Quarter involvement

One quarter Two quarters Three quarters All

No % No % No % No %

22 18 81.8% 4 18.2% 0 0% 0 0%

* No. of the animals mentioned refers to the animals that have affected udder

pattern with one, two, three or four affected udder quarter/s without isolation

of bacterial pathogen, even if the animal has additional affected quarter/s

with isolated pathogen.



RESULTS

68

Antibiogram of the recovered pathogens was performed by agar disc diffusion method (shown in photo 6).

Photo (6): Shows the inhibition zones of different antibiotic

discs

- Results with eighteen antibiotics are shown as the following tables (15-21).



RESULTS

69

- Table (15): Antibiogram of Staphylococcus aureus (23 isolates) recovered from cows with chronic bovine mastitis to antimicrobial agents.

No Antibacterial Agent

Conc. µg disc Symbol

I P

S I R

No % No % No %

1 Ampicillin 10 AMP 18 75 1 9 4 16

2 Amoxicillin & Clavulanic acid 30 AMC 16 69 2 8 5 21

3 Amoxicillin 10 AML 15 65 1 4 7 30

4 Penicillin 10 units P 5 22 0 0 18 78

5 Gentamicin 10 CN 21 92 1 4 1 4

6 Kanamycin 30 K 20 87 0 0 3 13

7 Streptomycin 10 S 19 82.7 0 0 4 17.3

8 Neomycin 30 N 18 78 4 17.3 1 4.3

9 Cephalexin 30 C L 15 65 3 13 5 21

10 Cefoxitin 30 FOX 14 60 4 17 5 21

11 Cefotaxime 30 CTX 12 52.2 8 34.7 3 13

12 Ciprofloxacin 5 CIP 23 100 0 0 0 0

13 Norfloxacin 10 NOR 22 96 1 4 0 0

14 Doxycycline 30 DO 17 74 2 9 4 17.3

15 Tetracycline 5 TE 7 30 5 21.7 11 47.8

16 Chloramphenicol 30 C 19 82.6 2 8.6 2 8.6

17 Rifampin 5 RA 18 89 4 17 1 4.3

18 Erythromycin 15 E 13 57 6 26 4 17.3

IP: interpretation S: sensitive R: resistant I: intermediate



Results

70

Graphic (2) : Susceptibility pattern of Staphylococcus aureus recovered from cows with chronic bovine mastitis against different antibacterials.

Am

picillin

Am

oxicillin & C

lavulanic acid

Am

oxicillin

Penicillin

Gentam

icin

Kanam

ycin

Streptomycin

Neom

ycin

Cephalexin

Cefoxitin

Cefotaxim

e

Ciprofloxacin

Norfloxacin

Doxycycline

Tetracycline

Chloram

phenicol

Rifam

pin

Erythromycin

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Series1



RESULTS

71

Table (16): Antibiogram of Staphylococcus epidermidis (25 isolates) recovered from cows with chronic bovine mastitis to antimicrobial agents.



I P S I R

No % No % No %


2 Ampicillin 10 AMP 5 20 5 20 15 60



5 Gentamicin 10 CN 22 88 3 12 0 0

6 Streptomycin 10 S 20 80 3 12 2 8

7 Kanamycin 30 K 18 72 6 24 1 4

8 Neomycin 30 N 17 68 5 20 3 12

9 Cefoxitin 30 FOX 11 44 4 16 10 40

10 Cephalexin 30 C L 9 36 4 16 12 48

11 Cefotaxime 30 CTX 8 32 5 20 12 48



14 Doxycycline 30 DO 16 64 7 28 2 8

15 Tetracycline 5 TE 14 56 5 20 6 24

16 Chloramphenicol 30 C 16 64 3 12 6 24

17 Rifampin 5 RA 9 36 3 12 13 52

18 Erythromycin 15 E 4 16 11 44 10 40




Results

72

Graphic (3): Susceptibility pattern of Staphylococcus epidermidis recovered from

cows with chronic bovine mastitis against different antibacterials

Am

oxicillin & C

lavulanic acid

Am

picillin

Am

oxicillin

Penicillin

Gentam

icin

Streptomycin

Kanam

ycin

Neom

ycin

Cefoxitin

Cephalexin

Cefotaxim

e

Norfloxacin

Ciprofloxacin

Doxycycline

Tetracycline

Chloram

phenicol

Rifam

pin

Erythromycin

0102030405060708090

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Series1



RESULTS

73

Table (17): Antibiogram of Staphylococcus saprophyticus ( 19 isolates ) recovered from cows with chronic bovine mastitis to antimicrobial agents.



I P S I R

No % No % No %


2 Ampicillin 10 AMP 10 52 1 5 8 42

3 Amoxicillin 10 AML 8 42.2 1 5.3 10 52.6

4 Penicillin 10 units P 1 5.3 0 0 18 94.7

5 Gentamicin 10 CN 18 95 1 5 0 0

6 Kanamycin 30 K 14 74 4 21 1 5

7 Neomycin 30 N 14 74 4 21 1 5

8 Streptomycin 10 S 13 68 3 16 3 16

9 Cefoxitin 30 FOX 9 47 4 21 6 31

10 Cephalexin 30 C L 9 47 1 5 9 47

11 Cefotaxime 30 CTX 6 31.5 3 15.7 10 52.6

12 Norfloxacin 10 NOR 16 84 1 5 2 10


14 Doxycycline 30 DO 16 84 3 16 0 0

15 Tetracycline 5 TE 8 42 5 26.3 6 31.5

16 Chloramphenicol 30 C 12 63 4 21 3 15.7

17 Erythromycin 15 E 8 42 6 32 5 26

18 Rifampin 5 RA 6 32 5 26.3 8 42




Results

74

Graphic (4): Susceptibility pattern of Staphylococcus saprophyticus recovered

from cows with chronic bovine mastitis against different antibacterials

Am

oxicillin & C

lavulanic acid

Am

picillin

Am

oxicillin

Penicillin

Gentam

icin

Kanam

ycin

Neom

ycin

Streptomycin

Cefoxitin

Cephalexin

Cefotaxim

e

Norfloxacin

Ciprofloxacin

Doxycycline

Tetracycline

Chloram

phenicol

Erythromycin

Rifam

pin

0102030405060708090

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Series1



RESULTS

75

Table (18): Antibiogram of 3 isolates Streptococcus sp. recovered from cows with chronic bovine mastitis to antimicrobial agents.



I P S I R

No % No % No %

1 Ampicillin 10 AMP 2 66.6 1 33.3 0 0

2 Amoxicillin & Clavulanic acid 30 AMC 1 33.3 1 33.3 1 33.3

3 Penicillin 10 units P 1 33.3 1 33.3 1 33.3

4 Amoxicillin 10 AML 1 33.3 0 0 2 66.6

5 Gentamicin 10 CN 3 100 0 0 0 0

6 Kanamycin 30 K 3 100 0 0 0 0

7 Streptomycin 10 S 3 100 0 0 0 0

8 Neomycin 30 N 2 66.6 1 33.3 0 0

9 Cefoxitin 30 1 2 66.6 0 0 1 33.3

10 Cephalexin 30 C L 2 66.6 0 0 1 33.3

11 Cefotaxime 30 CTX 2 66.6 0 0 1 33.3


13 Norfloxacin 10 NOR 2 66.6 1 33.3 0 0

14 Doxycycline 30 DO 3 100 0 0 0 0

15 Tetracycline 5 TE 3 100 0 0 0 0

16 Erythromycin 15 E 2 66.6 1 33.3 0 0

17 Chloramphenicol 30 C 2 66.6 1 33.3 0 0

18 Rifampin 5 RA 2 66.6 1 33.3 0 0




Results

76

Graphic(5): Susceptibility pattern of Streptococcus sp. recovered from cows

with chronic bovine mastitis to different antibacterials.

h85 A

mpicillin

Am

oxicillin & Clavulanic acid

Penicillin

Am

oxicillin

Gentam

icin

Kanam

ycin

Streptomycin

Neom

ycin

Cefoxitin

Cephalexin

Cefotaxim

e

Ciprofloxacin

Norfloxacin

Doxycycline

Tetracycline

Erythromycin

Chloram

phenicol

Rifam

pin

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Series1



RESULTS

77

Table (19): Antibiogram of Bacillus cereus (24 isolates) recovered from cows with chronic bovine mastitis to antimicrobial agents.



