Content uploaded by Hari Mohan Saxena
Author content
All content in this area was uploaded by Hari Mohan Saxena on Sep 08, 2020
Content may be subject to copyright.
Content uploaded by Hari Mohan Saxena
Author content
All content in this area was uploaded by Hari Mohan Saxena on Jun 04, 2020
Content may be subject to copyright.
ACTA SCIENTIFIC MICROBIOLOGY (ISSN: 2581-3226)
Volume 3 Issue 6 June 2020
Effects of Bacteriophage Therapy on Host Immune Responses in Brucellosis Affected Cattle
Anju Mohan and Hari Mohan Saxena*
Department of Veterinary Microbiology, College of Veterinary Science, Guru Angad
Dev Veterinary and Animal Sciences University, Ludhiana, India 141001.
*Corresponding Author: Hari Mohan Saxena, Department of Veterinary
Microbiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal
Sciences University, Ludhiana, India .
Research Article
Received: April 10, 2020
Published: May 28, 2020
© All rights are reserved by Anju Mohan and
Hari Mohan Saxena.
Abstract
Keywords: Phage Therapy; Brucellosis;_Antibody Titers; Leukocytes; Lymphocytes; Neutrophils
Introduction
increase in total serum proteins from 0D to 45D, 60D and 75D and from 30D to 45D and a decrease from 45D to 90D, respectively.
in Brucellosis stimulates immune responses in cattle initially which decline slowly after 45 days.
Brucellosis is a major bacterial zoonosis of global importance.
About 500,000 cases of human brucellosis are estimated to occur
worldwide every year.
Bovine brucellosis is endemic in all states of India. In India, the
occurrence of brucellosis is to the extent of 10% in the marginal
of the disease was estimated to run over Rs. 500 crores annually.
In Punjab, overall 17.7% prevalence of brucellosis was reported in
cattle and buffaloes [1,2]. It causes heavy economic loss to the ani
retention of placenta and temporary or permanent infertility [3]
in females and orchitis and epididymitis in males, with excretion
of organisms in semen, uterine discharges and in milk [4]. Once
infected, the animal may continue to shed bacteria and remains a
source of infection to others for long period[2]. Sometimes the vac
cinated animals may also suffer from Brucellosis due to inadequate
immunity induced by the vaccine [5]. Phage therapy may be effec
tive in chronic carriers infected with antibiotic resistant bacteria.
However, little data are available in the published literature on the
effects of phage therapy on host immune response to natural in
fection of Brucellosis in cattle. The present study was, therefore,
undertaken to explore this aspect of phage therapy of bovine bru
cellosis.
Materials and Methods
Ethical approval
All the experimental protocols performed on cattle were ap
mals were kept in IAEC approved facilities and received feed and
water ad libitum.
Infected cattle
Six naturally infected brucellosis positive adult cattle, which had
been vaccinated during calf hood with B. abortus_strain 19 vaccine
Citation: Anju Mohan and Hari Mohan Saxena. “Effects of Bacteriophage Therapy on Host Immune Responses in Brucellosis Affected Cattle".
Microbiology
147
Effects of Bacteriophage Therapy on Host Immune Responses in Brucellosis Affected Cattle
Dairy Farm were included in the study.
Preparation of phage for therapy
Phage preparation was produced employing standardized opti
mum conditions. The eluted phage preparation in SM diluent was
the therapy. The phage count in the preparation was made to the
required dose of 108 pfu/ ml and stored at 4°C until use.
Sterility testing of phage preparation
Sterility test was carried out as per the Indian Pharmacopoeia
Section 2.2.11. A loopful of the phage preparation was suspended
in 5ml BHI broth as well as streaked on BHI and blood agar plates
followed by incubation at 37°C. The broth and the plates were ex
amined upto 48 hours for any microbial growth.
Safety test of phage in mice
Safety test was conducted by injecting 0.5 ml of phage prepa
ration through subcutaneous, intramuscular and intraperitoneal
route into each of the three mice. The mice were observed for any
untoward reactions or mortality till 7th day of inoculation.
