Studies on microbial causes of mastitis in buffaloes Thesis Presented by Badia Saad EL-Sayed ...

Studies on microbial causes of mastitis in buffaloes Thesis Presented by

Badia Saad EL-Sayed Moshref

Under the supervision of

Prof. Dr. Mohamed Kamal Refai

Professor of Microbiology Faculty of Veterinary Medicine

Cairo University

Prof. Dr. Salah Abd-EL-Hamid Youssef Dr. Nagat Ahmed Saleh

Prof. of Pharmacology Chief Researcher Faculty of Veterinary Medicine Buffalo Diseases Research Depart Cairo University Animal Health Research Institute

2004

The aim of the present work was: 1. To estimate the prevalence of various

microbial causes of clinical and subclinical mastitis in buffaloes.

2. To detect virulence factors of the most important bacteria isolated from mastitic milk.

3. To Select an effective antimicrobial agents for achieving successful treatment using some recent fluoroquinolone antimicrobial (as enrofloxacin, norfloxacin, and flumequine ) and other commonly used antibacterials.

MATERIAL AND METHODS Animals: A total of 157 dairy buffaloes in 4 herds from different farms were examined for mastitis. According to clinical observation and California mastitis test (Schalm et al., 1971), the mastitic cases (104) were classified into clinical (47) and subclinical (57). Samples: A total of 393 individual quarter milk samples were collected from 104 lactating buffaloes, 169 quarter milk samples from clinically infected animals (47 buffaloes) and 224 samples from subclinically mastitic animals (57 buffaloes).

The diagnostic “O” sera 51 vials

(polyvalent, 8 vials and monovalent , 43 vials)

Polyvalent 1: O1, O26, O86A, O111, O119, O127A , O128. Polyvalent 2: O44, O55, O125, O126 and O146, O166. Polyvalent 3: O18, O114, O142, O151, O157, O158. Polyvalent 4: O6, O27, O78, O148, O159, O168. Polyvalent 5: O20, O25, O63, O513, O167. Polyvalent 6: O8, O15, O115, O169. Polyvalent 7: O28ac, O112AC, O124, O136, O144. Polyvalent 8: O29, O143, O152, O164

Dry spotkit (staphy tect plus) (Oxoid , DR100M): Latex slide agglutination test for the identification of staphylococci which possess clumping factor:

Components of the kit (DR 100M)

1-DR101M Dry spot staphy tect plus reagent cards. 2-Blue latex particles coated with both porcine fibrinogen and rabbit IgG together with specific polyclonal antibodies raised against capsular polysaccharides of Staphylococcus. aureus. 3-Four pouches each containing 10 cards, there are 3 test and 3 control reaction areas on each card (120 tests in total).

Antibiotic sensitivity discs (Bio-Merieux): Reagents and products of such discs containing the following antibiotics: amikacin (30µg), streptomycin(10µg), chloramphenicol (30µg), cefotaxim (30µg), colistin sulphate (25µg), norfloxacin (10µg), flumequine (30µg), gentamicin (10µg), enrofloxacin (10µg) , kanamycin (30µg), cefacetril (30 µg) and nitrofurantoin (300 µg).

Laboratory animals Infant mice: 2-4 day old

albino baby mice were used for enterotoxin assay.

White New Zealand rabbits: 2-2.5 kg body weights were used for invasiveness assay

Methods Rapid diagnostic tests: • Modified White Side test (MWT): This test depends directly or indirectly on the presence of leucocytes, which cause a precipitate on adding sodium hydroxide solution. • California mastitis test (CMT) Schalm et al. (1971) The CMT reagents consist of a detergent plus pH indicator (brown cresol purple). The reaction between the detergent and DNA of cell nuclei is a measure of the number of somatic cell in the milk Bacteriological examination of milk samples (Quinn et al. , 1994): Mycological examination of milk samples (Refai, 1988) • Identification of the isolates Studies of virulence factors of the isolates

Test

S.a

gal

acti

ae

S.d

ysa

gal

acti

ae

S.f

aeca

lis

S.u

ber

is

S. p

neu

mo

nia

e

S. p

yo

gen

es

Catalase - - - - - -

Oxidase - - - - - - Trehalose + + + + + +

Sorbitol - - + + - - Mannitol - - + + - v

Salicin (+) - + + v +

Lactose + + + + + +

Raffinose - - - - + -

Inulin - - - + + - Aesculin hydrolysis - - + + (+) -

Na.hippurate hydrolysis + - V + - - Growth in 6.5% Nacl - - + .(+) - -

Growth on MacConkcy - - + - - -

Litmus milk AC B NAC NAC AC A

Gelatin liquefaction - - D - - -

Bile solubility - - - - + -

Arginine hydrolysis + + - + + -

CAMP test + - - - - -

Biochemical reactions for the identification of Streptococcus and Enterococcus

Differentiation of members of family Enterobacteriaceae

Test

Esc

her

ich

ia

coli

Kle

bsi

ella

p

neu

mo

ni

ae

En

tero

bac

ter

ae

rog

enes

Ser

rati

a m

arce

scen

s

Catalase + + + +

Oxidase - - - -

Indole production + - - -

MR( Methyl red) + (-) - (-)