I P S I R

No % No % No %

1 Amoxicillin & Clavulanic acid 30 AMC 13 54 2 8 9 37.5

2 Amoxicillin 10 AML 10 42 1 4 13 54

3 Ampicillin 10 AMP 9 38 4 16 11 45

4 Penicillin 10 units P 2 8.3 0 0 22 91.7

5 Gentamicin 10 CN 22 92 1 4 1 4

6 Streptomycin 10 S 21 88 1 4 2 8

7 Neomycin 30 N 20 83 3 12.6 1 4.3

8 Kanamycin 30 K 14 58 7 29 3 12.5

9 Cefoxitin 30 FOX 16 66 4 16 4 16

10 Cephalexin 30 C L 15 62.5 3 12.5 6 25

11 Cefotaxime 30 CTX 7 29.2 9 37.5 8 33.3


13 Ciprofloxacin 5 CIP 22 91.7 2 8.3 0 0

14 Doxycycline 30 DO 21 88 1 4 2 8

15 Tetracycline 5 TE 13 54 6 25 5 20.8

16 Chloramphenicol 30 C 15 62.5 5 20.8 4 16.6

17 Rifampin 5 RA 13 54 3 12.5 8 33

18 Erythromycin 15 E 12 48 6 24 7 28




Results

78

Graphic(6): Susceptibility pattern of Bacillus cereus recovered from cows

with chronic bovine mastitis todifferent antibacterials.

Am


Am

oxicillin

Am

picillin

Penicillin

Gentam

icin

Streptomycin

Neom

ycin

Kanam

ycin

Cefoxitin

Cephalexin

Cefotaxim

e

Norfloxacin

Ciprofloxacin

Doxycycline

Tetracycline

Chloram

phenicol

Rifam

pin

Erythromycin

5442 38

8.3

92 88 83

5866 62.5

29.2

96 91.7 88

5462.5

5448

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Series1



RESULTS

79

Table (20) Antibiogram of Escherichia coli (25 isolates)

recovered from cows with chronic bovine mastitis to

antimicrobial agents.



I P S I R

No % No % No %



3 Ampicillin 10 AMP 0 0 4 16 21 84


5 Gentamicin 10 CN 21 84 4 16 0 0

6 Kanamycin 30 K 16 64 5 20 4 16

7 Neomycin 30 N 14 56 5 20 6 24

8 Streptomycin 10 S 6 24 5 20 14 56

9 Cefoxitin 30 FOX 7 28 8 32 10 40

10 Cephalexin 30 C L 7 28 5 20 13 52

11 Cefotaxime 30 CTX 6 24 8 32 11 44

12 Doxycycline 30 DO 15 60 3 12 7 28

13 Tetracycline 5 TE 11 48 0 0 13 52

14 Norfloxacin 10 NOR 21 84 1 4 3 12



17 Rifampin 5 RA 5 20 5 20 15 60

18 Erythromycin 15 E 0 0 8 32 17 68




Results

80

Graphic (7): Susceptibility pattern of Escherichia coli recovered from cows with

chronic bovine mastitis to different antibacterials.

Am


Am

oxicillin

Am

picillin

Penicillin

Gentam

icin

Kanam

ycin

Neom

ycin

Streptomycin

Cefoxitin

Cephalexin

Cefotaxim

e

Doxycycline

Tetracycline

Norfloxacin

Ciprofloxacin

Chloram

phenicol

Rifam

pin

Erythromycin

0102030405060708090

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18



RESULTS

81

Table (21): Antibiogram of two isolates Klebsiella

pneumoniae recovered from cows with chronic bovine

mastitis to antimicrobial agents

NO Antibacterial Agent


I P

S I R

No % No % No %



3 Ampicillin 10 AMP 0 0 1 50 1 50


5 Gentamicin 10 CN 2 100 0 0 0 0

6 Kanamycin 30 K 0 0 1 50 1 50

7 Neomycin 30 N 0 0 1 50 1 50

8 Streptomycin 10 S 0 0 1 50 1 50

9 Cephalexin 30 C L 0 0 1 50 1 50

10 Cefotaxime 30 CTX 0 0 1 50 1 50

11 Cefoxitin 30 FOX 0 0 1 50 1 50



14 Doxycycline 30 DO 0 0 1 50 1 50

15 Tetracycline 5 TE 0 0 0 0 2 100


17 Erythromycin 15 E 0 0 0 0 2 100

18 Rifampin 5 RA 0 0 0 0 2 100




Results

82

Graphic (8): Susceptibility pattern of Klebsiella pneumoniae recovered from cows with

chronic bovine mastitis to different antibacterials.

Am

oxicillin

Am


Am

picillin

Penicillin

Gentam

icin

Kanam

ycin

Neom

ycin

Streptomycin

Cephalexin

Cefotaxim

e

Cefoxitin

Ciprofloxacin

Norfloxacin

Doxycycline

Tetracycline

Chloram

phenicol

Erythromycin

Rifam

pin

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18



RESULTS

83

Antibiogram of Arcanobacterium pyogenes recovered from

cows with chronic bovine mastitis to antimicrobial agents:

The recovered isolate was sensitive to gentamicin,

kanamycin, neomycin, cefotaxime, ciprofloxacin and rifampin.

Resistance to amoxicillin, amoxicillin & clavulanic acid, ampicillin,

cephalexin, cefoxitin, doxycycline, chloramphenicol and

erythromycin was recorded.

Antibiogram of Pseudomonas aeruginosa recovered from

cows with chronic bovine mastitis to antimicrobial agents:

The recovered isolate was Resistant to all used eighteen

antibiotics.



DISCUSSION



DISCUSSION

84

V. DISCUSSION

Mastitis is a worldwide disease causing high economic

losses in dairy industry. Losses in milk yield, increased

replacement heifer costs, loss of antibiotic contaminated milk,

drug and veterinary costs, extra labour and loss of cows with

high genetic value from the herd are among the aforementioned

losses (Gulliemette et al., 1996).

Three degrees of mastitis are commonly recognized; acute,

chronic (the clinical mastitis forms) and subclinical mastitis.

Chronic mastitis is less severe than acute mastitis and is seen in

most dairy herds (Labib, 1994)

Bovine mastitis often becomes chronic, and it is important

to identify quickly the new clinical cases in order to control

infection in the herd (Yousef, 2005).

The current study was planned to investigate the bacterial

etiology of chronic mastitis in bovine. One hundred fifty samples

were obtained from clinical cases of seventy-nine chronic

mastitic cows. From the data of Table (8), incidence of the

affected chronic quarters per each cow was 1.88 quarter/cow.

The higher incidence was recorded in Sues–farm and Sharkia-



DISCUSSION

85

farm (2 quarter/cow), while the lower incidence was recorded in

Fayiom- farm (0.66 quarter/cow).

From the data given in table (9), it is evident that out of

150 samples collected from chronic mastitic cows, 123 (82%)

bacterial isolates were recovered. The ratio of the total bacterial

isolates was nearly similar to that reported for all forms of

clinical mastitis by Odongo and Ambanim (1989) and Sargeant et

al. (1998) who recorded rates of 83.9% and 82.3%. On the other

hand, Wilson et al.(1991) and Benites et al. (2003) recorded

lower incidences (79.8% and 67% respectively).

The possible reasons for the failure to isolate pathogens

from mastitic quarters include spontaneous bacteriological

recovery with a persistent state of chronically changed mammary

tissues, too few pathogens to be detected, pathogens inhibited by

antibiotic residues, pathogens killed after the samples was taken

and before culturing (Miltenburg et al., 1996)

Out of these 123 isolates, 95 (77.2%) were Gram positive

bacteria and 28 (22.8%) isolates were Gram negative organisms.

Several factors have been reported to influence the

prevalences of pathogens. A correlation of Gram-positive bacteria



DISCUSSION

86

with the chronic udder inflammation was suggested, while Gram-

negative bacteria were associated with acute catarrhal

inflammation of alveoli (Inui et al., 1979). Other factors

including the definition of the culture-positive result and the

procedure used for freezing of the samples. Freezing has a

significant effect on the survival of some pathogenic organisms

causing mastitis. With the longer period of freezing there was a

significant decrease in of Escherichia coli and Arcanobacterium

pyogenes positive samples, while an increase in number of

sample positive for coagulase-negative staphylococci. On the

other hand, freezing had no effect on the Staphylococcus aureus

and Streptococci (Schukken et al., 1989). During the present

investigation all of the examined samples were kept frozen as

recommended by Murdough et al. (1996) because immediate

culturing of samples was not possible.

The predominant Gram positive bacteria were Staphylococcus

sp. [67 isolates (54.5%)]. The study result was higher than that

reported by Hanselmann (1978) and Wilson et al. (1991) (45%

and 7.3% respectively).

The coagulase negative staphylococci were predominating

[44 isolates (35.8%)]. The recorded ratio is nearly similar to that

reported by Benites et al. (2003) who reported 35.7%. This ratio



DISCUSSION

87

was higher than that recorded by Lafi et al. (1994), Miltenburg et

al. (1996), Kudinha and Simango (2002), Malinowski et al.(2003)

and who recorded 16%, 4.2%, 22.9% and 15% respectively.

Results in table (10) showed that out of these isolates,

coagulase negative Staphylococcus epidermidis constituted

20.3% of the total isolates. These result was nearly similar with

that reported for all forms of clinical mastitis by Mahbub-E-

Elahi et al.(1996) who identified 18.0% of the isolates as

Staphylococcus epidermidis. On the other hand Twardona et

al.(1999) recorded higher incidence (34.9%) while lower incidences

were recorded by Inui et al. (1979) and Beytut et al. (2002) who

identified 11.8%, 8.33% of the isolates, respectively as

Staphylococcus epidermidis.