Treatment trials in cattle
Treatment trials of the phage preparation were carried out on
naturally Brucellosis infected Holstein Friesian crossbred adult
cattle at the University Dairy Farm, GADVASU, Ludhiana. Six Bru
cellosis positive adult cattle were treated with 2 ml of 108 pfu/
ml_brucellaphage preparation through subcutaneous route.
Collection of blood
Blood samples were collected from cattle through jugular vein
blood was collected for separating leukocytes while sera were sep
studying the immune response of the treated animals.
Analysis of immune responses
Estimation of total protein levels in serum samples
The total protein content in serum samples of phage treated
cattle at 0, 30, 45, 60, 75 and 90 days post treatment were deter
Estimation of total globulin levels in serum samples
Total globulin levels in the serum samples of treated cattle at 0,
30, 45, 60, 75 and 90 days post treatment were estimated by sub
tracting albumin content from the total protein level determined
by using Vitros TP slides, Vitros ALB DT slides and the Vitros DTSC
Estimation of antibody titers
treated cattle at 0, 30, 45, 60, 75 and 90 days post treatment were
methods followed have been described earlier [5].
Estimation of total leukocyte counts (TLC) in the blood of
treated cattle
Levels of total leukocytes in blood samples of phage treated Bru
cellosis affected cattle at 0, 30, 45, 60, 75 and 90 days post treat
diluted blood was loaded into Neubeauer’s counting slide and a
cover slip was put over it. The number of cells in the WBC counting
chamber was counted under a microscope.
Estimation of differential leukocyte counts (DLC) in the blood
of treated cattle
Levels of leukocyte subsets in blood samples of phage treated
Brucellosis affected cattle were determined at 0, 30, 45, 60, 75
and 90 days post treatment. Blood smear from treated cattle was
prepared on a clean microscopic slide and the slide was air dried.
Fixation was done by immersing the slide in methanol for around
minutes. Afterwards it was washed in running water and the slide
was kept for drying. The slide was observed under a microscope
and the total numbers of cells were counted upto 100 cells.
Statistical analysis of data
test.
Results and Discussion
Bacteriophages are viruses that infect and multiply inside the
bacteria. Bacteriophages that infect the Brucella species are called
Brucellaphages. Use of lytic bacteriophages to treat Brucellosis can
be a cheap and effective alternative to antibiotics for control of the
disease. Lytic phages grow in the viable bacterial cells, reproduce
[6].
make them a safe alternative to antibiotics for treatment of bac
terial diseases of animals [6]. Our present study was undertaken
Citation: Anju Mohan and Hari Mohan Saxena. “Effects of Bacteriophage Therapy on Host Immune Responses in Brucellosis Affected Cattle".
Microbiology
148
Effects of Bacteriophage Therapy on Host Immune Responses in Brucellosis Affected Cattle
to investigate the effect of brucellaphage therapy on immune re
sponses in cattle naturally infected with Brucella abortus.
A broad acting phage lytic to Brucella organisms isolated in our
laboratory was used for the present study. Brucella abortus strain
19 organism was used for the revival and propagation of phage.
ence of phage by secondary streaking. The brucellaphage lysed
Brucella abortus strain 19, Brucella abortus strain 99 and Brucella
melitensis
the heterologous species tested viz. Staphylococcus aureus, Salmo-
nella species, Escherichia coli and Pasteurella multocida.
5.05 to 8.15 in 45 days and then slowly declined to 6.39 by 90 days
proteins from 0D to 45D, 60D and 75D and from 30D to 45D fol
lowed by a decrease from 45D to 90D, respectively was very sig
to 4.86 in one and a half month and thereafter gradually declined
crease in serum globulin level from 0D to 45D and 60D was very
90th
3 months of therapy. The rise in IHA titers from 0D to 45D and
Sl. No. 0 D 30D 45 D 60 D 75 D 90 D
1 5.6 7.3 7.6 7.5 6.6 6.6
2 5.8 6.7 6.7 6.8 6.2 5.6
3 6.0 6.9 9.0 8.0 7.1 6.7
4 3.9 6.2 9.3 8.1 7.7 7.1
5 4.0 5.8 8.2 7.5 6.7 5.8
6 5.0 6.2 8.1 7.2 7.1 6.6
Mean ± SD 5.05 ±
0.91
6.51 ±
0.54
8.15 ±
0.94
7.51 ±
0.48
6.89 ±
0.51
6.39 ±
0.57
Table 1: Total serum protein levels in Brucellosis
affected cattle after phage therapy.