VP(Voges Proskauer ) - + 4- +

Citrate - + + +

TSl A/A A/A A/A A/A

Hzs - - - -

Mannose + + + +

Lactose + + + -

Glucose(gas) + + + +

Sucrose D + 4- +

Dulcitol D d - -

Maltose + + + +

Mannitol + + + +

Xylose + + + -

Urease test - + - (-)

EMB Metallic sheen - - -

Mucoid colonies (-) + + -

Detection of virulence factors of Staphylococcus aureus: 1. Serum soft agar technique 2. Coagulase tests 3. Detection of haemolysin titre 4. Detection of enterotoxins producing

isolates (Igarashi et al. , 1986) 5. Pathogenicity in mice (Yoshida and

Takuchi,1970) 6. Capsular polysaccharide Assay

(Tollesuran et al. , 2000). 7. SDS-PAGE of Capsular polysaccharide

Detection of virulence factors of E. coli isolates: 1. Haemagglutination test (HA Evans et al. , 1977) 2. Serum resistance 3. Survival of E. coli in serum 4. Growth of E.coli in serum 5. Detection of heat stable enterotoxin (STa) of

E.coli 6. Verocytotoxin assay ( Emery et al. , 1991 ) 7. Invasiveness assay: (Bailey and Scott, 1990) 8. Extraction and purification of LPS 9. LPS lethality (Johns et al. , 1983) 10. Susceptibility of the isolated strains to various

antibiotics

Antimicrobial agents Disk potency Diameter of inhibition

Resistant Sensitive

Enrofloxacin 5 µg <12 >16

Norfloxacin 5 µg <15 >21

Amikacin µg < 14 > 17

Cefacetrile 30 µg < 14 >23

Gentamicin 10 µg < 12 > 15

Nitrofurantoin 300 µg <14 > 17

Cefataxime 30 µg < 14 > 23

Streptomycin 10 µg < 11 > 15

Chloramphenicol 30 µg < 12 > 18

Colistin sulphate 10 µg < 8 > 11

Flumequine 30 µg <16 > 16

Kanamycin 30 µg < 13 > 18

Interpretation of zone diameter of growth inhibition

Zone diameter Interpretative standard and approximate (MIC) correlates

Determination of minimum inhibitory concentration (MIC )

Antimicrobial agents Disk

content

Zone diameter Approximate MIC correlates

µg/ ml

R S R S

Amikacin 30 < 14 > 17 > 32 < 16

Chloramphenicol 30 < 12 > 18 > 25 < 12.5

Colistin sulphate 10 < 18 > 11 > 4 < 4

Gentamicin 10 < 12 > 15 > 8 < 4

Nitrofurantoin 300 < 14 > 17 > 100 < 25

Streptomycin 10 < 11 > 15 > 32 < 32

Results

No of

animals

examined

No of

mastitic

animals

%

Types of mastitis

Clinical Subclinical

No % No %

157 104 66.24 47 29.94 57 36.30

Table (7): Prevalence of mastitis among buffaloes

Table 8: Distribution of positive quarters among mastitic buffaloes

Types of

mastitis

No of

animals

Distribution of infected quarters

One quarter

Two quarters

Three

quarters

Four

quarters No of

examined

quarters

No of

positive

quarter

s

%

No % No % % No %

Clinical 47 5 10.6 8 17.02 18 38.29 16 34.04 169 139 82.25

Subclinic

al 57 13 22.81 21 36.84 15 26.32 8 14.03 224 132 58.93

Total 104 18 17.30 29 27.88 33 31.73 24 23.07 393 271 68.96

TABLE 9: MICROORGANISMS ISOLATED FROM MASTITIC BUFFALOES

TYPES OF

MASTITIS BACTERIA YEASTS MOULDS

TOTAL

NUMBER OF

ISOLATES

CLINICAL

169 QUARTERS 147 48 17 212

SUBCLINICAL 109 52 21 182

TOTAL 256

(64.97%)

100

(25.38%)

38

(9.64%)

394

(100%)

TABLE (10): BACTERIA ISOLATED FROM MASTITIC BUFFALOES

TYPES OF ISOLATES CLINICAL SUBCLINICAL TOTAL

NO. % NO. % NO. %

E. COLI

STAPHYLOCOCCI

S. AUREUS

S. EPIDERMIDIS.

S. INTERMEDIUS

S. HYICUS

STREPTOCOCCI

S. DYSGALACTIAE

S. AGALACTIAE

60

62

40

11

8

3

25

13

12

40.82

42.18

27.21

8.16

5.44

2.04

17.01

8.84

7.48

45

41

14

13

11

3

23

13

10

41.28

37.61

12.84

11.93

9.17

2.75

21.10

11.93

10.09

105

103

54

24

19

6

48

26

22

41.02

40.23

21.09

9.38

7.42

2.34

18.75

10.16

8.59

TOTAL 147 57.42 109 42.58 256 100

TABLE 11: PREVALENCE OF SINGLE AND MIXED BACTERIA FROM MASTITIC BUFFALOES

TYPES OF ISOLATES

CLINICAL

(139)

SUBCLINICAL

(132)

TOTAL

(271)