The coagulase positive Staphylococcus aureus represented

18.7% of the total isolates. The recorded ratio was nearly similar

to that recorded by Wilson et al. (1991) and Kudinha and Simango

(2002) who reported 18.4% and 17.1%, respectively. On the other

hand, higher incidences were recorded by Erer et al. (1996),

Twardona et al. (1999) and Beytut et al. (2002) who reported 47.3%,

29.1% and 43.75% respectively. Lower incidences were recorded

by Benites et al.(2003) and Lafi et al. (1994) who identified



DISCUSSION

88

12.2% and 14% of the isolated pathogens as Staphylococcus

aureus.

Β-haemolytic streptococci and B. cereus represented 2%

and 16% respectively. On the other hand, Inui et al. (1979)

identified higher ratio of isolates (5.9%) as Β-haemolytic

streptococci, while Erer et al. (1996), Mahbub-E-Elahi et

al.(1996) and Beytut et al. (2002) identified lower incidences of

the pathogens as Gram positive Bacillus cereus (1.1%, 2.08%

and 4.6% of the total isolates respectively).

One isolate of Arcanobacterium pyogenes was recovered

with a prevalence of 0.8% that was exceeded by the higher

incidences which were recorded by Inui et al.(1979), Erer et al.

(1996) and Beytut et al.(2002), who identified 16.3%, 16.3% and

19.79% of mastitic isolates, respectively as Arcanobacterium

pyogenes

Gram negative bacteria were also recovered. The

predominant Gram negative Escherichia coli were 25isolates

(16.7%). The recorded prevalence was higher than that reported

by Jha et al.(1994) and Miltenburg et al. (1996) who identified

16.8% and 16.7% of the isolates recovered from cases of clinical

mastitis (two forms) respectively as Escherichia coli . Too lower



DISCUSSION

89

incidences were recorded Samborski et al. (1992) and by Beytut et

al. (2002) who identified 0.9% and 7.29% of the isolated

pathogens, respectively as Escherichia coli.

Two isolates of Klebsiella pneumoniae (1.3%) were

recovered. Higher incidence was recorded by Inui et al. (1979) and

Erer et al. (1996) who identified 5.9% and 3.8% of the isolated

mastitic pathogens, respectively as Klebsiella pneumoniae.

Prevalence of Escherichia coli and Klebsiella sp. on

whole-herd surveys may be evaluated in lower ratios. These may

be due to the relatively short duration of intramammary

infections caused by Gram-negative bacteria (Sears et al., 1993)

compared with agents that usually persist longer in the mammary

gland. Wilson et al.(1997) suggested that Cram-negative bacteria

remain a less common cause of bovine mastitis regarding to the

Gram-positive pathogens.

One isolate of Pseudomonas aeruginosa (0.7%) was

recovered. On the other hand Wilson et al. (1997) recorded

lower incidence (0.1%) while the result recorded by Inui et al,

(1979) was higher (5.9%).



DISCUSSION

90

Results in Table (11) showed that in case of Alexandria-

farm, Staphylococcus epidermidis was the higher prevalent

organism (22.85%) followed by Staphylococcus aureus (20%)

while the Streptococcus sp., Arcanobacterium pyogenes,

Pseudomonas aeruginosa and Klebsiella pneumoniae were the

least (2.86% for each).

Bacillus cereus was isolated in the higher ratio in Sues-

farm (26.67%) followed by Escherichia coli (22.22%) while the

lower ratio was reported for Streptococcus sp. (4.44%).

In case of the Sharkia-farm Staphylococcus epidermidis

together with Escherichia coli constituted the higher ratios of the

isolated organisms (21.6% for each).

In Assuit, the isolated pathogens were Staphylococcus

aureus, Staphylococcus epidermidis and Escherichia coli. They

were equally isolated (33.3% for each).

Staphylococcus aureus, Klebsiella pneumoniae and

Bacillus cereus were equally isolated (33.3%) in Fayiom –farm.

Distribution of chronic mastitis according to the affected

quarter involvement was shown in Table (12). Animals were

categorized according to number of affected quarters (whether



DISCUSSION

91

the animal was infected with one pathogen or more). Results

revealed that animals with one mastitic quarter were the greatest

group (50.6%). The recorded ratio was much higher than that

reported by Mahbub-E-Elahi et al. (1996) which was 34% and

based on data of the clinical mastitis (two forms of the clinical

mastitis). The second group was those which had two affected

quarters (22.8%). That ratio was nearly similar to that obtained

by Mahbub-E-Elahi et al. (1996) who recorded 24.67%. The

third group was the affected whole udder (15.2%) followed by

those of three mastitic quarters (10.2%).

Data in Table (12) represent the distribution of chronic

mastitis in the affected quarters for each pathogen. 95.25% of the

Staphylococcus aureus infections were found in only one

affected quarter per cow. On the same time, neither two quarter

infections nor, four-udder infections with Staphylococcus

aureus were reported at the same cow.

Each of 85.75% Staphylococcus epidermidis isolates and

80% Staphylococcus saprophyticus isolates were isolated in one

quarter infections and they were not isolated from the all udder

four quarters of any cow.



DISCUSSION

92

Although twelve cases of four-quarter infections seen in

table (12) were found with multiple pathogens at the same cow,

none of them were infected with one pathogen in its four quarters

as seen in Table (13).

The data in Table (15) represent the applied antibiogram

study on 23 isolates of Staphylococcus aureus The results of the

sensitivity tests with the tested members of β-lactam group of

antibiotics revealed that the higher incidence of sensitivity was

with ampicillin (78.2% of the isolates were sensitive) followed

by the combination of amoxicillin and clavulanic acid (69%)

while the lower incidence was recorded in case of penicillin

(22%). The result was nearly agreed with that recorded by Binde

and Gjul (1988) while Winter and Duetz (1998) recorded higher

incidences.

In case of tested elements of the aminoglycosides group, the

higher incidence was with gentamicin (92%) followed by

kanamycin (87%) while the lower incidence was with neomycin

(78%).

In case of the tested members of the cephalosporins group,

Staphylococcus aureus represented the higher incidence of



DISCUSSION

93

sensitivity with cephalexin (65%). followed by cefoxitin (60%)

The lower incidence was (52.2%) in case of cefotaxime.

On the other hand, all the isolates of Staphylococcus aureus

were sensitive for ciprofloxacin and it was 96% in case of

norfloxacin while the incidence of the sensitive isolates was 74%

for doxycycline, it was 30% for tetracycline. Higher incidences

were reported for rifampin (89%) and chloramphenicol (82.6%)

while it was 57% for Erythromycin.


study on 25 isolates of Staphylococcus epidermidis. The results

of the sensitivity tests with the tested members of β-lactam group

of antibiotics revealed that the higher incidence of sensitivity was

with combination of amoxicillin and clavulanic acid (24% of the

isolates were sensitive) followed by ampicillin (20%) while the

lower incidence was recorded with penicillin (8 %).

In case of tested elements of the aminoglycosides group, the

higher incidence was with gentamicin (88%) followed with

streptomycin (80%) while the lower incidence was with

neomycin (68%).



DISCUSSION

94

In case of the tested members of the cephalosporins group,

Staphylococcus epidermidis represented the higher incidence of

sensitivity with cefoxitin (44%), followed by cephalexin (36%).

The lower incidence was (32%) in case of cefotaxime.

On the other hand, 92% the isolates of Staphylococcus

epidermidis were sensitive for norfloxacin and it was 88% in

case of ciprofloxacin while the incidence of the sensitive isolates

was 64% for doxycycline, it was 56% for tetracycline.

The incidence with chloramphenicol, rifampin and

erythromycin were 64%, 36% and 16% respectively.


study on 19 isolates of Staphylococcus saprophyticus. The

results of the sensitivity tests with the tested members of β-

lactam group of antibiotics revealed that the higher incidence of

sensitivity was with combination of amoxicillin and clavulanic

acid (68% of the isolates were sensitive) followed by ampicillin

(52%) while the lower incidence was recorded with penicillin

(5.3 %).



DISCUSSION

95

For aminoglycosides group, the higher incidence was with

gentamicin (95%) followed with kanamycin (74%) while the

lower incidence was with streptomycin (68%).

With the tested members of cephalosporins group, 47% of

the isolates of Staphylococcus saprophyticus were sensitive in

case of cefoxitin and cephalexin. Lower incidence (31.5%) was

recorded in case of cefotaxime.

On the other hand, 84% the isolates of Staphylococcus

saprophyticus were sensitive for norfloxacin and it was 74% in

case of ciprofloxacin while the incidence of the sensitive isolates

was 84% for doxycycline, it was 42% for tetracycline.

