Transcript Infected - PublicationsList.org

Nissim Silanikove
Department of Animal Physiology,
Agricultural Research Organization,
The Volcani Center, Israel
Interactions between bacteria type,
caseinolysis and physico-chemical
properties in caprine, ovine and bovine milk
Cork 2005
Milk quantity:
Yield of milk, fat, total proteins,
casein and curd.
Milk quality:
Internal bacterial contamination,
somatic cell count,
secreted enzymes.
Cork 2005
Bacterial infection may affect
caseinolysis and micelle properties
by three main routes:
1. directly, by secreting extracellular enzymes
different bacteria will cause different "type"
of physico-chemical damage to the milk
Cork 2005
2. activate the host innate immune system
milk from different type of bacteria with similar
SCC will result in similar damage to the milk
3. a combination of 1 and 2
Cork 2005
Hypothesis:
CASEINOLYSIS INDEX
Infected gland
~ 3,000,000
Cell depended
Healthy gland
~ 20,000 Cows
~ 800,000 goats and sheep
Bacteria and Cells depended
Cork 2005
Somatic cell count and gross composition as bases
for grading milk quality in sheep and goats
Aim: to calculate the losses of milk and cheese loss as
related to the level of subclinical udder infection in a herd.
Elucidated the major factors that influence milk yield and,
consequently, curd yield in Assaf sheep and Saanen and
Shami × Anglo-Nubian goats,
The LS Means of CMT and log SCC in uninfected and infected
udders and their different significance level (P [F]).
Bacteriological
Status
Sheep
Goats
CMT
Log SCC
CMT
Log SCC
Uninfected
0.65 b
5.33 b
0.91 b
5.51 b
Infected
2.23 a
6.38 a
1.59 a
6.12 a
P [F]
0.0001
0.0001
0.0001
0.0001
The LS Means of fat, protein and lactose in uninfected and
infected udders and their different significance level (P [F]).
Bacteriological
Status
Sheep
Goats
Fat
Protein
Lactose
Fat
Protein
Lactose
Uninfected
55.9
49.0 b
49.4 a
37.4 a
38.1
45.9 a
Infected
56.9
50.3 a
42.9 b
35.1b
38.3
44.0 b
P [F]
NS
0.0001
0.0001
0.04
NS
0.0001
Quantifying the damage caused by IMI with CNS
From data collected in the present study and those published recently
two equations could be developed to calculate milk yield loss and
total curd yield loss.
These equations combine milk loss and reduction in curd yield per litre of
milk in sheep or goats with sub clinical IMI:
Milk yield loss (%) = 100 - [C × 100 + (100-C) × IUY]/100
Total curd yield loss (%) = 100 - [C × 100 + (100-C) × (IUY-ICY × D)]/100
where:
C = % uninfected udders;
IUY = percentage to which milk production is reduced
by sub clinical udder infection;
ICY = percentage of curd lost because of sub clinical udder
infection;
D = litres of milk needed to produce 1 kg of cheese
(30 %moister)
Calculated percent milk and curd loss in sheep and goats herd due
to rate of infection with CNS according to the equations
Infection
rate
Projected
SCC
Milk loss (%)
Total curd loss (%)
Half-udder
model
Herd
Half-udder
model
Herd
Sheep
25
760,000
12
8
17
12
50
1,300,000
25
15
34
24
75
2,100,000
38
23
51
36
Goat
25
640,000
8
3
21
16
50
920,000
15
6
41
32
75
1,300,000
23
8
62
48
The model: Each goat, sheep or cow tested had at leas
one uninfected quarter (NBF) and one of
the other quarters infected with one of the
following bacteria:
Bacteria
+
-
Number
NBF
33
Streptococci
23
CNS
11
E. Coli
8
S. aureus
9
Cork 2005
Lactose concentration: sheep or goat with one
gland infected with CNS specie and the
contra-lateral being free
Sheep - 25.1%, P < 0.0001
Goat - 11.3%, P < 0.004
50
45
40
35
30
25
20
15
10
5
0
Sheep
Goat
Uninfected
Infected
Milk yield (half) of sheep or goat infected with CNS
specie in one gland and the contra-lateral being free.
Open bars – S; Hatched bars – G
Milk yield (kg/day)
2.0
1.5
1.0
0.5
0.0
Uninfected
Infected
The ratio in the reduction in milk yield between
goats and sheep in comparison to the ratio of
reduction in lactose concentration
0.6
0.5
0.4
0.3
0.2
0.1
0
Ratio of
reduction in
MY between
goats and
sheep
Ratio of
reduction in
lactose
between
goats and
sheep
Conclusion