NO. % NO. % NO. %

SINGLE INFECTION

E. COLI

S. AUREUS

S. DYSGALACTIAE

S. AGALACTIAE

S. EPIDERMIDIS.

S. INTERMEDIUS

S. HYICUS

22

17

9

6

5

3

1

15.83

12.23

6.47

4.32

3.60

2.16

0.72

19

4

11

6

4

10

2

14.39

3.03

8.33

4.55

3.03

7.58

1.52

41

21

20

12

9

13

3

15.13

7.75

7.38

4.43

3.32

4.80

1.11

TOTAL 63 45.32 56 42.42 119 43.91

MIXED INFECTION

S. AGALACTIAE + E. COLI

S. AUREUS + E. COLI

S. INTERMEDIUS + E. COLI

S. DYSGALACTIAE + E. COLI

6

8

3

2

5.76

4.32

2.16

1.45

4

0

1

1

3.03

0.0

0.76

0.76

12

6

4

3

4.43

2.21

1.48

1.11

TOTAL 19 13.67 6 4.55 25 9.23

TABLE 12: PREVALENCE OF BACTERIAL ISOLATES FROM CLINICAL MASTITIC MILK SAMPLES IN SINGLE AND MIXED INFECTION

TYPES OF

ISOLATES SINGLE MIXED TOTAL

NO % NO % NO %

E. COLI

S. AUREUS

S.DYSGALACTIA

E

S. AGALACTIAE

STAPH

EPIDERMIDIS

S. INTERMEDIUS

S. HYICUS

22

17

9

6

5

3

1

34.92

26.98

14.29

9.52

7.94

4.76

1.59

38

23

4

6

6

5

2

45.24

27.38

4.76

7.14

7.14

5.95

2.38

60

40

13

12

11

8

3

40.82

27.21

8.84

8.16

7.48

5.44

2.04

TOTAL 63 42.86 84 57.14 147 100

TABLE 13: PREVALENCE OF BACTERIAL ISOLATES FROM SINGLE AND MIXED INFECTION OF SUBCLINICAL MASTITIS IN BUFFALOES

TYPES OF

ISOLATES SINGLE MIXED TOTAL

NO % NO % NO %

E. COLI

S. AUREUS

S.EPIDERMIDIS

S.DYSGALACIAE

S. INTERMEDIUS

S. AGALACTIAE

S. HYICUS

19

11

10

6

4

4

2

33.93

19.64

17.86

10.71

7.14

7.14

3.57

26

3

3

7

7

6

1

49.06

5.66

5.66

13.21

13.21

11.32

1.89

45

14

13

13

11

10

3

41.28

12.84

11.93

11.93

10.09

9.17

2.75

TOTAL 56 51.38 53 48.62 109 100

FUNGI CLINICAL SUBCLINICAL TOTAL

NO. % NO. % NO. %

YEASTS

MOULDS

48

17

73.85

26.15

52

21

71.23

28.77

100

38

72.46

27.54

TOTAL 65 100 73 100 138 100

Table (14): Fungi isolated from mastitic buffaloes

TYPES OF

ISOLATES

CLINICAL SUBCLINICAL TOTAL

NO % NO % NO %

Yeasts

Candida albicans

Candida tropicalis

Candida parapsillosis

Candida krusei

Saccharomyces

C.rugosa

Trichosporon

Rodotorulla

12

11

4

6

5

5

3

2

25.0

22.92

8.33

12.5

10.42

10.42

6.25

4.17

15

10

9

7

4

3

2

2

28.85

19.23

17.30

13.46

7.69

5.77

3.85

3.85

27

21

13

13

9

8

5

4

27.0

21.0

13.0

13.0

9.0

8.0

5.0

4.0

Total 48 48.0 52 52.0 100 100.0

Moulds

A. fumigatus

A. flavus

A . niger

Mucor

7

4

4

2

41.18

23.53

23.53

11.76

4

5

5

7

19.05

23.81

23.81

33.33

11

9

9

9

28.95

23.68

23.68

23.68

Total 17 44.74 21 55.26 38 100

Overall total 65 47.10 73 52.9 138 100

Table (15) : Prevalence of fungi isolated from both clinical and

subclinical mastitis in buffaloes

TYPES OF ISOLATES SINGLE TOTAL

NO % NO % NO %

Yeast Candida albicans Candida tropicalis Candida krusei C.rugosa Saccharomyces Candida parapsillosis Trichosporon spp. Rhodotorulla spp.

6 2 0 0 0 2 1 0

54.55 18.18 0.00 0.00 0.00

18.18 9.09 0.00

6 9 6 5 5 2 2 2

16.22 24.32 16.22 13.51 13.51 5.41 5.41 5.41

12 11 6 5 5 4 3 2

25.0 22.9 12.5 10.42 10.42 8.33 6.25 4.17

Total 11 22.92 37 77.08 48 100

Moulds A. fumigatus A. flavus A . niger Mucor spp.

2 0 0 0

100.0 0.00 0.0 0.0

5 4 4 2

33.33 26.67 26.67 13.33

7 4 4 2

41.18 23.53 23.53 11.76

Total 2 11.76 15 88.24 17 100

Overall total 13 20.0 52 80.0 65 100

Table (16): Identification and prevalence of fungi isolated from

clinical mastitic milk samples in single and mixed infection

Types of isolates Single Mixed Total

No % No % No %

Yeasts

Candida albicans

Candida tropicalis

Candida parapsillosis

Candida krusei

Saccharomyces spp.