The incidence with chloramphenicol, erythromycin and

rifampin were 63%, 42% and 32% respectively.


study on 3 isolates of Streptococcus sp. The results of the



with ampicillin (66.6% of the isolates were sensitive) followed

by the other three tested members (33.3% for each).



DISCUSSION

96

For aminoglycosides group, all isolates of Streptococcus

sp. were sensitive (100%) for kanamycin, streptomycin and

gentamicin.

With the tested members of cephalosporins group, the

incidence the sensitive isolates of Streptococcus sp was 66.6%

for each of cefoxitin, cephalexin and cefotaxime.

On the other hand, all of the isolates of Streptococcus sp

were sensitive for ciprofloxacin and it was 66.6% in case of

norfloxacin. All of the isolates were sensitive to doxycycline and

tetracycline.

The incidence with chloramphenicol, erythromycin and

rifampin were 66.6% for each.


study on 24 isolates of Bacillus cereus The results of the



with the combination of amoxicillin and clavulanic acid (54% of

the isolates were sensitive) followed by amoxicillin (42%) while

the lower incidence was recorded with penicillin (8.3 %).



DISCUSSION

97


gentamicin (92%) followed with streptomycin (88%) while the

lower incidence was with kanamycin (58%).

With the tested members of cephalosporins group, Bacillus

cereus represented the higher incidence of sensitivity with

cefoxitin (66%) followed by cephalexin (62.5%). The lower

incidence was (29.2.5%) in case of cefotaxime.

On the other hand, 96% the isolates of Bacillus cereus

were sensitive for norfloxacin and it was 91.7% in case of

ciprofloxacin.

The incidence of the sensitive isolates was 88% for

doxycycline and 54% for tetracycline.

The incidence with chloramphenicol, rifampin and

erythromycin were 62.5%, 54% and 48% respectively.

Only one isolate of Arcanobacterium pyogenes was identified

and was resistant for the tested members of β-lactam group,

streptomycin, cephalexin, norfloxacin, tetracycline, doxycycline,

chloramphenicol and erythromycin.



DISCUSSION

98

Contrarily, it was sensitive for gentamicin, kanamycin,

neomycin, cefotaxime, ciprofloxacin and rifampin.


study on 25 isolates of Escherichia coli. In case of β-lactam

group of antibiotics, the higher incidence of sensitive isolates

was with combination of amoxicillin and clavulanic acid (28 %

of the isolates were sensitive) followed by amoxicillin (12%)

while no isolates were sensitive for ampicillin or penicillin. The

present result nearly agreed with Sogaard (1982) and Sikorski

(1986) who reported that all isolates were sensitive.


gentamicin (84%) followed with kanamycin (64%) while the

lower incidence was with streptomycin (24%).

With the tested members of cephalosporins group, 28% of

the isolates of Escherichia coli were sensitive in case of

cefoxitin and cephalexin while the incidence was (24.5%) in case

of cefotaxime.

On the other hand, 84% the isolates of Escherichia coli

were sensitive for norfloxacin and it was 20% in case of

ciprofloxacin while the incidence of the sensitive isolates was



DISCUSSION

99

60% for doxycycline. The recorded percentage in case

tetracycline was 48%.

The incidence with chloramphenicol and rifampin were

52%, 20% respectively. No isolates were sensitive to

erythromycin.

From the data given in Table (21), only two isolates of

Klebsiella pneumoniae were picked and were sensitive (100%)

for gentamicin and ciprofloxacin. One isolate (50%) was

sensitive for the combination of amoxicillin and clavulanic acid,

norfloxacin and chloramphenicol. For other tested antibiotics, all

isolates were resistant.

Only one isolate of Pseudomonas aeruginosa was picked

and showed resistance for all used antibiotics.



SUMMARY



SUMMARY

100

VI. SUMMERY

Mastitis is still implicated as one of the major disease

problems in dairy animals. From the economical point of view,

chronic mastitis results in eradication of the cow that became

unfit to be kept as milk producing cow cconstituting great losses

to dairy producers. Due to the little number researches about

the causes of the chronic mastitis, the present study was

performed.

The present study was planned to discuss the following:

1- To detect the prevalence of the most important bacterial

pathogens isolated from the clinically chronic mastitic

cows.

2- Purification as well as morphological, cultural and

biochemical identification of the recovered isolates.

3- An antibiogram study on the recovered isolates was

undertaken to explore the sensitivity patterns of the

recovered isolates to eighteen antibiotics.

The study revealed that the bacterial pathogens were

isolated from (82%) of chronically mastitic quarters. The Gram

positive bacteria constituted 77.2% of the isolates while the

Gram negative organisms were 22.8%. On one hand

Staphylococcus sp. were the predominant of the Gram positive



SUMMARY

101

bacteria (54.5%). On the other hand Escherichia coli was the

predominant of the Gram negative isolates (20.3%).

Staphylococcus epidermidis and Escherichia coli were the

predominant organism (20.3% each) followed by Bacillus

cereus (19.5%), Staphylococcus aureus (18.7%) and

Staphylococcus saprophyticus (15.4%). Both of Streptococcus

sp., Arcanobacterium pyogenes, Klebsiella pneumoniae and

Pseudomonas aeruginosa were recovered in low prevalences

(2.4%, 0.8%, 1.6% and 0.8% respectively).

The coagulase negative Staphylococcus sp. represented the

widely spreading organism in-between quarters in a same one

cow (infected two quarters in the same one cow represented

22.9% of the isolates while in case of infected three quarter was

11.4 %. Staphylococcus aureus was the next spreading

organism (infection in all quarters by 4.75 % of the isolates).

Sensitivity test of eighteen antibiotics showed that the

norfloxacin and gentamicin were the most effective antibiotic in

case of Staphylococcus aureus, Staphylococcus epidermidis,

Staphylococcus saprophyticus, Bacillus cereus and

Escherichia coli.



SUMMARY

102

In addition, ciprofloxacin was effective in case of

Staphylococcus aureusو Staphylococcus epidermidis and

Bacillus cereus, doxycycline for Staphylococcus saprophyticus

and kanamycin for Escherichia coli.

Ciprofloxacin and gentamicin were effective in case of

Klebsiella pneumoniae.

The Pseudomonas aeruginosa was resistant to all of the

tested antibiotics.



Recommendations



RECOMMENDATIONS

103

VII. RECOMMENDATIONS

- The wide spreading contagious pathogens (Staphylococcus

aureus and Coagulase negative staphylococcus) comparing

to the environmental pathogens (Escherichia coli,

Klebsiella pneumoniae and Pseudomonas aeruginosa)

indicate the need to follow rigorous program of teat dipping

and dry cow antibiotic therapy (intramammary antibiotic

treatment at drying off).

- Improvement of the used dipping programs during the

milking process to combat the wide spreading of the

contagious pathogens isolated in case of chronic mastitis by

periodical estimation of the validity of used agents and the

continuous change of used materials

- Heifers can be given intramammary antibiotic treatment

during gestation.

- Giving attention to the clinical use of some antibiotics to

which most of the chronic mastitis-causing pathogens

showed sensitivity (norfloxacin, gentamicin, ciprofloxacin

and doxycycline).



Abstract



ABSTRACT

104

VIII. ABSTRACT

Examination of 150 chronically mastitic quarter milk samples collected

from 79 machine milked Friesian cows from five dairy herds. Animals had

chronic mastitis and showed developing fibrosis and were assigned as chronic

mastitic cases. The bacterial pathogens were isolated from 82% of the samples.

The Gram positive bacteria constituted 77.2% of the isolates while the Gram

negative organisms were 22.8%. Staphylococcus sp. represented 54.5% of the

total isolates. Staphylococcus epidermidis (20.3%), Staphylococcus aureus

(18.7%), Staphylococcus saprophyticus (15.4%), Escherichia coli (20.3%),

Streptococcus sp. (2.4%), Arcanobacterium pyogenes (0.8%), Klebsiella

pneumoniae (1.6%) and Pseudomonas aeruginosa (0.8%) were isolated..

coagulase negative Staphylococcus sp represented the most spreading organisms

in-between quarters in a same one cow followed by Staphylococcus aureus. An

antibiogram of the isolates revealed that norfloxacin, gentamicin, ciprofloxacin,

and doxycycline were the most effective antibiotics in case of Staphylococcus

aureus, Staphylococcus epidermidis, Bacillus cereus, Staphylococcus

saprophyticus and Escherichia coli. The Pseudomonas aeruginosa was resistant

to all of the tested antibiotics.



REFERANCES



REFERANCES

105

IX. REFERENCES

Armenteros, M.; Peña, J.; Pulido, J. L. and Linares, E. (2002):

Characterization of the situation in dairy herds affected by

bovine mastitis.

Revista de Salud Animal 24 (2): 99-105. Cited from http://

www.ncbi. nlm. nih.gov/pubmed

Barto, J.; Czokoly, P. and Hlinka, D. (1984): Classification of

bovine mastitis caused by group L streptococci in an

infected herd.

Veterinárhacekstvi, 34 (9): 395-396.

Bauer, A. W.; Kirby W. M. M.; Sherris, J. C. and Turk, M.

