Biological Control of Bovine Mastitis in Cows Using
Bacteriophages
Report on the Animal Trial
Executive Summary
The primary aim of this study was to identify lytic bacteriophages that are specific to mastitis
causing S. aureus and to test these phages in an in vivo setting against mastitis infections in
dairy cows.
Three phages, SaBp1, SaBp2 and SaBp3, obtained from Dr. I. H. Basdew, were re-isolated and
screened against four antibiotic- resistant S. aureus, all three phages showed to still have the
potential and viable the four antibiotic-resistant S. aureus. The three potential phages were mixed
into a cocktail, to make a concentration of 1.2 x 10 8 PFU/ml, and these concentration was applied
to cows as treatment in the Southdown dairy farm. Six cows with mastitis were assigned for this
experiment, three cows treated with phage cocktail in 40% glycerol, and three cows untreated
cows with 40% glycerol. The treatment was applied by spraying in the teat of each cow during
morning milking daily. Milk from each cow was collected daily before treatment, milk samples
were sent to the laboratory for further analysis. The experiment was carried for 3 weeks.
The treated cows showed to have higher Somatic cell count (SCC) compared to cows treated
with 40% glycerol (Untreated). The treated cows with phages showed to have lower CFU/ml,
which was declining during the period of 3 weeks (21 days). The untreated cows showed to have
the higher CFU/ml compared to treated cows, this results indicate the presence of phages which
were able inhibit and kill the targeted bacteria. The untreated cows showed to have lower PFU/ml
compared to the treated cows, this results indicate that the phages were able to multiply and
disperse to all mastitis infected areas with the cows and to the host bacterium to produce more
phages.
To test the presence of S. aureus in the collected milk samples, a total of 92 samples milk
samples analysed and 61 were positive for S. aureus. A total of 122 potential isolates (2 per
samples) were subcultured and further analysed, however, only 35 isolates satisfied all the
identification criteria and were used for spot-testing and for subsequent analysis. Phage cocktail
(1.2 x 108) was spot-plated on TSB plates containing one of the 35 positive identified isolates of
S. aureus, this was done to check if the cocktail phages are able to inhibit the new isolated S.
aureus. The phages were able to form zones of clearing in the spot-test plates, thus indicate the
presence of plaques and that the phages are viable against the new S. aureus isolates, isolated
from the experimental cows.
In conclusion, phages were able to reduce or control S. aureus from Southdown dairy farm cows,
but there are many factors that still need to be considered before making sure that phages can
completely control S. aureus in herds.
Materials and Methods
Milk analysis. Milk samples were aseptically collected from individual quarters of the study cows.
Samples were transported to the laboratory within 2 h after collection and stored in -80° C until
analysis. Each quarter sample (10 µl) were plated onto TSA containing 5% sheep blood and
incubated for overnight at 37°C. The plates were then examined for the presence of bacteria
Staphylococcus aureus. Preliminary determinations of bacterial identity were made by colony
morphology and hemolysis. The number and type of colonies found per 10 µl aliquot were
counted and used to calculate the CFU/ml.
Pre-trial. A preliminary trial was conducted (October – November 2015), with mastitis, lactating
cows to assess the effects of phage infusion on milk quality. The animals were milked twice daily
and housed in a tie-stall arrangement at the Southdown Dairy farm. Aseptic quarter milk samples
were collected from candidate cows 3 days prior to treatment and will be examined for SCC and
bacterial content, as described above. Treatments were administered by spraying at the cow’s
normal milking time by the farm workers. At milking time, a milk samples were aseptically
collected from each quarter to be treated. The cows were milked out normally and the teat end
was disinfected with 70% Ethanol. The treatment was then aseptically sprayed to the teat canal
using spray bottle. Following spraying, the mammary gland was massaged upwards briefly to
distribute the treatment into the gland. Individual quarters of six mastitis, lactating cows will be
randomly assigned to one of two treatments (Three per treatments): purified phage in 40%
glycerol or 40% glycerol. The purified phage concentration was 1.2. X 108 PFU/ml. Treatments
were randomized among cows. Each teats received a single 10-ml spray in the manner
described above. Milk samples were collected aseptically immediately before treatment, at the
following five days consecutive, and at 14 days post-spraying. Milk samples were then analyzed
for SCC and bacteriology. All milk samples were also assayed for the presence and the amount
of bacteriophage by spotting and plating the samples on a lawn of S. aureus, as described
above.