Candida rugosa

Trichosporon spp.

Rhodotorulla

2

6

2

0

2

1

0

2

13.33

40.0

13.33

0.0

13.33

6.67

0.0

13.33

13

4

7

7

2

2

2

0

35.14

10.81

18.92

18.92

5.41

5.41

5.41

0.0

15

10

9

7

4

3

2

2

28.85

19.23

17.31

13.46

7.69

5.77

3.85

3.85

Total 15 28.85 37 71.15 52 100

Moulds

Mucor spp.

A . niger

A. flavus

A. fumigatus

5

2

4

3

35.71

14.29

28.57

21.43

2

3

1

1

28.57

42.86

14.29

14.29

7

5

5

4

33.33

23.81

23.81

19.05

Total 14 66.67 7 33.33 21 100

Overall total 29 39.73 44 60.27 73 100

Table (17): Identification and prevalence of fungi isolated from subclinical mast

itic milk samples in single and mixed infection

Types of infection No %

Double infection

E. coli +C. albicans

S. aureus +C. tropicalis

S. epidermidis + C. krusei

S. aureus + Sacchromyces spp.

S. aureus A. fumigatus

S. aureus + A. niger

E. coli + A. niger

E. coli + C. parapsillosis

E. col+ A. fumigatus

E. coli + C. rugosa

S. epidermidis + C. rugosa

S.hyicus + C. krusei

E. coli+ C. tropicalis

S.aureus +C.rugosa

Triple infection

E. coli + C. tropicalis+ A. flavus

S. dysgalactiae + Trichosporon + Mucor spp.

S. aureus + Rhodotorulla + Saccharomyces spp.

S. intermedius + E. coli + C.albicans

4

4

4

3

3

2

2

2

2

2

2

2

1

1

4

2

2

2

2.88

2.88

2.88

2.16

2.16

1.44

1.44

1.44

1.44

1.44

1.44

1.44

0.72

0.72

2.88

1.44

1.44

1.44

Overall total 44 31.65

Table (18): Prevalence of mixed infection of bacteria and fungi isolated from clinical mastitis

Types of infection No %

Double infection

E. coli + C.krusei

E. coli +C. albicans

S. agalactiae + C. albicans

S. dysgalactiae + C. parapsillosis

S. epidermidis + C. parapsillosis

S. aureus +C. tropicalis

S. intermedius + C. albicans

E.coli + A.niger

E.coli + Saccharomyces spp.

S. intermedius + C. rugosa

S. intermedius + C. parapsillosis

E. coli + A. fumigatus

Triple infection

E. coli + Trichosporon spp. + Mucor spp.

S. hyicus + A. flavus++C. tropicalis

7

5

5

3

3

3

3

3

2

2

1

1

2

1

5.30

3.79

3.79

2.27

2.27

2.27

2.27

2.27

1.52

1.52

0.76

0.76

1.52

0.76

Overall total 41 31.06

Table 19: Prevalence of mixed infection of bacteria and fungi isolated from subclinical mastitis

Staphylococcal

species

Clinical mastitis Subclinical

mastitis

Total

Staphylococcal

isolates

No % No % No %

S. aureus 40 64.52 14 34.15 54 52.43

S. epidermidis 11 17.74 13 31.71 24 23.30

S. intermedius 8 12.9 11 26.83 19 18.45

S. hyicus 3 4.84 3 7.32 6 5.82

Total 62 60.19 41 39.81 103 100

Table (20) : Prevalence of Staphylococcal species isolated from mastitic animals.

Source of isolates Total

Growth pattern

Diffuse growth Compact growth

No. % No. %

Clinical mastitis 40 26 65 14 35.7

Subclinical

mastitis 14 5 35.7 9 64.3

Total 54 31 57.4 23 42.6

Table (21): Growth pattern of S. aureus strains isolated from clinical

and subclinical mastitic milk in S.S.A

Source of

isolates

Clumping factor Tube coagulase

Positive Negative Positive Negative

No. % No. % No. % No. %

Clinical

mastitis 14 35.7 26 65 32 80 8 20

Subclinical

mastitis 9 64.3 5 35.7 10 71.4 4 28.57

Total 23 42.59 31 57.4 42 77.77 12 22.22

Table (22): Coagulase reaction (clumping factor , tube coagulase) of

S. aureus strains isolated from clinical and subclinical mastitic

milk samples

Source

of

isolates

Haemolysin titer

1/32 1/64 1/128 1/256 1/520 1/1024 Total

positive

Clinical

mastitis

1/40 3/40 7/40 8/40 9/40 12/40 36 /40

2.5 % 7.5% 17.5% 20% 22.5% 30% 90 %

Subclinical

mastitis

1/14 2/14 5/14 4/14 1/14 0/14 10 /14

7.1% 14.2% 35.7% 28.5% 7.1% 0% 71.43

Total 2 /54 5 /54 12 /54 12/54 10/54 12/54 46 /54

5.6% 12.9% 22.2 % 22.2% 18.5% 22.2% 85.2%

Table(23): Haemolysin titer of S. aureus isolates recovered from clinical

and subclinical mastitic milk samples

Source of

isolates Total

Toxigenic

strains

Types of enterotoxin

A B C

Clinical mastitis 40 27

(67.5%)