(1966): Antibiotic susceptibility testing by standardized

single disc method.

American Journal of Clinical Pathology. 45 (493-496).

Benites, N. R.; Melville, P. A. and Costa E. O. (2003): Evaluation

of the microbiological status of milk and various structures

in mammary glands from naturally infected dairy cows.

Tropical Animal Health Production 35 (4): 301-307.



REFERANCES

106

Bertoldini, G.; Burnner, F. and Redaellin, G. (1985): Further

studies on the antibiotic sensitivity of streptococci and

staphylococci associated with bovine mastitis.

Archivio Veterinario Italiano 36 (4): 49-101. Cited from

http://ezproxy.htu.se/menu

Beytut, E.; Aydn, F.; Özcan, K. and Genc, O. (2002):

Pathological and bacteriological investigations on bovine

mastitis in Kars Region and its surrounds.

Kafkas Üniversitesi Veteriner Fakültesi Dergisi 8 (2):111-

122.

Binde, M. and Gjul, G. G. (1988): Penicillin resistance of S.

aureus strains isolated from bovine mammary glands with

acute and chronic mastitis.

Norsk Veterinaertidsskrift 100 (5): 345-348.

Blood, D. C. and Handerson, J. A. (1986): veterinary medicine.

3rd Edition. Baillierc, Tindall and Gassel, London

Böhmer, N. and Schneider, P. (1999): The isopathic-homeopathic

mastitis therapy: practical example.

Ganzheitliche Tiermedizin 13 (2): 80-82.




REFERANCES

107

Brouillette, E.; Grondin, G.; Shkreta, L.; Lacasse, P. and

Talbot, B. G. (2003): In vivo and in vitro demonstration

that Staphylococcus aureus is an intracellular pathogen in

the presence or absence of fibronectin-binding proteins.

Microbial Pathogenesis 35 (4):159-168.

Brouillette, E.; Martinez, A.; Boyll, B. J.; Alien, N. E. and

Malouin, F. (2004): Persistence of a Staphylococcus

aureus small-colony variant under antibiotic pressure in

vivo.

FEMS Immunology and Medical Microbiology 41 (1): 35-

41.

Buddle, B. M.; Herceg, M.; Ralston, M. J. and Pulford, H. D.

(1987): Reinfection of bovine mammary glands following

dry-cow antibiotic therapy.

Veterinary Microbiology 15 (3): 191-199.

Cheesbrough, Monica (1984): Medical laboratory manual for

tropical countries.

2 nd Edition. Tropical health technology and Butterworth.

CLSI (2005): Clinical and Laboratory Standards Institute

Performance Standards Antimicrobial susceptibility Testing.



REFERANCES

108

Collee, J. G.; Fraser, A. G.; Marmion, B. P. and Simmons, A.

(1996): Practical Medical Microbiology.

14th Edition. Churchill, LivingStone, New York.

Costa, E. O.; Benites, N. R.; Guerra, J. L. and Melville, P. A.

(2000): Antimicrobial susceptibility of Staphylococcus spp.

isolated from mammary parenchymas of slaughtered dairy

cows.

J. Vet. Med, Infectious Disease Vet Public Health 47(2): 99-

103.

Costa, E. O.; Ribeiro, A. R.; Watanabe, E. T. and Melville, P.

A. (1998): Infectious bovine mastitis caused by

environmental organisms.

Zentralbl Veterinarmed B., 45(2): 65-71. Cited from

http://ezproxy.htu.se/menu.

Cruickshank, R.; Dugid, J. p.; Mormion, B. P. and Swain, R.

H. A. (1975): The practice of medical microbiology.

12th Edition. Churchill Livingstone, London, U.K...

DeOliveira A. P.; Watts, J. L.; Salmon, S. A. and Aarestrup

FM.(2000):Antimicrobial susceptibility of Staphylococcus

aureus isolated from bovine mastitis in Europe and the

United States.

Journal of Dairy Science 83 (4): 855- 862.




REFERANCES

109

Diarra, M. S.; Petitclerc, D.; Deschênes, É.; Lessard, N.

Grondin, G.; Talbot, B. G. and Lacasse, P. (2003):

Lactoferrin against Staphylococcus aureus Mastitis:

Lactoferrin alone or in combination with penicillin G on

bovine polymorphonuclear function and mammary

epithelial cells colonization by Staphylococcus aureus

Veterinary Immunology and Immunopathology 95 (1-

2):33-42.

Dogan, B.; Klaessig, S.; Rishniw, M.; Almeida, R. A.; Oliver, S.

P.; Simpson, K. and Y.H. Schukken (2006): Adherent

and invasive Escherichia coli are associated with persistent

bovine mastitis.

Journal of Veterinary Microbiology 116 (4): 270-282.

Dopfer, D.; Almeida, R. A.; Lam, T. J.; Nederbragt, H. ;

Oliver, S. P. and Gaastra, W. (200٠): Adhesion and

invasion of Escherichia coli from single and recurrent

clinical cases of bovine mastitis in vitro.


Döpfer, D.; Nederbragt, H.; Almeida, R. A. and Gaastra, W. (2001):

Studies about the mechanism of internalization by mammary epithelial

cells of Escherichia coli isolated from persistent bovine mastitis.




REFERANCES

110

Doymaz, M. Z.; Sordillo, L. M.; Oliver, S. P. and Guidry, A. J.

(1988): Effects of staphylococcus aureus mastitis on

bovine mammary gland plasma cell populations and

immunoglobulin concentrations in milk.

Veterinary Immunology and Immunopathology 20 (1): 87-

93.

El Kholy, A. M.; Hosein, H. I. and Thabet, A. El-R(1994):

Chemical and cytobacteriologicals studies for the detection

of clinical mastitis.

Assuit Vet. Med. J., 30 (60): 154-164

Erer, H.; Ates, M.; Kran, M. M.; Ciftci, M. K. and Kaya, O.

(1996): Pathological and bacteriological studies on bovine

mastitis.

Veteriner Bilimleri Dergisi, 12 (123-133).

Fang, W. (1996): A novel fluorometric method for evaluation of

the post antibiotic effect of antibacterial drugs on mastitis-

causing Staphylococcus aureus and Escherichia coli •

Journal of Microbiological Methods 26 (1-2): 151-159

Finegold, S. D.; and Martin W. J. (1982): Diagnostic

Microbiology.

6th Edition. the Mosby company, USA



REFERANCES

111

Garg, D. N. and Kapoor, P. K. (1986): Isolation and characterization of

Rhodococcus (Corynebacterium) equi from cows with mastitis.

Indian Journal of Comparative Microbiology Immunology and

Infectious Diseases 7 (23): 91-95.

Gonzalez, R. N. (1996): Prototheca, Yeast, and Bacillus as a Cause of

Mastitis.

National Mastitis Council Annual Meeting Proceedings (1996) p.

82.

Gronlund, U. ; Hallen, S. C. and Persson, W. K. ; (2005): Haptoglobin

and serum amyloid A in milk from dairy cows with chronic sub-

clinical mastitis.

Vet. Res. 36 (2): 191-198.

Grunert, E. and Weight, U. (1979): Enterkrnkheiten aus uiatrik,

kurzgefabte D Arstelling. Verlag. und H hapter, 3. Uberarbeitete

und erweiterte und erweiterte Auflage. Abstract in English

language was Cited from http://ezproxy.htu.se/menu.

Gulliemette, J. M.; Bouchard, E. And Bigraspoulin, M. (1996): Mastitis

and its control. Increases in somatic cell count.

Producteur de lait Quebecois 16 (6):24-27. Abstract in English

language was cited from http://ezproxy.htu.se/menu.





REFERANCES

112

Hanselmann, R. (1978): Epidemiology of chronic mastitis.

Clinical and microbiological studies on the course of the

disease on a herd basis.

Zur Enzootologie der chronischen Mastitis Klinische und

mikrobiologische Verlaufsuntersuchungen auf Betriebsbasis.,

1978, p. 77pp., 32 ref.

Harmon, V. R. (1995): Mastitis and milk quality

Milk Quality edited by F. Harding.

Hartmann, H. (1990): Rates of resistance of mastitis pathogens

from cows in Switzerland.

Schweiz Arch Tierheilkd 132 (6): 325-9. Abstract in

English Cited from http://www.ncbi.nlm.nih.gov/pubmed

Hensen, S.; Pavicic, M.;, Lohuis J.; DeHoog J. and Poutrel,

B.(2000): Location of Staphylococcus aureus within the

experimentally infected bovine udder and the expression of

capsular polysaccharide type 5 in situ.

Journal of Dairy Science 83 (9):1966-1975.

Hill, A. W. and Shears, A. L. (1979): Recurrent coliform mastitis

in dairy cow.

Vet. Rec., 105: 299-301.


http://www.ncbi.nlm.nih.gov/pubmed


REFERANCES

113

Hussain, S. A.; Willayat, M. M.; Munshi, Z. H. and Asifa, N.