The greater reduction in lactose
concentration in infected glands of
sheep than in goats, explains the
higher loss of milk yield in sheep
Fat concentration: sheep or goat with one gland
infected with CNS specie and the
contra-lateral being free
Sheep - 5%, NS
Goat - 0.03%, NS
70
60
50
40
Speep
Goat
30
20
10
0
Uninfected
Infected
Protein concentration: sheep or goat with one
gland infected with CNS specie
and the contra-lateral being free
Sheep - 9%, P < 0.0009
Goat + 2.3%, P <0.07
60
50
40
Sheep
Goat
30
20
10
0
Uninfected
Infected
Casein concentration: sheep or goat with one
gland infected with CNS specie
and the contra-lateral being free
Sheep - 12%, P < 0.0002
Goat + 0.003%, NS
50
45
40
35
30
25
20
15
10
5
0
Sheep
Goat
Uninfected
Infected
Whey concentration: sheep or goat with one
gland infected with CNS specie
and the contra-lateral being free
Sheep + 7.5%, P < 0.07
Goat +11.5%, P < 0.0001
14
12
10
8
Sheep
Goat
6
4
2
0
Uninfected
Infected
CONCLUSIONS


In goats the increase in total protein
concentration relates to increase in total
whey concentration
In sheep the reduction in total protein
concentration relates to a decrease in
casein concentration, which overweighs
the increase in whey concentration
Proteose-peptone concentration: sheep or goat
with one gland infected with CNS specie
and the contra-lateral being free
Sheep + 247%, P < 0.0001
Goat +151%, P < 0.0001
2.5
2
1.5
Sheep
Goat
1
0.5
0
Uninfected
Infected
Ca activity: sheep or goat with one gland
infected with CNS specie and the
contra-lateral being free
Sheep - 30.1%, P < 0.002
Goat -14.2%, P < 0.002
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Speep
Goat
Uninfected
Infected
Conclusions



In both goats and sheep, infection is
associated with increased casein degradation
The increase in casein degradation is greater
in sheep then in goats
Measurement of Ca activity is potentially a
convenient and cheap method to track casein
degradation
Plasminogen activator activity: sheep or goat
with one gland infected with CNS specie
and the contra-lateral being free
Sheep + 239%, P < 0.002
Goat +128%, P < 0.05
4500
4000
3500
3000
2500
Sheep
Goat
2000
1500
1000
500
0
Uninfected
Infected
Plasminogen activity: sheep or goat with one
gland infected with CNS specie
and the contra-lateral being free
Sheep - 32.2%, P < 0.001
Goat 0%, NS
100
90
80
70
60
50
40
30
20
10
0
Sheep
Goat
Uninfected
Infected
Conclusions

Plasminogen activator activity in goats is
unusually high and consequently all the
plasminogen in the gland is converted to
plasmin
Plasmin activity: sheep or goat with one gland
infected with CNS specie and the
contra-lateral being free
Sheep + 73.7%, P < 0.0007
Goat + 195%, P < 0.0003
60
50
40
Sheep
Goat
30
20
10
0
Uninfected
Infected
Conclusions



The basal level of PL activity is higher in
sheep than in goats, which explains the
higher basal level of proteose-peptones
PL activity in infected glands is higher in
sheep than in goats, which explain the
higher increase in proteose-peptones
In sheep, the source of increased PL activity
in the infected gland is accelerated
conversion of plasminogen to plasmin,
whereas in goats the source is external
Question: How comes that in goats accelerated
degradation of casein is not reflected in casein
concentration, whereas in sheep it does?