7

(17.5%)

5

(12.5%)

15

(37.5%)

Subclinical

mastitis

14 7

(50%)

1

(7.1%)

2

(14.2%)

4

(28.57%)

Total 54 34

62.96%

8

(14.8%)

7

(12.96%)

19

(35.19%)

Table (24): Frequency percentage of toxigenic S. aureus isolates

recovered from clinical and subclinical

mastitic milk samples

Source of

isolates

Total

No

. of

ino

cula

ted

mic

e No dead

mice

Mortality

rate

Clinical

mastitis 40 240 176 73.7%

Subclinical

mastitis 14 64 44 68.3%

Total 54 304 220 72.37%

Table (25) : Pathogenicity of S. aureus isolates recovered from clinical

and subclinical mastitic milk samples in mice

Virulence

attributes

Clinical mastitis Subclinicalmastits Total

(54)

No. % No %

Diffuse growth 26 65 5 35.7

31

(57.4%)

Compact growth 14 35 9 64.28

(23)

(42.59%)

Clumping factor 14 35 9 64.28

23

(42.59%)

Tube coagulase 32 80 10 71.42

42

(77.77%)

Hemolysin 36* 90 10 71.43

46

(85.19%)

Toxin production 27 67.5 7 50

34

(62.96%)

Pathogenicity to mice 31** 77.5 7 50

38

(70.37%)

Table (26) : Collective virulence attributes of S. aureus recovered

from clinical and subclinical mastitis

STRAINS NO. COLONIAL MORPHOLOGY

ON S.S.A. C.F. COAGULASE HEMOLYSIN TOXIN PRODUCTION

MORTALITY RATE

(%)

1 DIFFUSE - - 1/256 B 83.3

2 DIFFUSE - - 1/521 C 100

3 DIFFUSE - - 1/256 C 100

4 DIFFUSE - - 1/64 - 66.6

5 DIFFUSE - - 1/1024 C 83.3

6 DIFFUSE - - 1/32 C 83.3

7 DIFFUSE - - 1/521 C 100

8 DIFFUSE - - 1/64 A 83.3

9 DIFFUSE - + 1/128 C 100

10 DIFFUSE - + 1/521 B 100

11 DIFFUSE - + 1/1024 A 100

12 DIFFUSE - + 1/521 - 83.3

13 DIFFUSE - + 1/128 C 100

14 DIFFUSE - + 1/521 A 100

15 DIFFUSE - + 1/521 C 83.3

16 DIFFUSE - + 1/1024 A 83.3

17 DIFFUSE - + 1/256 B 83.3

18 DIFFUSE - + 1/521 A 83.3

19 DIFFUSE - + 1/64 C 83.3

20 DIFFUSE - + 1/1024 - 83.3

TABLE (27) : RELATIONSHIP OF COLONIAL MORPHOLOGY IN S.S.A , CLUMPING FACTOR, TUBE COAGULASE , HEMOLYSIN , TOXIN PRODUCTION AND MORTALITY RATE IN MICE OF S.AUREUS ISOLATES RECOVERED FROM CLINICAL MASTITIC MILK

TABLE (27) CONTINUED: RELATIONSHIP OF COLONIAL MORPHOLOGY IN S.S.A , CLUMPING FACTOR, TUBE COAGULASE , HEMOLYSIN , TOXIN PRODUCTION AND MORTALITY RATE IN MICE OF S.AUREUS ISOLATES RECOVERED FROM CLINICAL MASTITIC MILK

21 DIFFUSE - + 1/1024 A 83.3

22 DIFFUSE - + 1/1024 C 100

23 DIFFUSE - + 1/128 B 83.3

24 DIFFUSE - + 1/1024 C 100

25 DIFFUSE - + 1/256 C 100

26 DIFFUSE - + 1/1024 C 100

27 COMPACT + + 1/1024 A 66.6

28 COMPACT + + 1/256 B 50

29 COMPACT + + 1/521 - 50

30 COMPACT + + 1/256 - 33.3

31 COMPACT + + 1/1024 A 33.3

32 COMPACT + + 1/128 - 16.6

33 COMPACT + + 1/256 - 16.6

34 COMPACT + + 1/521 - 16.6

35 COMPACT + + 1/128 - 33.3

36 COMPACT + + 1/256 C 83.3

37 COMPACT + + 1/1024 C 66.6

38 COMPACT + + 1/128 - 33.3

39 COMPACT + + 1/128 - 16.6

40 COMPACT + + 1/1024 C 66.6

STRAINS

NO.

COLONY

MORPHOLOGY IN

S.S.A.