(2002): Isolation and antibiogram of coagulase positive

Staphylococci from bovine mastitic milk.

Indian Journal of Comparative Microbiology Immunology

and Infectious Diseases 23 (2): 191-192.

Inui, S.; Kume, T. Hiramune, T. and Murase, N. (1979):

Pathological survey of bovine mastitis.

Bulletin of the National Institute of Animal Health (78): 25-

38.

Jain, N. C. (1979): Common factors infectious mastitis

Journal of Dairy Science 62 (1): 128-134.

Jha, V. C.; Thakur, R. P. and Yadav, J. N.(1994): Bacterial

species isolated from clinical bovine mastitis and their

antibiotic sensitivity patterns

Veterinary Review (Kahamandu) 9 (1): 21-23

Kerig, N. R. and Holt, J. G. (1984): Bergey’s Manual of Systemic

Bacteriology.

8th Edition.Williams and Willkins, Baltimore, London.



REFERANCES

114

Koneman, E. W.; Allen, S.D.; Janda, W. M. Schreckenberger,

P. C. and Winn, W. C. (1992): Introduction to diagnostic

Microbiology.

4th Edition. J. B. Lippincott Company Philadelphia, USA.

Krzywoszynski, W. (1977): Value of residual milk in the

diagnosis of chronic mastitis in cows.

Polskie Archiwum Weterynaryjne 20 (1): 105-120.

Kudinha, T. and Simango, C. (2002): Prevalence of coagulase-

negative staphylococci in bovine mastitis in Zimbabwe.

Journal of the South African Veterinary Association 73 (2):

62-65.

Labib, Sahar, R. M. (1994): Bacteriological studies on Recurrent

Mastitis in Friezian cows.

Thesis of M. V. Sc., Cairo University, Faculty of Veterinary

Medicine, Department of microbiology.

Lafi, S. Q.; Al-Rawashdeh, O. F; Ereifej, K. I. and Hailat, N. Q.

(1994): Incidence of clinical mastitis and prevalence of

subclinical udder infections in Jordanian dairy cattle.

Preventive Veterinary Medicine 18 (2): 89-98.



REFERANCES

115

Larsen, H. D.; Sloth, K. H.; Elsberg, C.; Enevoldsen, C.;

Pedersen, L. H.; Eriksen, N. H. R.; Aarestrup, F. M.

and Jensen, N. E.(2000): The dynamics of Staphylococcus

aureus intramammary infection in nine Danish dairy herds.

Veterinary Microbiology 71 (1-2): 89-101.

Lehtolainen, T.; Shwimmer, A.; Shpigel, N.; Honkanen-

Buzalski, T. and Pyorala, S.(2003): n vitro antimicrobial

susceptibility of Escherichia coli isolates from clinical bovine

mastitis in Finland and Israel.

Journal of Dairy Science 86 (12): 3927- 3932.

Liu, P. V. and Mercer, C. B. (1963): Virulence of Pseudomonas

aeruginosa.

Journal Hygiene 61:485.

Mahbub-E-Elahi, A. T. M.; Rahman, M. A.; Rahman, M. M.;

Rahman, M. M.; Rahman, M. M. and Prodhan, M. A.

M. (1996): Isolation and identification of bacteria from

different quarters of mastitis affected dairy cows in

Bangladesh.

Bangladesh Veterinary Journal 30 (1/2): 63-65. Cited from

Veterinary Bulletin 68 (4): 329 "Abstract 2213”.



REFERANCES

116

Makovec, J. A .and Ruegg, P. L. (2003): Results of milk samples

submitted for microbiological examination in Wisconsin from

1994 to 2001.

Journal of dairy science, 86 (11): 3466-3472.

Malinowski, E.; Klossowska, A.; Kaczmarowski, M.; Kotowski, K.;

Nadolny, M. and Kuzama, K. (2003): Health status of mammary

glands and etiological agents of mastitis in herds with a high

somatic cell count.

Medycyna Weterynaryjna, 59 (2): 128-132.

Malouin, F.; Brouillette, E.; Martinez, A.; Boyll, B. J.; Toth, J. L.;

Gage, J. L. and Allen, N. E. (2005): Identification of

antimicrobial compounds active against intracellular

Staphylococcus aureus.

FEMS Immunology and Medical Microbiology 45 (2, 1): 245-252.

Matsunaga, T.; Kamata, S. I.; Kakiichi, N. and Uchida, K. (1993):

Characteristics of Staphylococcus aureus isolated from peracute,

acute and chronic bovine mastitis.

Journal of Veterinary Medical Science, 55 (2): 297-300.

Melchior, M. B.; Vaarkamp, H. and Fink-Gremmels, H. (2006):

Biofilms: A role in recurrent mastitis infections?

The Veterinary Journal 171 (3): 398-407.



REFERANCES

117

Merck (2006): The Merck Veterinary Manual Cited from the site:

http://www.merckvetmanual.com/mvm/index.jsp

Service of Merck & Co., Inc.,

Miltenburg, J. D.; de Lange, D.; Crauwels, A. P. P.; Bongers, J. H.;

Tielen, M. J. M.; Schukken, Y. H. and Elbers, A. R. W (1996):

Incidence of clinical mastitis in a random sample of dairy herds in

the southern Netherlands.

Veterinary Record 139 (9): 204-207.

Murdough, P. A.; Deitz, K. E. and Pankey, J. W. (1996): Effects of

freezing on the viability of nine pathogens from quarters with

subclinical mastitis.

Journal of Dairy Science, 79 (2): 334 – 336.

Myllys, V.; Ridell, J.; Bjorkroth, J.; Biese, I. and Pyorala, S. (1997):

Persistence in bovine mastitis of Staphylococcus aureus clones as

assessed by random amplified polymorphic DNA analysis,

ribotyping and biotyping.

Vet. Microbiol, 57 (2-3): (245-51).

Nemeth, J.; Muckle, C. A. and Gyles, C. L. (1994): In vitro comparison

of bovine mastitis and fecal Escherichia coli isolates.



http://www.merckvetmanual.com/mvm/index.jsp


REFERANCES

118

Odongo, M. O. and Ambanim A. I. A. (1989): Microorganisms

isolated from bovine milk samples submitted to the

veterinary diagnostic laboratory.

Bulletin of animal health and production in Africa 37

(2): 195-196.

Orm, S.; Olesen, L. and Madsen, K.K. (1989): Antibiotic

resistance of pathogenic bacterial strains in the udder.

Dansk Veterinaertidsskrift 72 (12): 668-673. Cited from

Owens, W.; Ray, C.; Watts, J. and Yancey, R. (1997):

Comparison of success of antibiotic therapy during

lactation and results of antimicrobial susceptibility tests for

bovine mastitis.

J. Dairy Sci. 80 (2):313-317.

Oxoid manual, (1990): the oxoid manual.

6th Edition. Unipath ltd., united kingdom.

Oz, H. H.; Fransworth, r. j. and Larson, V. L. (1985):

Environmental Mastitis.

Vet. Bull. 55 (11): 829-841.



REFERANCES

119

Pitkala, A.; Haveri, M.; Pyorala, S.; Myllys, V. and Honkanen-

Buzalski, T. (2001): Bovine mastitis in Finland 2001 prevalence,

distribution of bacteria, and antimicrobial resistance.

J Dairy Sci. 87 (8): 2433-2441

Quinn, P. J.; Markey, B. K.; Carter, M. E.; Donnelly, W. J. C.;

Leonard, F. C. and Maguire, D. (2002): Veterinary

Microbiology and Microbial Diseases.

Blackwell Scientific Publication, Oxford, London.

Rachid, S.; Ohlsen, K. ; Witte, W. ; Hacker, J. ; and Ziebuhr, W.

(2000) : Effect of Subinhibitory Antibiotic Concentrations on

Polysaccharide Intercellular Adhesion Expression in Biofilm-

Forming Staphylococcus epidermidis

Antimicrobial Agents and Chemotherapy 44 (12): 3357-3363.

Raimundo, O.; Deighton, M.; Capstick, J.; and Gerraty, N. (1999):

Molecular typing of Staphylococcus aureus of bovine origin by

polymorphisms of the coagulase gene.

Vet. Microbiol 66 (4): 275-284.

Saikia, G. K.; Barman, N. N.; Boro, B. R. and Das, J. (1989):

Bacteriological investigation on chronic mastitis on cattle

farm.



REFERANCES

120

Indian Journal of Comparative Microbiology Immunology and

Infectious Diseases 10 (3):153-155.

Samborski, Z.; Twardona, J.; Bielas, W.; Fronczek, T. and

Kaniowska, F. N. (1992): Aureomycin and Syntarpen in

the treatment of subclinical and clinical chronic mastitis in

cows. Medycyna Weterynaryjna, 48 (2): 76-78.

Sanchez, M. ; Ford, C. and Yancey, R. J. (1988): Evaluation of

antibiotic effectiveness against Staphylococcus aureus

surviving within the bovine mammary gland macrophage.