Results
Prevalence of S. aureus in tested milk samples
A total of 92 milk samples were analysed and 61 were positive for S. aureus (Table 1, 2, 3). A
total of 122 potential isolates (2 per sample) were subcultured and further analysed. However,
only 35 isolates satisfied all the identification criteria and were used for subsequent analysis.
These constituted a total of 35 S. aureus isolates. S. aureus was obtained from each of the 61
samples taken in the farm, giving a prevalence of S. aureus of 67% for the 91 samples. The
results demonstrated the presence of S. aureus in 61 milk samples, regardless of the farm
setting. Table 4 and 5 show the average colony forming units and the average plaque forming
units found in each isolate from each experimental animal.
Table 1.
Cow ID No.
Streaking
Results
9133
8029
11287
10255
Growth
Growth
No Growth
Growth
10255
11129
8029
9133
11287
13537
Growth
Growth
No Growth
Growth
No Growth
No Growth
10255
13537
11129
11287
9133
8029
Growth
No Growth
No Growth
No Growth
Growth
Growth
11287
11129
13537
No Growth
No Growth
No Growth
Somatic Cell
Count x 103
Spot-Test
Results (Phage
assays)
23 October 2015
855
Clear Zones
685
No Clear Zones
448
No Clear Zones
439
Clear Zones
26 October 2015
247
Clear Zones
201
Clear Zones
222
No Clear Zones
964
Clear Zones
315
Clear Zones
28
No Clear Zones
27 October 2015
423
Clear Zones
Clinical
No Clear Zones
Clinical
No Clear Zones
Clinical
No Clear Zones
1675
Clear Zones
1296
Clear Zones
28 October 2015
Clinical
No Clear Zones
228
No Clear Zones
6
No Clear Zones
Presence of
Staphylococcus
aureus
+
+
+
+
+
+
+
+
+
-
8029
9133
10255
Growth
Growth
Growth
9133
10255
11287
13537
8029
11129
Growth
Growth
No Growth
No Growth
Growth
No Growth
13537
10255
11129
9133
11287
8029
No Growth
Growth
No Growth
Growth
No Growth
Growth
406
Clear Zones
661
Clear Zones
1109
Clear Zones
29 October 2015
1500
Clear Zones
588
Clear Zones
641
No Clear Zones
22
No Clear Zones
2235
Clear Zones
203
No Clear Zones
30 October 2015
32
No Clear Zones
4085
Clear Zones
1284
No Clear Zones
5374
Clear Zones
494
No Clear Zones
9999
Clear Zones
+
+
+
+
+
+
+
+
Table 2.
Cow ID No.
Streaking
11129
8029
11287
10255
9133
13537
Growth
Growth
Growth
Growth
Growth
No Growth
11129
8029
11287
10255
9133
13537
Growth
Growth
Growth
Growth
Growth
No Growth
11129
8029
11287
10255
Growth
Growth
Growth
Growth
Somatic Cell
Count (x103)
Presence of
Staphylococcus
aureus
02 November 2015
3672
+
7056
+
1428
1413
+
2219
+
44
03 November 2015
2902
+
3081
+
66
3552
+
5338
+
71
04 November 2015
1487
+
1464
+
786
9999
+
Spot-Test
Results (Phage
assays)
Clear Zones
Clear Zones
No Clear Zones
Clear Zones
Clear Zones
No Zones
Clear Zones
Clear Zones
Clear Zones
Clear Zones
Clear Zones
No Zones
Clear Zones
Clear Zones
No Clear Zones
Clear Zones
9133
13537
Growth
No Growth
11129
8029
11287
10255
9133
13537
Growth
Growth
Growth
Growth
Growth
No Growth
11129
8029
11287
10255
9133
13537
Growth
Growth
Growth
Growth
Growth
No Growth
1187
54
05 November 2015
4301
2446
523
1971
5220
37
06 November 2015
5830
9999
1333
7289
4251
102
+
-
Clear Zones
No Zones
+
+
+
+
+
-
Clear Zones
Clear Zones
Clear Zones
Clear Zones
Clear Zones
No Clear Zones
+
+
+
+
-
Clear Zones
Clear Zones
Clear Zones
Clear Zones
Clear Zones
No Clear Zones
Table 3.