Sl. No. 0 D 30D 45 D 60 D 75 D 90 D
1 2.1 2.3 4.7 4.4 4.0 3.8
2 2.4 2.8 3.7 3.7 3.0 2.6
3 3.0 3.1 6.6 4.5 3.8 3.8
4 2.4 4.9 6.0 5.0 4.0 3.6
5 1.0 1.7 4.4 3.5 3.2 2.0
6 2.8 3.6 3.8 3.7 3.3 2.6
1.01
1.18
0.58
0.43
0.76
Table 2: Serum globulin levels in Brucellosis
affected cattle after phage therapy.
Figure 1: Serum proteins and globulin levels in
Brucellosis affected cows treated with phage.
The decline in titers from 0D to 60D and 75D and from 45D to 60D
Total leukocyte count in the peripheral blood of Brucellosis
affected cattle increased from 5766.66 to 8150 by one month fol
lowed by a decline to 6883.33 till 60 day and then gradual increase
Citation: Anju Mohan and Hari Mohan Saxena. “Effects of Bacteriophage Therapy on Host Immune Responses in Brucellosis Affected Cattle".
Microbiology
149
Effects of Bacteriophage Therapy on Host Immune Responses in Brucellosis Affected Cattle
Sl. No. 0 D 30D 45 D 60 D 75 D 90 D
1 2.204 2.505 2.806 2.806 2.806 2.806
2 1.602 2.204 2.806 2.806 1.903 1.903
3 1.602 2.505 2.806 2.204 1.903 1.602
4 2.204 2.505 2.806 2.806 2.806 2.204
5 1.903 2.204 2.505 2.505 2.505 2.505
6 2.204 2.505 2.806 2.505 2.204 1.903
0.29
0.15
0.12
0.24
0.41
2.153
Table 3: Antibody titers by standard tube agglutination test
Sl. No. 0 D 30D 45 D 60 D 75 D 90 D
1 1.602 2.204 3.408 3.408 3.408 3.408
2 1.903 3.107 3.709 3.709 1.903 1.903
3 1.602 1.903 4.311 3.408 2.806 2.204
4 1.602 1.903 3.408 3.408 3.408 1.903
5 1.903 2.204 3.408 3.408 3.408 3.408
6 2.204 3.709 4.311 2.204 1.903 1.602
0.24
0.73
0.44
0.52
0.73
0.80
Table 4: Antibody titers by microtitre plate agglutination test
Sl.No. 0 D 30D 45 D 60 D 75 D 90 D
1 2.505 3.107 3.709 3.709 3.709 3.709
2 1.903 3.107 3.408 2.505 2.204 1.903
3 1.602 1.903 3.709 3.408 2.505 2.204
4 1.903 2.505 4.311 4.311 4.311 3.709
5 2.204 3.408 3.709 3.709 3.709 3.709
6 2.204 3.709 4.311 4.311 3.107 1.903
0.31
0.65
0.36
0.67
0.80
0.94
Table 5: Antibody titers by indirect haemagglutination test
test in brucellosis affected cattle treated with phage.
siderably from 54.5 to 35.33 by 1 month and then rose to 60 by
45 day, followed by slight decline reaching 54.83 by 90 day post
to 3.16 by 30 day and then declined to 1.66 by 45 day, maintained
Sl. No. 0 D 30D 45 D 60 D 75 D 90 D
1 1.453 1.434 1.331 0.784 0.754 1.100
2 0.610 1.292 1.245 0.704 0.500 0.756
3 1.293 1.422 1.176 0.671 0.753 0.780
4 0.496 1.303 0.691 0.511 0.574 0.562
5 1.408 1.347 1.001 0.685 0.709 0.788
6 1.473 1.367 1.174 0.733 0.822 0.832
0.44
0.05
0.20
0.09
0.12
0.17
Table 6:
Brucellosis affected cattle treated with phage.
followed by increase from 30D to 45D, 60D, 75D and 90D was very
Figure 2: Antibody titers in Brucellosis affected
cows after phage therapy.