J. Antimicrob Chemother. 21 (6): 773-786.

Sargeant, J. M.; Scott, H. M.; Leslie, K. E.; Ireland, M. J. and

Bashiri, A. (1998): Clinical mastitis in dairy cattle in

Ontario: Frequency of occurrence and bacteriological

isolates.

Canadian Veterinary Journal 39 (l): 33-38. Cited from

Veterinary Bulletin 68 (5): 533 "Abstract 3090”.

Schalm, O. W.; Carroll, E. J. and Jain, N. C. (1971): bovine

mastitis.

Lea and Fibiger, Philadelphia.

Cited in J. Dairy Research ( 1981), 48:167



REFERANCES

121

Schalm, O. W. ; Lasmanis, J. (1968): The leukocytes origin and

function in mastitis.

Amr. J. Vet. Res. Ass., 135: 1688

Schalm, O. W.; Lasmanis, J. and Carroll, E. J. (1964):

Pathogenises of coli form mastitis in cattle.

Amr. J. Vet. Res., 25: 75

Schukken, Y. H.; Smith, J. A. H.; Grommers, F. J.; Van de

Geer, D. and Brand, A. (1989): Intramammary infections

and risk factors for clinical mastitis in herds with low somatic

cell counts in bulk milk.

Vet. Rec., 125: 393-396.

Seadawy, Hala, M. A. (2004): Bacteriological and mycological

studies on subclinical mastitis in bovines

M. V. Sc. Thesis, Department of microbiology, Faculty of

Veterinary Medicine, Cairo University

Sears, P. M.; Gonzalez, R. N.; Wilson, D. J.; and Han, H. R.

(1993): Procedures for mastitis diagnosis and control.

Veterinary Clinics of North America 9 (3): 449.



REFERANCES

122

Sikorski, J. (1986): Treatment of acute bovine mastitis by a

combination of colistin and ampicillin.

Zur Therapie der akuten mastitis des Rindes mit einem

Kombinationspräparat von Colistin und Ampicillin., p.

131pp., 130 ref.

Smith, R. E. and Hagstad, H. V. (1985): Infection of the bovine

udder with coagulase-negative staphylococci.

Kieler Milchwirtschaftliche Forschungsberichte 37 (4):

611-614. Cited from http://www.ncbi.nlm.nih.gov/pubmed.

Smith, G.; Lyman, R. and Anderson, K. (2006): Efficacy of

vaccination and antimicrobial treatment to eliminate

chronic intramammary Staphylococcus aureus infections in

dairy cattle.

J. Am. Vet. Med Assoc. 228 (3): 422-425.

Sogaard, H. (1982): In-vitro antibiotic susceptibility of E. coli

isolated from acute and chronic bovine mastitis with

reference to clinical efficacy.

Nord Vet Med. 34 (7-9): 248-54.


http://www.ncbi.nlm.nih.gov/pubmed


REFERANCES

123

Strandberg, Y.; Gray, C.; Vuocolo, T.; Donaldson, L.;

Broadway, M. and Tellam, R. (2005):

Lipopolysaccharide and lipoteichoic acid induce different

innate immune responses in bovine mammary epithelial

cells.

Cytokine 31 (1): 72-86.

Talbot, B. G. and Lacasse, P. (2005): Progress in the

development of mastitis vaccines.

Livestock Production Science 98 (1-2): 101-113.

Tamilselvam, B.; Almeida, R.; Dunlap, J. R.; and Oliver, S.

P. (2006): Streptococcus uberis internalizes and persists

in bovine mammary epithelial cells

Microbial Pathogenesis 40 (6): 279-285.

Taponen, S.; Simojoki, H.; Maarit, H.; Helle D.; Larsen and

Helsinki, S. P. (2006): Clinical characteristics and

persistence of bovine mastitis caused by different species of

coagulase-negative staphylococci identified with API or

AFLP.

Veterinary Microbiology 115(1-3): 199-207.



REFERANCES

124

Thorne, H. and Nilson, P. O. (1962): Pavisande avpeudomonas

aeruginosa imiolkprove.

Nord. Vet. Med., 14: 547.

Tollersrud, T.; Kenny, K.; Caugant, D. A. and Lund, A. (2000):

Characterization of isolates of Staphylococcus aureus from

acute, chronic and subclinical mastitis in cows in Norway.

APMIS 108 (9): 565-72.

Twardona, J.; Mordak, R.; Dejneka, G.; Dobrzynaska, Z. and

Dzieciol, M. (1999): Efficacy of Cloxa-coli (Virbac)

formulation in the treatment of subclinical and chronic

mastitis in lactating dairy cows.

Zdot overycie Weterynaryjne 74 (8): 402-403.

Vasudevan, P.; Nair, M. K. M.; Annamalai, T. and

Venkitanarayanan, K. S. (2003): Phenotypic and genotypic

characterization of bovine mastitis isolates of

Staphylococcus aureus for biofilm formation


Verheijden, J. H.; Vecht, U.; Van Leeuwen, W. and Van Miert

A. S. (1984): Spiramycin and antibacterial therapy of

mastitis.



REFERANCES

125

Tijdschr Diergeneeskd 109 (13): 554-556. Cited from http://

www. ncbi. nlm.nih.gov/pubmed

Vintov, J.; Aarestrup, F. M.; Zinn, C. E. and Olsen, J. E. (2003-

a): Association between phage types and antimicrobial

resistance among bovine Staphylococcus aureus from 10

countries •

Veterinary Microbiology 95 (1-2) 133-147.

Vintov J.; Aarestrup, F. M.; Zinn, C. E. and Olsen, J. E. (2003-

b): Phage types and antimicrobial resistance among Danish

bovine Staphylococcus aureus isolates since the 1950s.


Wilson, D. J.; Herer, P. S. and Sears, P. M. (1991): N-acetyl-

beta--D-glucosaminidase, etiologic agent, and duration of

clinical signs for sequential episodes of chronic clinical

mastitis in dairy cows.

Journal of Dairy Science 74 (5): 1539-1543.

Wilson, D. J.; Gonzalez, R. N. and Das, H. H. (1997): Bovine

mastitis pathogens in New York and Pennsylvania:

Prevalence and effect on somatic cell count and milk

production.

Journal of Dairy Science 80 (10): 2592 – 2598.



REFERANCES

126

Winter, P. and Duetz, A. (1998): In vitro susceptibility of bovine

mastitis pathogens.

Veterinary bulletin 69 (2): 126.

Yousef, Ashgan M. (2005): Molecular typing of major pathogens

from bovine mastitis.

Thesis of D. V. Sc., Cairo University, Faculty of Veterinary

Medicine, Department of microbiology (2005).

Zadoks, R. ; Gillespie, B.; Barkema, H.; Sampimon, O.; Oliver

S. and Schukken Y. (2003): Clinical, epidemiological and

molecular characteristics of Streptococcus uberis infections

in dairy herds.

Epidemiol. Infect. 130 (2):335-349.

Zadoks, R. N.; Tikofsky, L. L. and Boor, K. J. (2005):

Ribotyping of Streptococcus uberis from a dairy's

environment, bovine feces and milk.

Veterinary Microbiology 109 (3-4):257-265.

Zecconi, A.; Vicenzoni, G.; Facchin, E. and Ruffo, G. (1993):

Eradication of Streptococcus agalactiae mastitis.

Cytological, therapeutic and production aspects.

Obiettivie Documenti Veterinari, 14 (3): 43- 48.