Cow ID No.
Streaking
11129
8029
11287
10255
9133
13537
Growth
Growth
Growth
Growth
Growth
No Growth
11129
8029
11287
10255
9133
13537
Growth
Growth
Growth
Growth
Growth
No Growth
11129
8029
11287
10255
9133
Growth
Growth
Growth
Growth
Growth
Somatic Cell
Count (x103)
Presence of
Staphylococcus
aureus
09 November 2015
217
+
2661
+
566
+
76
+
3811
+
56
10 November 2015
1443
+
1345
+
1324
+
1892
+
823
+
33
11 November 2015
2437
+
5113
+
726
9694
+
2304
+
Spot-Test
Results (Phage
assays)
Clear Zones
Clear Zones
Clear Zones
Clear Zones
Clear Zones
No Zones
Clear Zones
Clear Zones
Clear Zones
Clear Zones
Clear Zones
No Zones
Clear Zones
Clear Zones
No Clear Zones
Clear Zones
Clear Zones
13537
No Growth
11129
8029
11287
10255
9133
13537
Growth
Growth
Growth
Growth
Growth
No Growth
11129
8029
11287
10255
9133
13537
Growth
Growth
Growth
Growth
Growth
No Growth
101
12 November 2015
1508
1145
1367
7968
1473
89
13 November 2015
6007
2568
763
6759
1822
161
-
No Zones
+
+
+
+
-
Clear Zones
Clear Zones
Clear Zones
Clear Zones
Clear Zones
No Clear Zones
+
+
+
+
+
-
Clear Zones
Clear Zones
Clear Zones
Clear Zones
Clear Zones
No Clear Zones
Table 4.
Cow ID No.
9133
8029
11287
10255
10255
11129
8029
9133
11287
13537
10255
13537
11129
11287
9133
8029
11287
11129
13537
Colony forming units/ml
23 October 2015
275
125
43
>300
26 October 2015
296
21
298
267
53
0
27 October 2015
292
1
19
54
269
298
28 October 2015
35
15
3
Plaque forming units/ml
55
39
2
65
69
0
35
88
3
1
71
5
3
41
81
21
43
9
1
8029
9133
10255
9133
10255
11287
13537
8029
11129
13537
10255
11129
9133
11287
8029
>300
255
286
29 October 2015
264
273
21
2
>300
15
30 October 2015
3
244
53
182
18
285
12
75
82
Colony forming units/ml
02 November 2015
289
288
145
266
217
2
03 November 2015
222
245
120
251
214
2
04 November 2015
204
188
99
201
179
0
05 November 2015
177
168
Plaque forming units/ml
77
87
15
0
16
8
2
89
0
73
12
6
Table 5.
Cow ID No.
11129
8029
11287
10255
9133
13537
11129
8029
11287
10255
9133
13537
11129
8029
11287
10255
9133
13537
11129
8029
21
33
51
92
69
0
25
15
43
98
76
2
11
14
48
101
66
1
16
9
11287
10255
9133
13537
11129
8029
11287
10255
9133
13537
90
154
155
0
06 November 2015
143
169
91
135
155
3
52
73
81
0
12
19
48
106
99
1
Figure 1. Graph showing treated (phages applied) and untreated cows (no phages) with the differences in their Somatic cell count
(SCC). The treated cow have high somatic cell count compared to untreated and for the green line of untreated showed to have
very low SCC compared to all cows, this is because this had no infection of mastitis.
Figure 2. Graph show the CFU/ml (x106) of treated (+ phage) and untreated cows (- phage) for 21 days. The treated cows as seen
on the graph has the lower CFU/ml compared to untreated cows, and as the time goes on the CFU/ml of the treated cows is
declining, which indicates the presence of phages in the treated cows.
Figure 3. This graph shows the PFU/ ml (x107) of the treated (+ phage) and untreated cows (- phage) from the Southdown dairy
farm for 15 days. The untreated cows shows to have lower PFU/ml compared to treated cows. Even though there is presence of
phages in the untreated cows, they indicate the presence of natural phages, which the tell us that S. aureus have it natural phages
that need to isolated and numerated, which can contribute in treating the bacteria.