Answer: In goats the reduction in casein
output (30%) is essentially similar to the
reduction in milk yield, whereas in sheep
the reduction in casein output (60%) is
higher than in milk volume (53%).
Thus, both in goats and sheep part of the
increased loss in casein yield is related to
increased degradation of casein
Effect of subclinical mastitis on curd yield Yc, and
clotting time Tc,
in milk of infected vs. uninfected udder halves of
.sheep
1200
1100
Seconds
1000
Infected
Uninfected
P < 0.0001
900
800
700
600
500
400
300
P < 0.0001
Grams
30
20
10
0
Curd yield
Clotting time
Effect of subclinical mastitis on curd
yield, Yc (a) and clotting time, Tc (b), in milk of infected vs.
uninfected udder halves of goats.
Open bars – uninfected; Hatched bars – infected
350
250
a
b
P < 0.0001
300
250
200
Clotting time (s)
Curd yield (g/L)
225
200
150
100
175
50
0
150
P < 0.02
SCC and cell differentiation according
to bacteria isolates in the milk
Bacteria
Number
SCC
(x1000)
PMN
(%)
CD8+
(%)
CD14+
(%)
NBF
33
116±20
c
32±3.3
c
4.4±0.8
b
5.0±1.1
Strep.
23
3379±350
a
58±5.2
ab
8.9±1.6
ab
9.4±1.6
CNS
11
543±129
b
54±8.5
ab
17.7±3.6
a
11.3±2.9
E. coli
9
4333±443
a
63±20.1
a
9.3±7.5
ab
10.3±7.2
S. aureus
9
1148±163
b
82±1.7
a
1.3±0.1
c
5.8±0.6
Cork 2005
No significant differences were fund among the
different bacteria in fat, protein and casein
Lactose concentrations was significantly
Lower in milk from all infected quarters
compared to milk from uninfected quarters,
regardless of type of bacteria
Cork 2005
Two parameters were tested using
the Optigraph© (Alliance Instruments)
according to bacteria isolates in the
quarter milk
Clotting time (in seconds)
Curd firmness (volts) 30 min after
coagulating enzyme additions
Cork 2005
Curd firmness
(volts)
Clotting time
(sec)
1
2
3
Cork 2005
Clotting time and Curd firmness
Bacteria
NBF
Strep.
CNS
Clotting time
(sec)
650±63
Curd firmness
(V)
6.58±0.2
2490±340
1255±468
1.02±0.3
3.80±0.8
1078±193
3.28±0.7
E. coli
S. aureus
Cork 2005
Effects of mammary gland infection on
plasminogen (PLG), plasminogen
activator (PA) and plasmin (PL)
Bacteria
PLG
(unit/mL)
PA
(unit/mL)
NBF
146.2
531
10.5
b
Strep.
167.8
518
23.3
a
CNS
174.4
530
18.8
E. coli
327
542
20.0
156.7
555
17.2
a
a
a
S. aureus
Plasmin
(unit/mL)
1 unit is the amount of PLG, PA or PL that produces a
Change of 0.1 in absorbance at 405 nm in 60 min
Cork 2005
Correlation matrix
Clotting time
(sec)
Curd firmness
(V)
log SCC
0.48; P = 0.020
- 0.46; P = 0.001
Lactose
- 0.64; P = 0.002
0.61; P = 0.003
Whey
0.83; P = 0.001
- 0.72; P = 0.001
pp
0.59; P = 0.005
- 0.60; P = 0.004
Plasmin
0.65; P = 0.001
- 0.65; P = 0.001
Casein
0.01; NS
0.08; NS
Cork 2005
50
Protein, r - NS
Casein, r - NS
40
30
30
Casein (g/L)
40
20
20
10
10
0
0
0
1
2
3
4
5
6
7
8
0
9
1
2
3
4
5
6
7
8
9
Curd firmness (A30 Optigraph)
Curd firmness (A30 Optigraph)
15
Whey, r = 0.83
Whey proteins (g/L)
Total protein (g/L)
50
10
5
0
0
1
2
3
4
5
6
7
8
9
Curd firmness (A30 Optigraph)
Cork 2005
log Somatic Cell Count
10000
SCC, r = 0.46
1000
100
10
0
1
2
3
4
5
6
7
8
9
Curd firmness (A30 Optigraph)
Cork 2005
log Somatic Cell Count
1000
Strep. dysgalactiae
S. aureus
100
0
1
2
3
4
5
6
Curd firmness (A30 Optigraph)
Cork 2005
CURD FROM HELTHY GLANDS
CURD FROM INFECTED GLAND
AFTER ONE DAY OF MATURATION
HEALTHY
INFECTED
AFTER 3 MONTH OF MATURATION
HEALTHY
INFECTED
SDS PAGE Tricine
52
35
28
21
14
0%
0%
50%
50%
100%
100%
FPLC
550.00
2395 - 100%
2395 - 0% (/2)
'‫א‬
500.00
450.00
400.00
350.00
300.00
mAU
'‫ב‬
'‫ג‬
'‫ד‬
'‫ה‬
250.00
200.00
150.00
100.00
50.00
6.00
8.00
10.00
12.00
14.00
ml
16.00
18.00
20.00
22.00
OPTYGRAPH
EFFECT OF ADDING VARIOUS P-P
FRACTIONS (O.5 MG/ML) TO BACTERIAL
FREE MILK
1
CONTROL
2
3
4
5
20
25
30
35
Clotting Time (min)
40
45
Hypothesis:
Infected glands
~ 200,000
Healthy gland
~ 20,000
Bacteria depended
Cork 2005
The results suggest that different
bacteria affects caseinolysis in a
different manner directly or
indirectly, influencing the quantity
and quality of milk products
Cork 2005
The main conclusions from this
study are: 2. Casein has low value
for predicting milk quality for cheese
production 3. Whey is a convenient
4. This study shows that in order to
understand milk quality it will be
important to study the interactions
among specific bacteria the innate
immune system and caeinolysis
Cork 2005
Thank you: I hope that this lecture
will contribute to our ability to
raise healthier cows and produce
better dairy products
BOLFA 2006