ARABIC

SUMMARY



امللخص العريب ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

1

الملخص العربـــي

التي تصـيب األبقـار من أهم األمراض االضرع واحد إلتهابيعتبر

وخاصـة الضـرع إلتهاب يؤدي النظر االقتصاديةة من وجهو. الحلوبة

يـتم كنتيجـة لـذلك وإدرار لـبن البقرة لقيمتها كحيوان فقدلمنه المزمن

لمنتجي فادحةديه مما يتسبب في خسائر اقتصاالقطيع الحيوان من استبعاد

تيار هذا الموضوع لقلة األبحاث في مصر وعالميا علـي إخوقد تم األلبان

.الضرع المزمن في االبقار إلتهابمسببات

:التاليةقد تم في هذه الدراسة دراسة النقاط و

فـي الضـرع المـزمن إلتهـاب لمرض البكتيريةتحديد المسببات - ١

إلتهـاب بمصـابه أرباعمن الحصول عليها تم البان لعينات االبقار

. مزمن ضرع

تنقيه وتصنيف العترات المعزولة طبقـا للصـفات المورفولوجيـه - ٢

.والمزرعيه والبيوكيميائيه

مـن الحـاالت المزمنـه لحساسيه للعترات المعزوله اختبارإجراء إ - ٣

.بكتيريثماني عشر مضاد ل تهالتحديد حساسي

عيناتمن % ٨٢ن مالمسببات البكتيريه عزل أظهرت الدراسه ولقد

وضـحت أوقـد .التي تم فحصهاالضرع المزمن إلتهابالمصابه ب رباعاأل

األكثـر الجرام هي البكتيريا ةصبغل ةموجبال ةن المسببات البكتيريأ نتائجال

وقـد تم عزلهـا البكتيريا التيجمالي إمن % ٧٧.٢ذ شكلت نسبه اانتشار

شكلت قدو. %)٢٢.٨( هالباقي الجرام النسبه ةصبغل ةسالبالشكلت البكتيريا



امللخص العريب ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

2

ـ ستافيلـوكوكساإلبكتيريا ةصـبغ ل ةالنسبه الغالبه من البكتيريـا الموجب

النسبه الغالبه مـن اإليشيريشيا كواليبكتيريا كانت و % ٥٤ ةبنسبالجرام

%).٢٠.٣(الجرام ةصبغلالبكتيريا السالبه

يدس مربيدياإلستافـيلوكوكس إد شكل كل من ميكروب فق وتحديدا

حيث لهعلي نسبه من الميكروبات المعزوأ اإليشيريشيا كواليميكروب و

ةبنسب سيريسباسيلس الميكروب جاءثم % ٢٠.٣ شكل كل منهم نسبه

ميكروبثم % ١٨.٧بنسبه سووريأ اإلستافيلوكوكسميكروب ثم % ١٩.٥

.%١٥.٤بنسبه ستافيلوكوكس سابروفيتيكس إلا

تيه بنسب صغيره وهي ميكـروب اآلالميكروبات كل منتم عزل ولقد

ــتريبتوكوكس ــدممالاالســ ــيل للــ ــروب %) ٢.٤( ســ وميكــ

ـ %) ٠.٨(ركانوباكتربيوجينزاأل %) ١.٦(بسيال نيمـوني ـوميكروب الكلي

%).٠.٨( السودوموناس ايروجينوزاوميكروب

جيوالزالكوآ ستافيلوكوكس السالبه إلختبارااإل اتميكروب تكان ولقد

فـي البقـره الضرع أرباعنتشار بين نسبه في اإل االعلي اتالميكروب يه

بقـار أها مـن تم عزله منه لمن العترات المعزو% ٢٢.٩ن أالواحده حيث

من % ١١.٤ تكانما نيب رباعثنين من األإبنفس الميكروب في همصاب

ميكـروب وكـان .الضرع أرباعثالثه إلتهاب بقار مصابه بأمن العترات

عترات تم المن % ٤.٧٥(نتشار التالي في اإل وه اإلستافيلوكوكس أوريوس

. )رباعضرع في جميع األ إلتهابببقار مصابه أعزلها من



امللخص العريب ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

3

ولقد تم اجراء اختبار الحساسيه للعترات المعزوله بإسـتخدام ثمـاني

تـأثيرا كثر هما األكان الجنتامايسين والنورفلوكساسين احيوي اعشر مضاد

كوكس أوريـوس و اإلستافــيلوكوكس اإلسـتافيلو اتميكروبمع كل من

ــابروفيتيكس ــتافيلوكوكس س ــدس واإلس ــيريس وإبيديرمي ــيلس س الباس

ـ ةرجفقد تم تسجيل د باالضافه لما سبقو. اإليشيريشيا كواليو مـن ةعالي

واإلسـتافيلوكوكس أوريـوس اتميكروبمع سيبروفلوكساسين للالحساسيه

كـذلك بالنسـبة والباسـيلس سـيريس واإلستافـيلوكوكس إبيديرميـدس

الكانامايسين و ،كروب اإلستافيلوكوكس سابروفيتيكسميمع لدوكسيسيكليينل

كما كـان ميكـروب الكليبسـيال نيمـوني اإليشيريشيا كواليميكروب مع

. حساسا للسيبروفلوكساسين والجنتامايسين

لكـل ةمقاومالمعزول يروجينوزا أميكروب السودوموناس ظهرأ وقد

.خدمهستالم ةويالمضادات الحي



ص العريب لخستامل ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

4

لص العربـــيستخالم

٧٩عينة من أرباع ابقار حلوبه مصابه بإلتهاب ضرع مزمن تم جمعها من ١٥٠تم فحص

أرباع االبقار المصابه كانت تظهـر عليهـا . بقرة فريزيان ضمن خمسة قطعان لألبقار الحلوبه

ة من وقد تم عزل المسببات البكتيري. عالمات التليف وتم تشخيصها كحاالت إلتهاب ضرع مزمن

وكانت نسبة المسببات البكتيرية الموجبة لصبغة الجرام هي . من العينات التي تم فحصها% ٨٢

بكتيريا وشكلت %). ٢٢.٨(البكتيريا السالبه لصبغة الجرام و إجمالي البكتيريا المعزولهمن % ٧٧.٢

وكوكس يـدس ولقد تم عزل كل من ميكروب اإلستافـيلوكوكس إبيديرم%. ٥٤نسبة اإلستافيـل

وميكـروب اإلسـتافيلوكوكس أوريـوس %) ١٩.٥(و ميكروب الباسيلس سيريس %) ٢٠.٣(

و ميكروب االسـتريبتوكوكس %) ١٥.٤(و ميكروب اإلستافيلوكوكس سابروفيتيكس %) ١٨.٧(

و وميكروب اإليشيريشيا كوالي %) ٠.٨(و ميكروب األركانوباكتر بيوجينز%) ٢.٤(المسيل للدم

و ميكـروب السـودوموناس ايروجينـوزا %) ١.٦(بسيال نيمـوني وميكروب الكلي%) ٢٠.٣(

)٠.٨.(%

ولقد كان ميكروبات ااإلستافيلوكوكس السالبه إلختبار الكوآجيوالز هـي الميكروبـات

. االعلي نسبه في اإلنتشار بين األرباع في البقره الواحده ثم ميكروب اإلستافيلوكوكس أوريوس

وقد اظهرتنتائج اختبار الحساسيه للعترات المعزوله بإستخدام ثماني عشر مضاد حيوي ان

النورفلوكساسين والجنتامايسين و السيبروفلوكساسين والدوكسيسيكليين االكثر تأثيرا مع كل من

ميكـروب ميكروب اإلستافــيلوكوكس إبيديرميـدس و ميكروب اإلستافيلوكوكس أوريوس و

. س و ميكروب اإلستافيلوكوكس سابروفيتيكس و وميكروب اإليشيريشيا كـوالي الباسيلس سيري

بينما اظهر ميكروب السودوموناس ايروجينوزا المعزول مقاومـه لكـل المضـادات الحيويـه

. المستخدمه



نبذه عن الباحث-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

-

ــ ــوزارة الدـف ــ ــ ــ ـــــ ــ ـاعــ ــ ــ

ــھ العسكریة االكادیمیھ الطبیــمعھد الصحھ والوبائیـ ـاتـ ـ

ــأك: االسم • ــ .رم محمد نبیھ السیدــ م ١٩٦٤ر عام ــاكتوب ١٨: تاریخ المیالد •ــالعباسی: محل المیالد • ــ ــالق –ھ ــ ــ ــ اھرةــــمصری :الجنسیھ • ــ ــ ھــــالعلوم الطبیھ البیط بكالوریوس: التأھیل • .هجامعھ القاھر/ریھ من كلیھ الطب البیطريــ

ــام دبلوم ــ ــ ــ ــ ــ ــ ــ ــ ــكلی /ان حیوالراض ـ . ةجامعھ القاھر /ھ الطب البیطري ـ

ــالمیكروبیولوجی ماجیستیر: الدرجة • ــ ــ ـــ ــ الطبیھ اـــة عن التھاب بكتیری اتدراس: عنوان الرسالھ • ــ ــ ــ ــالضرع المزمن في الماشی ولوجـی ــ ــ ــ ــ .ھـ

ــتحت اش محمود عصام حاتم/ االستاذ الدكتور :رافــ

ـاـاستاذ ورئیس قسم المیكروبیولوجی .امعھ القاھـرةـكلیھ الطب البیطري ج

ــفتحي ج/ دكتور واءـل ــ ابر عوضــ

مستشار رئیس ھیئھ االمداد والتموین ــوزاره الدف/ للخدمات البیطریھ ــ .ــاعـ

ـاـتامین عبد الف/ دكتور میدــع ح بدرـ

ــ ــمدیر معھد الصحھ والوقائی ــ ــ ـات ـــھ العسكری ــ ـــاالكادیمیھ الطبیـ ــ ــــ ــ ة ـ



الرحمـن الرحیمهللا بسم ا

والبح رأقل ـم م ن ش جرة ف ي االرض ول و أنمـ ـا ﴿

إن اهللا هللامانف دت كلم ت أبح ر ه م ن بع دة س بعة یم د

﴾عزيز حكيم

العظیمهللا صدق ا

)٢٧(سورة لقمان االیة



BACTERIOLOGICAL STUDIES ON CHRONIC MASTITIS IN BOVINE

Documents

Transcript of BACTERIOLOGICAL STUDIES ON CHRONIC MASTITIS IN BOVINE