Sl. No. 0 D 30D 45 D 60 D 75 D 90 D
1 5900 7500 5900 6100 5200 9200
2 4800 9200 8200 5200 6800 8100
3 6200 10200 7800 9800 7600 10200
4 6900 6500 5900 5800 9900 6500
5 5700 8300 9400 6900 8400 7300
6 5100 7200 8500 7500 8900 7800
Mean
758.06
1369.30
1430.26
1643.67
1660.12
8183.33
1331.79
Table 7:
affected animals treated with phage.
Citation: Anju Mohan and Hari Mohan Saxena. “Effects of Bacteriophage Therapy on Host Immune Responses in Brucellosis Affected Cattle".
Microbiology
150
Effects of Bacteriophage Therapy on Host Immune Responses in Brucellosis Affected Cattle
, monocytes
ing the period of 90 days after treatment with the phage.
Conclusion
Our present study indicates that phage therapy in Brucellosis
stimulates immune responses in cattle initially for about one and
Sl. No. 0 D 30D 45 D 60 D 75 D 90 D
1 1 3 2 2 1 3
2 4 4 3 4 2 3
3 4 2 1 1 3 2
4 4 4 1 1 2 1
5 1 3 2 1 4 2
6 3 3 1 1 2 2
1.47
0.75
0.81
1.21
1.21
0.75
Table 9: Blood monocyte counts in Brucellosis
affected animals treated with phage.
Sl. No. 0 D 30D 45 D 60 D 75 D 90 D
1 31 50 42 40 48 44
2 39 30 33 36 38 44
3 36 24 33 31 46 33
4 43 44 44 33 30 42
5 50 34 28 38 32 43
6 40 30 39 40 36 39
6.43
9.77
6.15
3.72
7.31
4.26
Table 10: Blood neutrophil counts in brucellosis
affected animals treated with phage.
Sl. No. 0 D 30D 45 D 60 D 75 D 90 D
1 66 50 55 56 50 51
2 54 30 62 58 58 50
3 55 24 64 65 49 62
4 50 44 53 63 65 55
5 46 34 68 60 60 53
6 56 30 58 57 58 58
6.74
9.77
5.69
3.54
6.12
4.53
Table 8: Blood lymphocyte counts in Brucellosis
affected animals treated with phage.
Figure 3: Total blood leukocyte counts in Brucellosis
affected animals treated with phage. Figure 4: Levels of different subsets of leukocytes in
Brucellosis affected animals treated with phage.
a half month which decline slowly after 45 days of initiation of
therapy.
Bibliography
1. Jain U., et al. “Outbreak of brucellosis in buffaloes aborted in
a
Veterinary World
2. Pandeya Y R., et al. “Seroprevalence of brucellosis in different
International Journal
of Infection and Microbiology
3. Kollannur JD., et al. “Epidemiology and economics of brucel
XIII International Congress in Animal Hygiene. International
4. Godfroid J., et alRevue Sci-
32.1
Citation: Anju Mohan and Hari Mohan Saxena. “Effects of Bacteriophage Therapy on Host Immune Responses in Brucellosis Affected Cattle".
Microbiology
151
Effects of Bacteriophage Therapy on Host Immune Responses in Brucellosis Affected Cattle
5. Mohan A., et al
tibodies of naturally infected and healthy vaccinated cattle
by standard tube agglutination test, microtiter plate agglu
tination test, indirect hemagglutination assay, and indirect
Veterinary World 9.7
6. Haq I U., et al. “Bacteriophages and their implications on fu
Virology Journal
• Prompt Acknowledgement after receiving the article
• Thorough Double blinded peer review
• Rapid Publication
•
• High visibility of your Published work
Assets from publication with us
Website:
Submit Article:
Email us:
Contact us: +91 9182824667
Citation: Anju Mohan and Hari Mohan Saxena. “Effects of Bacteriophage Therapy on Host Immune Responses in Brucellosis Affected Cattle".
Microbiology