C.F. COAGULASE HEMOLYSIN TOXIN

PRODUCTION

MORTALITY

RATE

(%)

1 DIFFUSE - - 1/521 B 66.6

2 DIFFUSE - - 1/256 C 100

3 DIFFUSE - - 1/128 A 66.6

4 DIFFUSE - - 1/256 C 83.3

5 DIFFUSE - + 1/128 C 83.3

6 Compact + + 1/256 - 16.6

7 Compact + + 1/64 - 33.2

8 Compact + + 1/128 C 82.3

9 Compact + + 1/64 - 16.6

10 Compact + + 1/128 - 16.6

11 Compact + + 1/256 B 66.6

12 Compact + + 1/128 - 33.3

13 Compact + + 1/23 - 0

14 Compact + + 1/256 C 66.6

TABLE (28) : RELATIONSHIP OF COLONIAL MORPHOLOGY IN S.S.A , CLUMPING FACTOR, TUBE COAGULASE , HEMOLYSIN , TOXIN PRODUCTION AND MORTALITY

RATE IN MICE OF S.AUREUS ISOLATES FROM SUBCLINICAL MASTITIC MILK

Types

of mastitis

Mortality per day

Total

mortality 1-3

days

3-6

days

6-9

days

9-12

days

12-

14 days

Clinical

mastitis 0 0 0 0 2 20%

Subclin

ical mastitis 0 0 0 1 0 10%

Table (29): Lethality of capsular polysaccharides of S.aureus in mice

Lane

Marker

Capsular

polysaccharides (1)

Capsular

polysaccharides (2)

M.W

(KDa) Amount

M.W.

(KDa) Amount

M.W.

(KDa) Amount

1

2

3

4

5

6

97

66

54

45

33

11

14.9

25

17.4

16.8

14.8

11.1

75.576

66.472

59.784

54.306

50.872

25.4

9.26

23

19.7

22.6

85.926

75.039

66.948

59.279

54.306

7.94

4.84

32.7

29

25.5

Table ( 30 ) : SDS PAGE pattern of C.P. extracted from S. aureus isolates

recovered from clinical and subclinical mastitic milk

E. coli

serogroups

Total

No. of

isolates

E. coli isolates recovered from

Clinical mastitic milk Subclinical mastitic

milk

No % No %

O114 13 7 11.67 6 13.33

O125 11 7 11.67 4 8.89

O44 10 5 8.33 5 11.11

O128 8 2 3.33 6 13.33

O 126 6 3 5.00 3 6.67

O 86 6 6 10.00 0 0.0

Untypable 51 30 50.00 21 46.67

Total 105 57.14 100 45 42.86

Table (31) : Incidence and distribution of pathogenic E. coli serovars

obtained from diseased cases

Table 32: Haemagglutination pattern of E.coli serogroups recovered from examined mastitic milk samples of buffaloes

RBCs species

Type of HA

Clinical mastitis Subclinical mastitis

Serogroups isolated Total (30) Serogroups isolated Total (24) O114

(7) O125

(7) O86 (6)

O44 (5)

O126 (3)

O128 (2)

No. % O114

(6) O125

(4) O44 (5)

O126 (3)

O128 (6)

No. %

Bovine

MSHA 4 6 6 5 1 1 23 76.7 4 4 3 1 3 15 62.5

MRHA

2 0 0 0 2 1 5 16.7 2 0 2 2 3 9 37.5

HA+ 6 6 6 5 3 2 28 93.3 6 4 5 3 6 24 100

Sheep

MSHA 0 0 0 1 0 0 1 3.3 0 0 0 0 0 0 0

MRHA

2 0 0 0 0 0 2 6.7 0 0 0 0 0 0 0

HA+ 2 0 0 1 0 0 3 10 0 0 0 0 0 0 0

Guinea pigs

MSHA 6 7 6 4 1 1 25 83.3 5 3 2 2 6 18 75

MRHA

1 0 0 0 0 1 2 6.7 0 0 3 0 0 3 12.5

HA+ 7 7 6 4 1 2 27 90 5 3 5 2 6 21 87.5

Chickens

MSHA 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

MRHA

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

HA+ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Table 33: Serum resistance of E.coli serogroups recovered from mastitic milk samples of buffaloes

E. c

oli

ser

og

rou

ps

Clinical mastitis (30) Subclinical mastitis (24)

No

. of

iso

late

s

Positive serum resistance

No

. of

sero

gro

up

s

Positive serum resistance

Survival in

serum

Growth in

serum Survival in

serum Growth in serum

N

No. %

N

No. %

O114 7 7 23.3 7 23.3 6 4 16.6 4 16.6

O125 7 6 20 5 16.7 4 4 16.6 4 16.6

O86 6 6 20 6 20 0 0 0 0 0

O44 5 4 13.3 4 13.3 5 5 20.8 4 16.6

O126 3 3 10 1 3.3 3 3 12.5 3 12.5

O128 2 1 3.3 1 3.3 6 3 12.5 2 8.3

Total 30 2

7

89.9

6 24 80 24 19 79.1 17 70.8

E.c

oli

ser

og

rou

ps


Total

No

. of

sero

gro

up

s

Enterotoxin +ve

No

. of

sero

gro

up

s Enterotoxin

+ve

No % No % No. %

O114 7 0 0 6 1 4.

17 1

1.8

5

O125 7 0 0 4 0 0 0 0

O86 6 1 3.33 0 0 0 1 1.8

5

O44 5 0 0 5 0 0 0 0

O126 3 0 0 3 0 0 0 0

O128 2 0 0 6 0 0 0 0

Total 30 1 3.33 24 1 4.

17 2 3.7

Table (34): Enterotoxigenic activity of E.coli serogroups isolated from examined mastitic milk samples of buffaloes using infant mouse assay

E.coli

serogroups


Total

(54)

No

. of

iso

late

s

Cytotoxic

activity

No

. of

iso

late

s

Cytotoxic

activity

No.

of +ve %

+ve

No. %

No.

tested

+ve

No. %

O114 7 0 0 6 0 0 13 0 0

O125 7 0 0 4 0 0 11 0 0

O86 6 1 3.33 0 0 0 6 1 1.85

O44 5 0 0 5 0 0 10 0 0

O126 3 1 3.33 3 1 4.1 6 2 3.7

O128 2 0 0 6 0 0 8 0 0

Total 3 0

0 2 66.6 24 1 4.1 54 3 5.56

Table (35) : Detection of cytotoxic activity of E.coli serogroups isolated from examined mastitic milk samples of buffaloes using vero-cells

E.c

oli

sero

gro

up

s


Total N

o. o

f

iso

late

s Positive

No

. of

iso

late

s Positive

No. % No. % No. %

O114 7 0 0 6 0 0 0 0

O125 7 0 0 4 0 0 0 0

O86 6 1 3.33 0 0 0 1 1.85

O44 5 0 0 5 0 0 0 0

O126 3 0 0 3 0 0 0 0

O128 2 0 0 6 0 0 0 0

Total 30 1 3.33 24 0 0 1 1.85

Table (36) : Invasive capacity of E.coli serogroups using Sereny test

Source of

isolates

Virulence

Attributes


No.

(30)

%

No.

(24)

%

HA activity using

Bovine RBCs

Sheep RBCs

Guinea pigs RBCs

Chicken RBCs

28

3

30

0

93.3

10

100

0

24

0

24

0

100

0

100

0

MRHA using

Bovine RBCs

Sheep RBCs

Guinea pigs RBCs

Chicken RBCs

23

1

27

0

76.7

3.3

90

0

15

0

18

0

62.5

0

75

0

Serum resistance

Survival in serum 27 89.9 18 75

Growth in serum 24 80 17 70.8

Heat stable enterotoxin 1 3.3 1 4.16

Verocytotoxin 2 6.66 1 4.16

Invasion using Sereny test 1 3.33 0 0

Table (37) : Virulence attributes of E.coli isolates recovered from clinical and subclinical mastitic milk samples

Source of isolates

virulence attributes

Clinical mastitis (30) Subclinical mastitis (24) Serogroups isolated

Total Serogroups isolated

Total O114 (7)

O125 (7)

O86 (6)

O44 (5)

O126 (3)

O128 (2)

O114 (6)

O125 (4)

O44 (5)

O126 (3)

O128 (6)

HA test using Bovine bovineHA* MSHA MRHA

7(100) 6(85.1) 6(100) 5(100) 3(100) 2(100) 28(93.3) 6(100) 4(100) 5(100) 3(100) 6(100) 24 (100) 4(57.1) 6(85.1) 6(100) 5(100) 1(33.3) 1(50) 23(76.7) 4(16.67) 4(100) 3(60) 1(33.3) 3(50) 15(62.5)

2(28. 0 0 0 2(66.6) 1(50) 5(16.7) 2(33.3) 0 2(40) 2(66.7) 3(50) 9(37.5)

Sheep HA+ MSHA MRHA

2(28.6) 0 0 1(20) 0 0 3(10) 0 0 0 0 0 0

0 0 0 1(20) 0 0 1(3.33) 0 0 0 0 0 0

2(28.6) 0 0 0 0 0 2(6.6) 0 0 0 0 0 0

Guinea pigs HA+ MSHA MRHA

7(100) 7(100) 6(100) 4(80) 1(33.3) 2(100) 27(90) 5(83.3) 3(75) 5(100) 2(66.7) 6(100) 21(87.5) 6(85.7) 7(100) 6(100) 4(80) 1(33.3) 1(50) 25(83.3) 5(83.3) 3(75) 2(40) 2(66.6) 6(100) 18(75) 1(14.3) 0 0 0 0 1(50) 2(6.6) 0 0 3(60) 0 0 3(12.5)

Chickens HA+ MSHA MRHA

0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0

Serum resistance Survival in serum

7(100) 6(85.7) 6(100) 4(80) 3(100) 1(50) 27(89.9) 4(66.6) 4(100) 5(100) 3(100) 3(50) 19(29.1)

Growth in serum 7(100) 5(71.4) 6(100) 4(80) 1(33.3) 1(50) 24(80) 4(66.6) 4(100) 4(80) 3(100) 2(33.3) 17(70.8) Heat stable enterotoxin production

0 0

1(16.7) 0 0 0 1(33.3) 1(16.6) 0 0 0 0 1(4.16)

Verocytotoxin 0 0 1(16.7) 0 1(33.3) 0 2(6.6) 0 0 0 0 1(16.6) 1(4.16) Invasion using Sereny test

0 0 1(16.7) 0 0 0 1(3.33) 0 0 0 0 0 0

Table 38: Main virulence attributes of E.coli serogroups recovered from clinical and subclinical mastitic milk samples of buffaloes

Lan

es b

and

s

M a r k e r s

O 1 1 4 O 1 2 5 O 4 4 O 1 2 8 O 1 2 6 O 8 6

MW

(K

.Da)

Am

ou

nt

MW

(K

.Da)

Am

ou

nt

MW

(K

.Da)

Am

ou

nt

MW

(K

.Da)

Am

ou

nt

MW

(K

.Da)

Am

ou

nt

MW

(K

.Da)

Am

ou

nt

MW

(K

.Da)

Am

ou

nt

1

2

3

4

5

6

97

66

54

45

33

11

16

20.8

15.7

19.3

14.4

13.9

12.9

9.9

58.5

41.5

13.2

11

9.15

12.9

37.1

5.5

14.0

11

80.5

19.5

11.692

9.93

19.1

86.9

11

9.34

46.3

53.7

15.545

12.944

10.778

2.32

45.7

52

Table 39: SDS PAGE pattern of LPS extracted from E. coli serogroups isolated from clinical and subclinical mastitic milk samples

Antibiotics

O groups

Ami Enroflo Chloram Coli Genta Nitrofur Strep

mic sy mic sy mic sy mic

sy mic sym mic sy mic sy

O114 25 I 0.3 S 12.5 S 25 R 3.1 S 3.1 S 100 R

O114 1.5 S 0.3 S 12.5 S 0.7 S 25 R 50 I 12.5 S

O114 0.7 S 0.1 S 50 R 1.5 S 3.1 S 100 R 25 I

O114 0.3 S 0.3 S 25 R 25 R 3.1 S 1.5 S 50 R

O114 0.3 S 0.3 S 25 R 1.5 S 25 R 100 R 12.5 S

O114 1.5 S 0.3 S 12.5 S 1.5 S 1.5 S 50 I 12.5 S

O114 0.3 S 0.3 S 12.5 S 0.7 S 3.1 S 6.25 S 25 I

O114 25 I 0.1 S 12.5 S 1.5 S 3.1 S 100 R 6.25 S

Table 40: Minimum inhibitory concentration (µg/ ml) of different antimicrobial agents on E. coli serogroups ( typable ) isolated from mastitic milk samples of buffaloes (a)

S : Sensitive R : Resistant I: Intermediate Sy : Symbol MIC : Minimum inhibitory concentration

Antibiotics

O groups

Ami Enroflo Chloram Coli Genta Nitrofu Strep

mic sy mic sy mic sy mic sy mic sym mic sy mic sy

O125 0.3 S 0.3 S 100 R 12.5 R 25 R 50 I 50 R

O125 0.7 S 0.1 S 6.25 S 3.1 S 3.1 S 1.5 S 12.5 S

O125 25 I 0.1 S 50 R 3.1 S 3.1 S 3.1 S 100 R

O125 6.25 S 0.3 S 6.25 S 3.1 S 0.7 S 100 R 100 R

O125 25 I 0.3 S 50 R 12.5 R 0.7 S 50 I 12.5 S

O125 3.1 S 0.3 S 6.25 S 3.1 S 3.1 S 100 R 50 R


Table 40 continued : Minimum inhibitory concentration (µg/ ml) of different antimicrobial agents on E. coli serogroups ( typable ) isolated from mastitic milk samples of buffaloes (a)

Antibiotics

O groups



O44 25 I 0.3 S 6.25 S 3.1 S 6.25 I 3.1 S 12.5 S

O44 50 R 0.3 S 6.25 S 25 R 50 R 50 I 25 S

O44 1.5 S 0.3 S 50 R 0.7 S 0.3 S 3.1 S 100 R

O44 3.1 S 0.1 S 3.1 S 3.1 S 1.5 S 100 R 25 S

O44 3.1 S 0.3 S 100 R 25 R 6.25 I 100 R 25 S



Antibiotics

O groups



O128 25 I 0.1 S 12.5 S 3.1 S 6.25 I 3.1 S 50 R

O128 3.1 S 0.3 S 6.25 S 25 R 3.1 S 100 R 12.5 S

O128 0.3 S 0.3 S 12.5 S 3.1 S 25 R 3.1 S 100 R

O126 3.1 S 0.3 S 50 R 12.5 R 1.5 S 100 R 50 R

O126 3.1 S 0.3 S 6.25 S 6.25 R 3.1 S 100 R 100 R

O126 3.1 S 0.3 S 6.25 S 12.5 R 3.1 S 100 R 100 R

O126 1.5 S 0.3 S 50 R 12.5 R 1.5 S 100 R 12.5 S

O126 25 I 0.1 S 6.25 S 6.25 R 0.3 S 100 R 50 R

O126 3.1 S 0.3 S 50 R 12.5 R 3.1 S 100 R 50 R

O86 3.1 S 0.1 S 50 R 6.25 R 6.25 I 1.5 S 100 R

O86 1.5 S 0.3 S 100 R 6.25 R 25 R 100 R 50 R

O86 3.1 S 0.3 S 12.5 S 3.1 S 0.3 S 50 I 50 R

O86 3.1 S 0.3 S 50 R 3.1 S 0.3 S 1.5 S 12.5 S

O86 1.5 S 0.1 S 50 R 12.5 R 6.25 I 100 R 100 R

O86 3.1 S 0.3 S 100 R 6.25 R 25 R 1.5 S 12.5 S



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Studies on microbial causes of mastitis in buffaloes Thesis Presented by Badia Saad EL-Sayed ...

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